阅读说明：本技术 用pd-1轴结合拮抗剂、抗代谢物和铂剂治疗肺癌的方法 (Methods of treating lung cancer with PD-1 axis binding antagonists, antimetabolites, and platinum agents ) 是由 G·香卡 A·桑德勒 D·S·陈 林伟瑜 于 2019-07-18 设计创作，主要内容包括：本公开提供用于治疗个体的肺癌(比如非小细胞肺癌,例如IV期非鳞状非小细胞肺癌)的方法。所述方法包括向所述个体施用PD-1轴结合拮抗剂(比如抗PD-L1抗体,例如阿特珠单抗)、抗代谢物(例如培美曲塞)和铂剂(例如顺铂或卡铂)。(The present disclosure provides methods for treating lung cancer (such as non-small cell lung cancer, e.g., stage IV non-squamous non-small cell lung cancer) in an individual. The methods comprise administering to the subject a PD-1 axis binding antagonist (such as an anti-PD-L1 antibody, e.g., atelizumab), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., cisplatin or carboplatin).)

1. A method of treating an individual having lung cancer, comprising administering to the individual an effective amount of an anti-PD-L1 antibody, an antimetabolite, and a platinum agent, wherein the treatment extends the Progression Free Survival (PFS) of the individual.

2. The method of claim 1, wherein the treatment extends Overall Survival (OS) of the individual.

3. A method of treating an individual having lung cancer, comprising administering to the individual an effective amount of an anti-PD-L1 antibody, an antimetabolite, and a platinum agent, wherein the treatment extends the Overall Survival (OS) of the individual.

4. The method of claim 1, wherein said treatment extends PFS of said subject by at least about 6 months.

5. The method of claim 2 or 3, wherein the treatment extends OS of the subject by at least about 15 months.

6. The method of any one of claims 1-5, wherein the anti-PD-L1 antibody comprises:

(a) heavy chain variable region (V)_H) Comprising HVR-H1 comprising the amino acid sequence of GFTFSDSWIH (SEQ ID NO:1), HVR-2 comprising the amino acid sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:2) and HVR-3 comprising the amino acid sequence of RHWPGGFDY (SEQ ID NO:3), and

(b) light chain variable region (V)_L) Comprising HVR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO:4), HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO:5), and HVR-L3 comprising the amino acid sequence of QQYLYHPAT (SEQ ID NO: 6).

7. The method of any one of claims 1-6, wherein the anti-PD-L1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:7 _H) And a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO 8_L)。

8. The method of any one of claims 1-7, wherein the anti-PD-L1 antibody is atezumab.

9. The method of any one of claims 1-8, wherein the antimetabolite is pemetrexed, 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, hydroxyurea, or methotrexate.

10. The method of claim 9, wherein the antimetabolite is pemetrexed.

11. The method of any one of claims 1-10, wherein the platinum agent is carboplatin.

12. The method of any one of claims 1-10, wherein the platinum agent is cisplatin.

13. According to the claimsThe method of any one of claims 1-11, wherein the anti-PD-L1 antibody is administered at a dose of 1200mg, wherein the platinum agent is carboplatin and is administered at a dose sufficient to achieve an AUC of 6mg/ml/min, and wherein the antimetabolite is pemetrexed and is 500mg/m²The dosage of (a).

14. The method of any one of claims 1-10 and claim 12, wherein the anti-PD-L1 antibody is administered at a dose of 1200mg, wherein the platinum agent is cisplatin and is at 75mg/m ²And wherein the antimetabolite is pemetrexed and is administered at 500mg/m²The dosage of (a).

15. The method of any one of claims 1-11 and claim 13, wherein the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and each 21-day cycle of cycles 1 to 4, wherein the anti-PD-L1 antibody is altlizumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²And wherein the platinum agent is carboplatin and is administered on day 1 at a dose sufficient to achieve AUC of 6 mg/ml/min.

16. The method of any one of claims 1-10, claim 12, and 14, wherein the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and in each 21-day cycle of cycles 1 to 4, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²And wherein the platinum agent is cisplatin and is administered at 75mg/m on day 1²The dosage of (a).

17. The method of any one of claims 15-16, wherein the anti-PD-L1 antibody, antimetabolite, and platinum agent are administered sequentially on day 1 of cycles 1 through 4.

18. The method of claim 17, wherein the anti-PD-L1 antibody is administered prior to the anti-metabolite on day 1 of cycles 1 to 4, and wherein the anti-metabolite is administered prior to the platinum agent.

19. The method of any one of claims 15-18, wherein the anti-PD-L1 antibody and the antimetabolite are further administered after cycle 4, each 21-day cycle of each cycle after cycle 4, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, and wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²The dosage of (a).

20. The method of claim 19, wherein the anti-PD-L1 antibody and the antimetabolite are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 4.

21. The method of claim 20, wherein the anti-PD-L1 antibody is administered prior to the antimetabolite on day 1 after cycle 4.

22. The method of any one of claims 1-11 and claim 13, wherein the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and each 21-day cycle of cycles 1 to 6, wherein the anti-PD-L1 antibody is altlizumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1 ²And wherein the platinum agent is carboplatin and is administered on day 1 at a dose sufficient to achieve AUC of 6 mg/ml/min.

23. The method of any one of claims 1-10, claim 12 and 14, wherein the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and each 21-day cycle of cycles 1 through 6, whereinThe anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²And wherein the platinum agent is cisplatin and is administered at 75mg/m on day 1²The dosage of (a).

24. The method of claim 22 or 23, wherein the anti-PD-L1 antibody, antimetabolite, and platinum agent are administered sequentially on day 1 of cycles 1 through 6.

25. The method of claim 24, wherein the anti-PD-L1 antibody is administered prior to the anti-metabolite on day 1 of cycles 1 to 6, and wherein the anti-metabolite is administered prior to the platinum agent.

26. The method of any one of claims 22-25, wherein the anti-PD-L1 antibody and the antimetabolite are further administered after cycle 6, each 21-day cycle of each cycle after cycle 6, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, and wherein the antimetabolite is pemetrexed and is 500mg/m on day 1 ²The dosage of (a).

27. The method of claim 26, wherein the anti-PD-L1 antibody and the antimetabolite are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 6.

28. The method of claim 27, wherein the anti-PD-L1 antibody is administered prior to the antimetabolite on day 1 of each 21-day cycle of each cycle after cycle 6.

29. The method of any one of claims 1-28, wherein the anti-PD-L1 antibody, the platinum agent, and the antimetabolite inhibitor are each administered intravenously.

30. The method of any one of claims 1-29, wherein the lung cancer is non-small cell lung cancer (NSCLC).

31. The method of claim 30, wherein the NSCLC is stage IV non-squamous NSCLC.

32. The method of claim 31, wherein the individual has never received treatment for stage IV non-squamous NSCLC.

33. The method of claim 31, wherein the individual has never received chemotherapy for stage IV non-squamous NSCLC.

34. The method of any one of claims 1-33, wherein the individual is asian.

35. The method of any one of claims 1-34, wherein the individual is at least 65 years old.

36. The method of any one of claims 1-35, wherein the individual is a never smoker.

37. The method of any one of claims 1-36, wherein the individual is PD-L1 high.

38. The method of any one of claims 1-36, wherein the individual is PD-L1 negative.

39. A method of treating an individual having stage IV non-squamous non-small cell lung cancer (NSCLC), comprising administering to the individual an effective amount of atuzumab, pemetrexed, and carboplatin, wherein the atuzumab is administered at a dose of 1200mg, and the pemetrexed is administered at 500mg/m²And said carboplatin is administered at a dose sufficient to achieve AUC of 6mg/ml/min, wherein said treatment extends Progression Free Survival (PFS).

40. The method of claim 39, wherein treatment extends Overall Survival (OS) of the individual.

41. The method of claim 39 or 40, wherein the atuzumab, pemetrexed, and carboplatin are administered in four 21-day cycles, and further atuzumab and pemetrexed are administered in a 21-day cycle after cycle 4; wherein the atelizumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 4 and 500mg/m on day 1 of each 21-day cycle of cycles 1 to 4 ²And carboplatin at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1 of each 21-day cycle of cycles 1 through 4, and wherein atezumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of each cycle after cycle 4 and 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 4²Further administered pemetrexed.

42. The method of claim 41, wherein the atuzumab, pemetrexed, and carboplatin are administered sequentially on day 1 of cycles 1 to 4.

43. The method of claim 42, wherein the atzezumab was administered prior to pemetrexed on day 1 of cycles 1 to 4, and wherein pemetrexed was administered prior to carboplatin.

44. The method of any one of claims 41-43, wherein the atuzumab and the pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 4.

45. The method of claim 44, wherein the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 4.

46. According to the claimsThe method of claim 39 or 40, wherein the atlizumab, pemetrexed, and carboplatin are administered in six 21-day cycles, and further atlizumab and pemetrexed are administered in a 21-day cycle following cycle 6, wherein the atlizumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 6 and 500mg/m on day 1 of each 21-day cycle of cycles 1 to 6 ²And carboplatin at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1 of each 21-day cycle of cycles 1 through 6, and wherein atezumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of each cycle after cycle 6 and 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 6²Further administered pemetrexed.

47. The method of claim 46, wherein the atuzumab, pemetrexed, and carboplatin are administered sequentially on day 1 of cycles 1 to 6.

48. The method of claim 47, wherein on day 1 of cycles 1-6, atuzumab is administered prior to pemetrexed, and wherein pemetrexed is administered prior to carboplatin.

49. The method of any one of claims 46-48, wherein the atuzumab and the pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 6.

50. The method of claim 49, wherein the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 6.

51. The method of any one of claims 39-50, wherein the atezumab, the pemetrexed, and the carboplatin are each administered intravenously.

52. The method of any one of claims 39-41, wherein treatment extends PFS of the subject by at least about 6 months.

53. The method of any one of claims 39-52, wherein treatment extends OS of the subject by at least about 15 months.

54. A method of treating an individual having stage IV non-squamous non-small cell lung cancer (NSCLC), comprising administering to the individual an effective amount of atuzumab, pemetrexed, and cisplatin, wherein the atuzumab is administered at a dose of 1200mg, and 500mg/m²Is administered at a dose of 75mg/m, and²wherein said treatment extends Progression Free Survival (PFS) in said subject.

55. The method of claim 54, wherein treatment extends the Overall Survival (OS) of the individual.

56. The method of claim 54 or 55, wherein the atuzumab, pemetrexed, and cisplatin are administered in four 21-day cycles, and further atuzumab and pemetrexed are administered in a 21-day cycle after cycle 4; wherein the atelizumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 4 and 500mg/m on day 1 of each 21-day cycle of cycles 1 to 4 ²And administering pemetrexed at a dose of 75mg/m on day 1 of each 21-day cycle of cycles 1 through 4²And wherein atelizumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of each cycle after cycle 4 and 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 4²Further administered pemetrexed.

57. The method of claim 56, wherein the atuzumab, pemetrexed, and cisplatin are administered sequentially on day 1 of cycles 1 through 4.

58. The method of claim 57, wherein on day 1 of cycles 1-4, atuzumab is administered prior to pemetrexed, and wherein pemetrexed is administered prior to cisplatin.

59. The method of any one of claims 56-58, wherein the atuzumab and the pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 4.

60. The method of claim 59, wherein the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 4.

61. The method of claim 54 or 55, wherein the atuzumab, pemetrexed, and cisplatin are administered in six 21-day cycles, and further atuzumab and pemetrexed are administered in a 21-day cycle after cycle 6; wherein the atelizumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 6 and 500mg/m on day 1 of each 21-day cycle of cycles 1 to 6 ²And administering pemetrexed at a dose of 75mg/m on day 1 of each 21-day cycle of cycles 1 through 6²And wherein the atelizumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of each cycle after cycle 6 and 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 6²Further administered pemetrexed.

62. The method of claim 61, wherein the atuzumab, pemetrexed, and cisplatin are administered sequentially on day 1 of cycles 1 through 6.

63. The method of claim 62, wherein on day 1 of cycles 1-6, atuzumab is administered prior to pemetrexed, and wherein pemetrexed is administered prior to cisplatin.

64. The method of any one of claims 61-63, wherein the atuzumab and the pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 6.

65. The method of claim 64, wherein the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 6.

66. The method of any one of claims 54-65, wherein the atuzumab, the pemetrexed, and the cisplatin are each administered intravenously.

67. The method of any one of claims 54-66, wherein treatment extends PFS of the subject by at least about 6 months.

68. The method of any one of claims 54-67, wherein treatment extends OS of the subject by at least about 15 months.

69. The method of any one of claims 39-68, wherein the individual has never received treatment for stage IV non-squamous NSCLC.

70. The method of any one of claims 39-69, wherein the individual has never received chemotherapy for stage IV non-squamous NSCLC.

71. The method of any one of claims 39-70, wherein the individual is Asian.

72. The method of any one of claims 39-71, wherein the individual is at least 65 years old.

73. The method of any one of claims 39-72, wherein the individual is a never smoker.

74. The method of any one of claims 39-73, wherein the individual is PD-L1 high.

75. The method of any one of claims 39-74, wherein the individual is PD-L1 negative.

76. The method of any one of claims 1-75, wherein the individual is a human.

77. A kit comprising an anti-PD-L1 antibody for use in combination with an antimetabolite and a platinum agent in the treatment of an individual having lung cancer according to the method of any one of claims 1-38 and claim 76.

78. A kit comprising atuzumab for use in combination with pemetrexed and carboplatin in treating an individual having lung cancer according to the method of any one of claims 39-53 and 69-76.

79. A kit comprising atuzumab for use in combination with pemetrexed and cisplatin in treating an individual having lung cancer according to the method of any one of claims 54-76.

80. An anti-PD-L1 antibody for use in a method of treating lung cancer in an individual, the method comprising administering to the individual an effective amount of an anti-PD-L1 antibody, an antimetabolite, and a platinum agent, wherein the treatment extends progression-free survival (PFS) and/or Overall Survival (OS) of the individual.

81. The anti-PD-L1 antibody according to claim 80 for use in a method according to any one of claims 1-38 and claim 76.

82. Composition comprising atelizumab for use in said compositionIn a method of treating stage IV non-squamous non-small cell lung cancer (NSCLC), the method comprising administering to the individual an effective amount of atuzumab, pemetrexed, and carboplatin, wherein the atuzumab is administered at a dose of 1200mg, at 500mg/m ²And administering said carboplatin at a dose sufficient to achieve AUC of 6mg/ml/min, and wherein said treatment extends Progression Free Survival (PFS) and/or Overall Survival (OS) of said individual.

83. The composition of claim 82, for use in the method of any one of claims 39-53 and 69-76.

84. A composition comprising atuzumab for use in a method of treating stage IV non-squamous non-small cell lung cancer (NSCLC), the method comprising administering to the individual an effective amount of atuzumab, pemetrexed, and cisplatin, wherein the atuzumab is administered at a dose of 1200mg, at 500mg/m²Is administered at a dose of 75mg/m, and²and wherein said treatment extends Progression Free Survival (PFS) and/or Overall Survival (OS) of said individual.

85. The composition of claim 84, for use in the method of any one of claims 54-76.

Technical Field

The present disclosure relates to methods of treating cancer by administering a PD-1 axis binding antagonist (e.g., atelizumab) in combination with an antimetabolite (e.g., pemetrexed) and a platinum agent (e.g., carboplatin or cisplatin).

Background

Lung Cancer remains the leading cause of Cancer death worldwide, being the most common Cancer in men, accounting for about 13% of all new cancers in 2008 (Jemal et al, (2011) CA Cancer j. clin 61: 69-90). It is estimated that 313,000 new lung cancer cases and 268,000 lung cancer deaths in europe in 2012 (GLOBOCAN (2012)). Estimated incidence of cancer: the global mortality and morbidity in 2012 can be obtained from the following websites: globocan (dot) iarc (dot) fr/Pages/fact _ sheets _ cameras. Similar data from the United states estimated that there would be 221,200 new cases of lung Cancer and 158,040 deaths of lung Cancer in 2015 (Siegel et al, (2015) CA Cancer J Clin.65: 5-29).

Non-small cell lung Cancer (NSCLC) is a major subtype of lung Cancer, accounting for approximately 85% of all cases (Molina et al, (2008) Mayo Clin Proc 83:584 (94); Howlader et al, (2011) SEE R Cancer standards review, 1975-. NSCLC can be divided into two major histological types: adenocarcinoma and squamous cell carcinoma (Travis et al, (2011) J Thorac Oncol 6: 244-85). Adenocarcinoma histology accounts for more than half of all NSCLCs, while squamous cell histology accounts for about 25% (Langer et al, (2010) J Clin Oncol28: 5311-20). The remaining NSCLC cases are represented by large cell carcinoma, neuroendocrine tumors, sarcomatoid carcinoma, and poorly differentiated histology.

The 5-year overall survival rate for advanced disease is 2% -4% depending on geographic location (Cetin et al, (2011) Clin epidemic 3: 139-48). Adverse prognostic factors for NSCLC patient survival include advanced disease at initial diagnosis, poor condition and a history of inadvertent weight loss. More than half of NSCLC patients are diagnosed with distant disease, which directly leads to poor survival prospects.

For locally advanced or metastatic NSCLC patients that are not accompanied by EGFR mutations or ALK gene rearrangements, platinum-based regimens remain standard one-line treatment regimens. In particular, for newly diagnosed advanced non-squamous NSCLC, the standard of care is to have cisplatin or carboplatin and taxane or pemetrexed platinum-containing dual drugs, with or without bevacizumab. However, current treatment regimens are associated with a large number of toxicities (e.g., febrile neutropenia, myelosuppression, nausea, hair loss, nephropathy, and neuropathy), and are generally poorly tolerated by the elderly and ill-conditioned patients. In addition, the survival benefits of cytotoxic chemotherapy have reached a plateau with an overall response rate of about 20% and a 1-year survival rate ranging from 31% to 36% (Schiller et al, (2002) N Engl J Med.346: 92-98).

There is a recognized difference in disease characteristics between adenocarcinoma and squamous NSCLC. First, squamous tumors are usually present in the central airways and usually remain localized in the bronchial epithelium (Hirsch et al, (2008) J Thorac Oncol.3:1468-1481), while non-squamous tumors are more commonly localized in the lung parenchyma distal to the central airways. Evaluation of NSCLC tumor tissue generally revealed a cytological difference between squamous cell types (keratinization, intracellular bridges and central necrosis) and adenocarcinomas (glandular structures). Immunohistochemical markers may support histological diagnosis in cases where tumor samples are poorly differentiated or available tissue is limited. Thyroid transcription factor-1 is expressed rarely in squamous cells and strongly in adenocarcinomas. In contrast, p63, CK5/6 and 34. beta.E 12 are strongly expressed in squamous cell carcinoma and less frequently in adenocarcinoma (Travis et al, (2011) J Thorac Oncol.6: 244-85).

Genetic changes of prognostic and/or predictive significance in NSCLC include mutations in the Epidermal Growth Factor Receptor (EGFR), rearrangements in the Anaplastic Lymphoma Kinase (ALK) gene, and mutations in the GTPase Kras (Kras) gene. The incidence of these mutations varies between squamous cell carcinoma and adenocarcinoma. For example, EGFR kinase domain mutations have been reported in 10% -40% of adenocarcinoma NSCLC patients, but are rarely seen in squamous NSCLC patients (Herbst et al, (2008) NEngl J Med.359: 1367-80). Similarly, an ALK fusion oncogene, which is considered to be the driver of pulmonary tumorigenesis, is observed in about 7% of adenocarcinoma patients, but is very rare in squamous histology (Herbst et al (2008) N Engl J Med.359: 1367-80; Langer et al (2010) J Clin Oncol 28: 5311-20). Furthermore, KRAS mutations are very rare in squamous NSCLC, and they can be observed in up to 30% of cases of adenocarcinoma NSCLC (Travis et al, (2011) J Thorac oncol.6: 244-85).

Genotype-directed therapy has the potential to significantly improve the balance between benefit and toxicity in selected NSCLC patients (primarily non-squamous histology) characterized by alterations that drive oncogenes, including sensitized EGFR mutations and ALK rearrangements. However, these mutations are more prevalent in adenocarcinoma NSCLC and are very rare in squamous NSCLC. Phase III randomization studies of gefitinib (IPASS), Erlotinib (EURTAC) and afatinib (LUX-Lung 3) showed significant improvement in PFS and ORR compared to platinum-containing dual-drug chemotherapy (Fukuoka et al, (2011) J Clin Oncol.29: 2866-2874; Rosell et al, (2012) Lancet Oncol.13: 239-246; Yang et al (2012) Lancet Oncol.13: 539-548). Similarly, the ALK inhibitors crizotinib and ceritinib also showed efficacy in NSCLC patients positive for ALK rearrangement as defined by fluorescence in situ hybridization (Crino et al, (2011) J Clin Oncol.29: Abstract 7514; Camidge et al, (2012) Lancet Oncol.13:1011- Abstract LBA1 PR; shaw et al, (2014) N Engl J Med.370: 2537-; XALKORIUSPI；ZYKADIA^TM USPI)。

Despite advances in new targeted therapies and new combinations of chemotherapy, the survival rate of advanced NSCLC disease remains low and the acquired resistance to targeted agents is a major clinical problem. Accordingly, there is a need in the art for alternative treatment options to improve the prognosis, e.g., extended survival, of patients with the disease.

All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot accession numbers, are incorporated by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference.

Disclosure of Invention

Provided herein are methods and uses of anti-PD-L1 antibodies for treating lung cancer patients. In particular, the methods and uses are based on atlizumab in the treatment of treatment-naive stage IV non-squamous non-small cell lung cancer (NSCLC) individuals (e.g., chemotherapy-naive individuals)Data from phase III randomized clinical studies combining pemetrexed and a platinum agent (e.g., carboplatin or cisplatin). This study showed that, compared to chemotherapy alone, Initial (first line) treatment of the combination of (atezumab) plus chemotherapy (pemetrexed + carboplatin or pemetrexed + cisplatin) reduced the risk of disease progression or death (PFS). In addition, compared to chemotherapy alone, receivingPatients with (atelizumab) plus chemotherapy (pemetrexed + carboplatin or pemetrexed + cisplatin) exhibited a numerical improvement in overall survival. TECENTRIQ andthe safety of the chemotherapy combination appears to be consistent with the known safety of the individual drugs, and no new safety signals were found for this combination.

In one aspect, provided herein is a method of treating an individual having lung cancer, comprising administering to the individual an effective amount of an anti-PD-L1 antibody, an antimetabolite, and a platinum agent, wherein the treatment extends the Progression Free Survival (PFS) of the individual. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, the treatment increases PFS in an individual by at least about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 months (including any range between these values) as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) who has received treatment with an antimetabolite (e.g., pemetrexed) and a platinum agent (e.g., carboplatin or cisplatin).

In another aspect, provided herein is a method of treating an individual having lung cancer, comprising administering to the individual an effective amount of an anti-PD-L1 antibody, an antimetabolite, and a platinum agent, wherein the treatment extends the Overall Survival (OS) of the individual. In some embodiments, Overall Survival (OS) is measured as the period of time from the start of treatment to death. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months. In some embodiments, the treatment increases OS in the individual by at least about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 months (including any range between these values) as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) and receiving treatment with an antimetabolite (e.g., pemetrexed) and a platinum agent (e.g., carboplatin or cisplatin).

In some embodiments, the anti-PD-L1 antibody comprises: (a) a heavy chain variable region (VH) comprising HVR-H1 comprising amino acid sequence GFTFSDSWIH (SEQ ID NO: 1), HVR-2 comprising amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and HVR-3 comprising amino acid RHWPGGFDY (SEQ ID NO: 3); and (b) a light chain variable region (VL) comprising HVR-L1 comprising amino acid sequence RASQDVSTAVA (SEQ ID NO: 4), HVR-L2 comprising amino acid sequence SASFLYS (SEQ ID NO: 5), and HVR-L3 comprising amino acid sequence QQYLYHPAT (SEQ ID NO: 6). In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 7 and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 8 (VL) in the amino acid sequence of seq id No. 8. In some embodiments, the anti-PD-L1 antibody is atelizumab.

In some embodiments, the antimetabolite is pemetrexed, 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, hydroxyurea, or methotrexate. In some embodiments, the antimetabolite is pemetrexed. In some embodiments, the platinum agent is carboplatin. In some embodiments, the platinum agent is cisplatin.

In some embodiments, the anti-PD-L1 antibody is administered at a dose of 1200mg, wherein the platinum agent is carboplatin and is administered at a dose sufficient to achieve an AUC of 6mg/ml/min, wherein the antimetabolite is pemetrexed and is 500mg/m²The dosage of (a). In some embodiments, the anti-PD-L1 antibody is administered at a dose of 1200mg, wherein the platinum agent is cisplatin and is at 75mg/m²And wherein the antimetabolite is pemetrexed and is administered at a dose of 500mg/m²The dosage of (a). In some embodiments, the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and each 21-day cycle of cycles 1 to 4, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1 ²Wherein the platinum agent is carboplatin and is administered on day 1 at a dose sufficient to achieve an AUC of 6 mg/ml/min. In some embodiments, at four 21 daysThe anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in cycles, and in each 21-day cycle of cycles 1 through 4, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²Wherein the platinum agent is carboplatin and is administered at 75mg/m on day 1²The dosage of (a). In some embodiments, the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered sequentially on day 1 of cycles 1 through 4. In some embodiments, the anti-PD-L1 antibody is administered prior to the anti-metabolite on day 1 of cycles 1 to 4, and wherein the anti-metabolite is administered prior to the platinum agent. In some embodiments, the anti-PD-L1 antibody and the anti-metabolite are further administered after cycle 4 for each 21-day cycle of each cycle after cycle 4, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the anti-metabolite is pemetrexed and is 500mg/m on day 1 ²The dosage of (a). In some embodiments, the anti-PD-L1 antibody and the antimetabolite are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 4. In some embodiments, the anti-PD-L1 antibody is administered prior to the antimetabolite on day 1 after cycle 4.

In some embodiments, the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and each 21-day cycle of cycles 1 to 6, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²Wherein the platinum agent is carboplatin and is administered at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1. In some embodiments, the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered in four 21-day cycles, and each 21-day cycle of cycles 1 to 6, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the antimetabolite is pemetrexed and is 500mg/m on day 1²Of (2)Amount administered wherein the platinum agent is cisplatin and at 75mg/m on day 1 ²The dosage of (a). In some embodiments, the anti-PD-L1 antibody, the antimetabolite, and the platinum agent are administered sequentially on day 1 of cycles 1 through 6. In some embodiments, the anti-PD-L1 antibody is administered prior to the anti-metabolite on day 1 of cycles 1 to 6, and wherein the anti-metabolite is administered prior to the platinum agent. In some embodiments, the anti-PD-L1 antibody and the anti-metabolite are further administered after cycle 6, for each 21-day cycle of each cycle after cycle 6, wherein the anti-PD-L1 antibody is atezumab and is administered at a dose of 1200mg on day 1, wherein the anti-metabolite is pemetrexed and is 500mg/m on day 1²The dosage of (a). In some embodiments, the anti-PD-L1 antibody and the antimetabolite are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 6. In some embodiments, the anti-PD-L1 antibody is administered prior to the antimetabolite on day 1 of each 21-day cycle of each cycle after cycle 6.

In some embodiments, the anti-PD-L1 antibody, the platinum agent, and the antimetabolite inhibitor are each administered intravenously. In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is stage IV non-squamous NSCLC. In some embodiments, the individual has not received treatment for stage IV non-squamous NSCLC. In some embodiments, the individual has not received chemotherapy for stage IV non-squamous NSCLC. In some embodiments, the individual is asian or asian descendants. In some embodiments, the individual is at least 65 years old. In some embodiments, the individual is a never-smoker. In some embodiments, the subject is high in PD-L1. In some embodiments, the individual is PD-L1 negative. In some embodiments, the individual is a human.

In another aspect, provided herein is a method of treating an individual having stage IV non-squamous non-small cell lung cancer (NSCLC), comprising administering to the individual an effective amount of atuzumab, pemetrexed, and carboplatin, wherein the atuzumab is administered at a dose of 1200mg, the pemetrexed is administered at 500mg/m²The carboplatin is administered at a dose sufficient to achieve an AUC of 6mg/ml/minWherein the treatment extends Progression Free Survival (PFS). In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, the treatment increases PFS in an individual by at least any one of (including any range between) about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 months, as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) who has received pemetrexed and carboplatin treatment. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, Overall Survival (OS) is measured as the period of time from the start of treatment to death. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months. In some embodiments, the treatment increases OS of the individual by at least any one of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 months (including any range between these values) as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) and receiving pemetrexed and carboplatin treatment. In some embodiments, the alemtuzumab, pemetrexed, and carboplatin are administered in four 21-day cycles, and further alemtuzumab and pemetrexed are administered in a 21-day cycle after cycle 4; wherein the atelizumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 4 and 500mg/m on day 1 of each 21-day cycle of cycles 1 to 4 ²Administering pemetrexed at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1 of each 21-day cycle of cycles 1 through 4, wherein 1200mg is administered on day 1 of each 21-day cycle of cycles 4Further administering atelizumab at 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 4²Further administered pemetrexed. In some embodiments, the attrituximab, pemetrexed, and carboplatin are administered sequentially on day 1 of cycles 1 through 4. In some embodiments, on day 1 of cycles 1 through 4, the atezumab was administered prior to pemetrexed, wherein pemetrexed was administered prior to carboplatin. In some embodiments, the attentizumab and pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 4. In some embodiments, the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 4. In some embodiments, the atuzumab, pemetrexed, and carboplatin are administered in six 21-day cycles, and further atuzumab and pemetrexed are administered in a 21-day cycle after cycle 6; wherein the atelizumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 6 and 500mg/m on day 1 of each 21-day cycle of cycles 1 to 6 ²Administering pemetrexed at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1 of each 21-day cycle of cycles 1 through 6, wherein atezumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 6, and 500mg/m on day 1 of each 21-day cycle of cycles 6²Further administered pemetrexed. In some embodiments, the attrituximab, pemetrexed, and carboplatin are administered sequentially on day 1 of cycles 1 through 6. In some embodiments, on day 1 of cycles 1 through 6, the atezumab was administered prior to pemetrexed, wherein pemetrexed was administered prior to carboplatin. In some embodiments, the attentizumab and pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 6. In some embodiments, the attrituximab is administered prior to the pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 6. In some embodiments, the atezumab, pemetrexed, and carboplatin are each administered intravenously.

In another aspect, the disclosure providesA method for treating an individual having stage IV non-squamous non-small cell lung cancer (NSCLC), comprising administering to the individual an effective amount of atzezumab, pemetrexed, and cisplatin, wherein the atzezumab is administered at a dose of 1200mg and the pemetrexed is administered at 500mg/m ²The cisplatin being administered at a dose of 75mg/m²Wherein the treatment extends Progression Free Survival (PFS) of the individual. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, the treatment increases PFS in an individual by at least any one of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 months (including any range between these values) as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) who has received pemetrexed and cisplatin treatment. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, Overall Survival (OS) is measured as the period of time from the start of treatment to death. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months. In some embodiments, the treatment increases OS of the individual by at least any one of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 months (including any range between these values) compared to an individual with lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) who has received pemetrexed and cisplatin treatment. In some embodiments, the atuzumab, pemetrexed, and cisplatin are administered in four 21-day cycles, and further atuzumab and pemetrexed are administered in a 21-day cycle after cycle 4; wherein in each 21-day cycle of cycles 1 through 4 Atlizumab was administered at a dose of 1200mg on day 1, 500mg/m on day 1 of each 21-day cycle of cycles 1 to 4²Is administered at a dose of 75mg/m on day 1 of each 21-day cycle of cycles 1 through 4²Is administered, wherein the atelizumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of each cycle after cycle 4 and 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 4²Further administered pemetrexed. In some embodiments, the attentizumab, pemetrexed and cisplatin are administered sequentially on day 1 of cycles 1 to 4. In some embodiments, on day 1 of cycles 1 through 4, the atuzumab is administered prior to pemetrexed, wherein pemetrexed is administered prior to cisplatin. In some embodiments, the attentizumab and pemetrexed are administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 4. In some embodiments, the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 4. In some embodiments, the atuzumab, pemetrexed, and cisplatin are administered in six 21-day cycles, and further atuzumab and pemetrexed are administered in a 21-day cycle after cycle 6; wherein the atezumab is administered at a dose of 1200mg on day 1 of each 21-day cycle of cycles 1 to 6 and pemetrexed is administered at 500mg/m on day 1 of each 21-day cycle of cycles 1 to 6 ²Cisplatin was administered at 75mg/m on day 1 of each 21-day cycle of cycles 1 through 6²Wherein atezumab is further administered at a dose of 1200mg on day 1 of each 21-day cycle of each cycle after cycle 6 and pemetrexed is administered at 500mg/m on day 1 of each 21-day cycle of each cycle after cycle 6²The dosage of (a) is further administered. In some embodiments, the attentizumab, pemetrexed and cisplatin are administered sequentially on day 1 of cycles 1 through 6. In some embodiments, on day 1 of cycles 1 through 6, the atezumab was administered prior to pemetrexed, wherein pemetrexed was administered prior to cisplatin. In some embodiments, wherein the atebrine is administered sequentially on day 1 of each 21-day cycle of each cycle after cycle 6Anti-pemetrexed and pemetrexed. In some embodiments, the atuzumab is administered prior to pemetrexed on day 1 of each 21-day cycle of each cycle after cycle 6. In some embodiments, the atelizumab, pemetrexed, and cisplatin are each administered intravenously.

In some embodiments, the individual has not received treatment for stage IV non-squamous NSCLC. In some embodiments, the individual has not received chemotherapy for stage IV non-squamous NSCLC. In some embodiments, the individual is asian or asian descendants. In some embodiments, the individual is at least 65 years old. In some embodiments, the individual is a never-smoker. In some embodiments, the subject is high in PD-L1. In some embodiments, the individual is PD-L1 negative. In some embodiments, the individual is a human.

In another aspect, a kit is provided comprising an anti-PD-L1 antibody for use in combination with an antimetabolite and a platinum agent in the treatment of an individual having lung cancer according to the methods described herein. In some embodiments, a kit is provided comprising atelizumab for use in combination with pemetrexed and carboplatin for treating an individual having lung cancer according to the methods described herein. In some embodiments, a kit is provided comprising atelizumab for use in combination with pemetrexed and cisplatin in treating an individual having lung cancer according to the methods described herein.

In another aspect, provided herein is a composition comprising an anti-PD-L1 antibody, for use in a method of treating lung cancer in an individual, the method comprising administering to the individual an effective amount of an anti-PD-L1 antibody, an antimetabolite, and a platinum agent, wherein the treatment extends the Progression Free Survival (PFS) of the individual. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, a composition comprising an anti-PD-L1 antibody is used in a method according to the disclosure herein.

In another aspect, provided herein is a composition comprising astuzumab for use in a method of treating stage IV non-squamous non-small cell lung cancer (NSCLC), the method comprising administering to an individual an effective amount of astuzumab, pemetrexed, and Carboplatin wherein atezumab is administered at a dose of 1200mg and pemetrexed at 500mg/m²Is administered at a dose sufficient to achieve AUC of 6mg/ml/min, and wherein said treatment extends the Progression Free Survival (PFS) of said subject. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, the composition is used in a method according to the disclosure herein.

In another aspect, provided herein is a composition comprising atuzumab for use in a method of treating stage IV non-squamous non-small cell lung cancer (NSCLC), the method comprising administering to an individual an effective amount of atuzumab, pemetrexed, and cisplatin, wherein the atuzumab is administered at a dose of 1200mg, the pemetrexed is administered at 500mg/m²Cisplatin at a dose of 75mg/m²And wherein the treatment extends Progression Free Survival (PFS) of the individual. In some embodiments, the treatment extends the Overall Survival (OS) of the individual. In some embodiments, the composition is used in a method according to the disclosure herein.

It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention are further described by the following detailed description.

Drawings

Figure 1 provides a schematic of the study design of the clinical trial described in example 1. 578 patients were enrolled. Group a included 292 patients and group B included 286 patients. Group a patients received treatment until Progressive Disease (PD) or loss of clinical benefit; patients in group B received treatment until PD.

FIG. 2 provides a Kaplan-Meier plot of Progression Free Survival (PFS) for patients in group A (Attuzumab + pemetrexed + carboplatin or cisplatin) versus group B (pemetrexed + carboplatin or cisplatin).

FIG. 3 provides a Kaplan-Meier plot of Overall Survival (OS) for patients in group A (Attributumab + pemetrexed + carboplatin or cisplatin) versus group B (pemetrexed + carboplatin or cisplatin).

Figure 4 provides a comparison of Overall Response Rates (ORR) confirmed for patients in group a versus group B (CR ═ complete response; CR/PR ═ complete response/partial response; SD ═ stable disease; PD ═ progressive disease.) ORR was evaluated according to RECIST v1.1 criteria.

Figure 5A provides a forest plot showing a subgroup analysis of PFS in patients with various baseline risk factors in group A (APP) versus group B (PP). (APP ═ attuzumab + pemetrexed + carboplatin or cisplatin; PP ═ pemetrexed + carboplatin or cisplatin.)

Figure 5B provides a forest plot showing a subgroup analysis of PFS in patients with various baseline risk factors in group A (APP) versus group B (PP). (APP ═ attuzumab + pemetrexed + carboplatin or cisplatin; PP ═ pemetrexed + carboplatin or cisplatin.)

FIG. 6 provides a forest map showing a subgroup analysis of OS for patients with various baseline risk factors in group A (APP) versus group B (PP). (APP ═ attuzumab + pemetrexed + carboplatin or cisplatin; PP ═ pemetrexed + carboplatin or cisplatin.)

FIG. 7A provides a Kaplan-Meier plot of Progression Free Survival (PFS) for "PD-L1 high" patients in group A (Attributumab + Pemetrexed + Carboplatin or cisplatin) versus group B (Pemetrexed + Carboplatin or cisplatin).

FIG. 7B provides a Kaplan-Meier plot of Progression Free Survival (PFS) for "PD-L1 Low" patients in group A (Attributumab + Pemetrexed + Carboplatin or cisplatin) versus group B (Pemetrexed + Carboplatin or cisplatin).

Figure 7C is a graph of Progression Free Survival (PFS) for "PD-L1 negative" patients in group a (attrituzumab + pemetrexed + carboplatin or cisplatin) versus group B (pemetrexed + carboplatin or cisplatin).

Detailed Description

I. Definition of

Before the present invention is described in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a", "an", "the" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" includes a combination of two or more such molecules, and the like.

The term "about" as used herein refers to the usual range of error for the corresponding value as readily known to those of skill in the art. References herein to "about" a value or parameter include (and describe) embodiments that refer to the value or parameter itself.

It is understood that the aspects and embodiments of the invention described herein include those aspects and embodiments that are referred to as "comprising," consisting of, "and" consisting essentially of.

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with its binding partner(s) to eliminate T cell dysfunction caused by the PD-1 signaling axis, which results in restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling resulting from the interaction of PD-1 with its one or more binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its one or more binding partners. In particular aspects, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and others that reduce, block, inhibit, eliminate, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist can reduce a negative costimulatory signal mediated by or through PD-1 signaling mediated by a cell surface protein expressed on the T lymphocyte, thereby rendering the dysfunctional T cell less dysfunctional (e.g., increasing effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided below.

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signaling resulting from the interaction of PD-L1 with its one or more binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In particular aspects, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and others that reduce, block, inhibit, eliminate, or interfere with signaling resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, a PD-L1 binding antagonist can reduce a negative costimulatory signal mediated by or through signaling of PD-L1 mediated by cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Specific examples of PD-L1 binding antagonists are provided below.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signaling resulting from the interaction of PD-L2 with its one or more binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its one or more binding partners. In particular aspects, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and others that reduce, block, inhibit, eliminate, or interfere with signaling resulting from the interaction of PD-L2 with its one or more binding partners (such as PD-1). In one embodiment, a PD-L2 binding antagonist can reduce a negative costimulatory signal mediated by or through signaling of PD-L2 mediated by cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., increasing effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

By "sustained response" is meant a sustained effect on the reduction of tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller than the size at the beginning of the dosing phase. In some embodiments, the duration of the sustained response is at least the same as the duration of treatment, at least 1.5X, 2.0X, 2.5X, or 3.0X the length of the duration of treatment.

The term "pharmaceutical formulation" refers to a preparation that is in a form effective to allow the biological activity of the active ingredient, and that is free of additional components having unacceptable toxicity to the subject to which the formulation is to be administered. Such formulations are sterile formulations. By "pharmaceutically acceptable" excipients (vehicles, additives) is meant excipients that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient used.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable therapeutic effects include reducing the rate of disease progression, slowing or alleviating the disease state, and ameliorating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing the proliferation of cancer cells (or destroying cancer cells), alleviating the symptoms caused by the disease, increasing the quality of life of a person suffering from the disease, reducing the dose of other drugs required to treat the disease, and/or prolonging the survival of the individual.

As used herein, "delaying the progression of a disease" refers to delaying, hindering, slowing, delaying, stabilizing and/or delaying the progression of a disease (e.g., cancer). Such delays may be of varying lengths of time, depending on the medical history and/or the individual to be treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually encompass prevention, as the individual will not suffer from the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

An "effective amount" is at least the minimum amount necessary to achieve a measurable improvement or prevention of a particular condition. An effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit an expected response in the individual. An effective amount is also an amount where any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or intended results include such things as elimination or reduction of risk, lessening of severity, or delaying the onset of disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that arise during the course of disease progression. For therapeutic use, beneficial or expected results include clinical results, such as reducing one or more symptoms caused by the disease, improving the quality of life of the patient, reducing the dosage of other drugs required to treat the disease, enhancing the effect of other drugs (such as by targeting, delaying disease progression, and/or prolonging survival). In the case of cancer or tumors, an effective amount of the drug may reduce the number of cancer cells; reducing tumor size; inhibit (i.e., slow to some extent or be expected to stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and expect to stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with the disorder to some extent. An effective amount may be administered one or more times. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient for direct or indirect prophylaxis or treatment. As understood in the clinical setting, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and administration of an effective amount of a single agent may be considered if the desired result can be achieved or achieved in combination with one or more other agents.

As used herein, "in conjunction with … …" means that one treatment modality is administered in addition to another treatment modality. As such, "in conjunction with … …" refers to administration of one treatment modality before, during, or after administration of another treatment modality to an individual.

A "disorder" is any condition that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation (whether malignant or benign), as well as all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive herein.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial diffuse melanoma, freckle-like malignant melanoma, acral freckle-like melanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma (including low grade/follicular non-hodgkin's lymphoma (NHL), Small Lymphocyte (SL) NHL, intermediate grade/follicular NHL, melanoma, and squamous cell lymphoma, Intermediate diffuse NHL, higher immunoblastic NHL, higher lymphoblastic NHL, higher small non-dividing cell NHL, large lumpy disease NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's macroglobulinemia), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular hyperplasia associated with nevus pustulosis, edema (e.g., associated with brain tumors), Meigs' syndrome, brain and head and neck cancer, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.

As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to a cell (e.g., causes cell death, inhibits proliferation, or otherwise impedes cell function). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu); a chemotherapeutic agent; a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents may be selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-a inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors. In one embodiment, the cytotoxic agent is a taxane. In one embodiment, the taxane is paclitaxel or docetaxel. In one embodiment, the cytotoxic agent is a platinum agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the antagonist of EGFR is N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (e.g., erlotinib). In one embodiment, the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib. In one embodiment, the cytotoxic agent is a PI3K inhibitor.

"chemotherapeutic agents" include compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (b)Genentech/OSI Pharm.), bortezomib (Millennium Pharm, disulfiram, epigallocatechin gallate, salinosporamide A (salinosporamide A), carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (A)AstraZeneca)、sunitib(Pfizer/Sugen), letrozole (C: (A)Novartis), imatinib mesylate (Novartis)、finasunate(Novartis), oxaliplatin (A)Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus,wyeth), lapatinib (GSK572016, Glaxo Smith Kline), Lonafami (SCH 66336), Sorafenib (R) (Bayer Labs), gefitinib (b)AstraZeneca), AG1478, alkylating agents such as thiotepa andcyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzotepa (benzodopa), carbaquinone (carboquone), metotepipa (meturedpa) and uredepa (uredpa); vinyl imines and methyl melamines, including hexamethylmelamine, triethylenemelamine (triethyleneemimine), triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphamide), and trimethylmelamine (trimethylmelamine); acetogenins (in particular bullatacin and bullatacinone); camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including adozelesin, carzelesin, and bizelesin synthetic analogs thereof); cryptophycins (especially cryptophycin 1 and cryptophycin 8); adrenal corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5 α -reductases including finasteride and dutasteride); vorinostat, romidepsin, pantoprazole, valproic acid, moxystadoxostat dolastatin (mocetinostat dolastatin); aldesleukin, talc duocarmycin (including the synthetic analogs KW-2189 and CB1-TM 1); (ii) an elutherobin; coprinus atrata base (pancratistatin); sarcodic tyin; spongistatin (spongistatin); nitrogen mustards such as chlorambucil, chlorambucil (chlorephazine), chlorophosphamide, estramustine, ifosfamide, mechlorethamine hydrochloride, melphalan, neomycin, benzene mustard cholesterol, prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil musard); nitrosoureas such as carmustine, chlorzotocin, temustine, lomustine, nimustine and ranimustine (ranimnustine); antibiotics, for example enediyne antibiotics (such as calicheamicin, in particular calicheamicin gamma 1I and calicheamicin omega 1I (Angew chem. Intl. Ed. Engl. 199433: 183-) -186; dynemicin, including dynemicin A; bisphosphonates, for example clodronate; esperamicin; and also neocarzinostain chromophores and related chromophoric proteins enediyne antibiotic chromophores), aclacinomycin (aclacinomycin), actinomycin, anthranomycin (Authramycin), azaserine (azaserine), bleomycin (bleomycin), cactinomycin, karabicin (caraubicin), carminomycin (caminomycin), carcinomycin (carzinophilin), tryptomycin (chromomycin), dactinomycin, daunomycin (torebixin), 6-5-diazo-5-norubicin), norubicin (carzinophilin), chromomycin (chromomycin), dactinomycin, daunomycin (6-5-norubicin), leucinomycin (leucin-5-norubicin), and leucinomycin, (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidine-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), isosbacin (esorubicin), idarubicin (idarubicin), Marcelomycin (marcelomycin), mitomycins such as mitomycin C, mycophenolic acid, nogomycin, olivomycin, pelomycin (pelomomycin), pristinamycin (porfiromycin), puromycin, triiron doxorubicin (quelamemycin), Rodocubicin (rodorubicin), streptonigrin (streptonigrin), streptomycin, tubercidin (tubicidin), ubenimexmeis, setastin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues, e.g. fludarabine, 6-mercaptopurineThiamiprine (thiamiprine), thioguanine; pyrimidine analogs, such as, for example, ancitabine (ancitabine), azacitidine, 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enoxistine (enocitabine), floxuridine (floxuridine); androgens such as calestrone, drotandrosterone propionate, epithioandrostanol, meindrotane, testolactone; anti-adrenalines, such as aminoglutamine, mitotane (mitotane), trilostane (trilostane); folic acid supplements, such as folic acid; acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatraxate; defofamine; dimecorsine (demecolcine); diazaquinone (diaziqutone); elfosmithine; ammonium etitanium acetate; epothilone (epothilone); ethydine (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguzon (mitoguzon); mitoxantrone (mitoxantrone); mopidarnol (mopidammol); nitrerine; pentostatin (pentostatin); phenamett; pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazine; methylbenzyl hydrazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizoxin (rhizoxin); sizofuran; germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecene toxins (particularly T-2 toxin, veracurin A, fisetin A (roridin A) and anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa (thiotepa); ramulus et folium taxi CuspidataeAlkanes, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.),(without cremophor), albumin engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and(docetaxel, docetaxel; Sanofi-Aventis); chlorambucil (chlorembucil);(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); norfloxacin (novantrone); (ii) teniposide; edatrexate (edatrexate); daunomycin (daunomycin); aminopterin; capecitabineIbandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Chemotherapeutic agents also include: (i) anti-hormonal agents which act to modulate or inhibit the action of hormones on tumors, e.g. anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including for example tamoxifen (includingTamoxifen citrate), raloxifene, droloxifene (droloxifene), iodoxyfene, 4-hydroxyttamoxifen, trooxifene (trioxifene), raloxifene hydrochloride (keoxifene), LY117018, onapristone (onapristone), and(toremifene citrate); (ii) aromatase inhibitors which inhibit aromatase, which regulates the estrogen production of the adrenal gland, e.g. 4(5) -imidazole, aminoglutethimide (aminoglutethimide),(megestrol acetate),(exemestane; Pfizer), formestane (formestanie), fadrozole (fadrozole),(vorozole)), (vorozole) (vorozole))), (letrozole; Novartis) and(anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin (buserelin), triptorelin (tripterelin), medroxyprogesterone acetate, diethylstilbestrol, bemeili, fluoxymesterone, all trans retinoic acid, tretinoamide (fenretinide), and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues); (iv) protein kinase inhibitors; (v) a lipid kinase inhibitor; (vi) antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as PKC- α, Ralf, and H-Ras; (vii) ribozymes, e.g. VEGF expression inhibitors (e.g. VEGF)) And inhibitors of HER2 expression; (viii) vaccines, e.g. gene therapy vaccines, such asAnd rIL-2; topoisomerase 1 inhibitors, e.g. And (ix) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Chemotherapeutic agents also include antibodies, such as alemtuzumab (Campath), bevacizumab (bGenentech); cetuximab (Imclone); panitumumab (A)Amgen), rituximab (rituximab), (b) Genentech/Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (trastuzumab) ((R)Genentech), tositumomab (tositumomab) (Bexxar, Corixia) and antibody drug conjugates, gemtuzumab ozogamicin (c: (r)Wyeth). In connection with the inventionOther humanized monoclonal antibodies with therapeutic potential for combination as agents include: aprezumab (apiolizumab), aselizumab (aselizumab), atilizumab (atlizumab), bapidizumab (bapineuzumab), mabuzumab (bivatuzumab mertansine), macrantuzumab (canuzumab mertansine), ceduzumab (cedenzab), polyethylene glycol-conjugated certuzumab (certolizumab pegol), cidfusizumab, cidtuzumab (daclizumab), daclizumab (daclizumab), eculizumab (eculizumab), efuzumab (epratuzumab), epratuzumab (epratuzumab), erilizumab (erbuzumab), eriolizumab (erbuzumab), fulizumab (erbuzumab), pantolizumab (feluzumab), tuzumab (feruzumab), gemtuzumab (gemtuzumab), gemtuzumab (zelizumab), gemtuzumab (gem, Omalizumab, palivizumab, paclobutrazumab, pecfuzumab, pertuzumab (petuuzumab), pelizumab (pelizumab), ralvizumab, ranibizumab (ranibizumab), relivizumab, rayleigh mab (resibizumab), resyvizumab (rovizumab), rovellizumab (rolizumab), lullizumab (ruplizumab), sibutrumab (sibutrumab), sibutrumab (siplizumab), solituzumab (sotuzumab), tacatuzumab (tacatuzumab), tacatuzumab (tetricuzumab), talibizumab (taluczumab), temab (teluzumab), tuzumab (tuzumab), tollizumab (torubukuzumab), taclizumab (taclizumab), temab (taclizumab), and (abcuizumab/or (abctuzumab/wutuzumab), and (tuzumab) and (tuzumab and (tuvutuzumab) and (tuzumab), this is a recombinant full-length IgG1 λ antibody specifically for human sequences that has been genetically modified to recognize the interleukin 12p40 protein.

Chemotherapeutic agents also include "EGFR inhibitors" which refer to binding to or interacting directly with EGFRCompounds that act to block or reduce their signaling activity are alternatively referred to as "EGFR antagonists". Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind EGFR include MAb 579(ATCC CRL HB 8506), MAb 455(ATCC CRL HB8507), MAb 225(ATCC CRL 8508), MAb 528(ATCC CRL 8509) (see U.S. patent nos. 4,943, 533; Mendelsohn et al) and variants thereof, such as chimeric 225(C225 or cetuximab;) And remodeled human 225(H225) (see WO 96/40210, Imclone Systems Inc.); IMC-11F8, fully human EGFR-targeting antibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. patent No. 5,891,996; human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (Panitumumab) (see WO98/50433, Abgenix/Amgen); EMD 55900 (Straglioto et al, Eur. J. cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody directed against EGFR, which competes with EGF and TGF- α for binding to EGFR (EMD/Merck); human EGFR antibody, HuMax-EGFR (genmab); fully human antibodies, designated E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3, and described in US 6,235,883; MDX-447 (Metarex Inc); MAb 806 or humanized MAb 806(Johns et al, J.biol.chem.279(29):30375-30384 (2004)). anti-EGFR antibodies can be conjugated to cytotoxic agents to produce immunoconjugates (see, e.g., EP659439a2, Merck Patent GmbH). EGFR antagonists include small molecules such as U.S. patent nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: compounds described in WO98/14451, WO98/50038, WO99/09016 and WO 99/24037. Specific small molecule EGFR antagonists include OSI-774(CP-358774, erlotinib, Genentech/OSI Pharmaceuticals); PD 183805(CI1033, 2-acrylamido, N- [4- [ (3-chloro-4-fluorophenyl) amino)]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]Dihydrochloride, Pfizer Inc.); ZD1839, gefitinib4- (3 '-chloro-4' -fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca); ZM 105180 (6-amino-4- (3-methylphenyl-amino) -quinazoline, Zeneca); BIBX-1382(N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5,4-d]Pyrimidine-2, 8-diamine, Boehringer Ingelheim); PKI-166((R) -4- [4- [ (1-phenylethyl) amino)]-1H-pyrrolo [2,3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenylethyl) amino group]-7H-pyrrolo [2,3-d]A pyrimidine; CL-387785(N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butynylamide); EKB-569(N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (dimethylamino) -2-butenamide (Wyeth)); AG1478 (Pfizer); AG1571(SU 5271; Pfizer); EGFR/HER2 tyrosine kinase dual inhibitors, e.g. lapatinib (A: (B))GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy]Phenyl radical]-6[5[ [ (2-methylsulfonyl) ethyl ] ethyl]Amino group]Methyl radical ]-2-furyl radical]-4-quinazolinamines).

Chemotherapeutic agents also include "tyrosine kinase inhibitors," including the EGFR-targeting drugs described in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); a dual HER inhibitor, such as EKB-569 (available from Wyeth), which preferentially binds EGFR, but simultaneously inhibits cells that overexpress both HER2 and EGFR; lapatinib (GSK 572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors, e.g. from ISIS pharmaceuticalals-derived antisense agent ISIS-5132, which inhibits Raf-1 signaling; non-HER targeted TK inhibitors, e.g. imatinib mesylate (Available from Glaxo SmithKline); multiple target tyrosine kinase inhibitors, e.g. sunitinib (C)Available from Pfizer); VEGF receptor tyrosine kinase inhibitors, such as vartanib (PTK787/ZK222584, available from Novartis/Schering AG); ci-1040, an inhibitor of MAPK extracellular regulated kinase I (available from Pharmacia); quinazolines, such as PD153035,4- (3-chloroanilino) quinazoline; a pyridopyrimidine; a pyrimidopyrimidine; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidine, 4- (phenylamino) -7H-pyrrolo [2,3-d ]A pyrimidine; curcumin (dieruloyl methane, 4, 5-bis (4-fluoroanilino) phthalimide); tyrosine containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g., molecules that bind to HER-encoding nucleic acids); quinoxaline (U.S. patent No. 5,804,396); tyrosine phosphorylation inhibitors (tryptophastins, U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787(Novartis/Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinib mesylatePKI 166 (Novartis); GW2016(Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxanib ((Semaxinib) Pfizer); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11(Imclone), rapamycin (sirolimus,) (ii) a Or in any of the following patent publications: U.S. Pat. No. 5,804,396, WO 1999/09016(American Cyanamid), WO 1998/43960(American Cyanamid), WO 1997/38983(Warner Lambert), WO 1999/06378(Warner Lambert), WO 1999 ^ or/and/or combinations thereof06396(Warner Lambert), WO 1996/30347(Pfizer, Inc), WO 1996/33978(Zeneca), WO 1996/3397(Zeneca), and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferon, colchicine, chlorpheniramine (metoprine), cyclosporin, amphotericin, metronidazole, alemtuzumab (alemtuzumab), alitretinoin (alitretinine), allopurinol (allopurinol), amifostine (amifostine), arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene (bexarotene), cladribine (cladribine), clofarabine (clofarabine), dyepoetin alpha (darbepoetin alfa), dinil interleukin (denileein), dexrazoxane (dexrazoxane), epoetin alpha (epoetin alfa), erlotinib (elotinib), filgrastim (filgrastim), histidinin acetate (histreetin acetate), irritin ibrinolide (irtuline), interferon alpha (interferon-2-interferon alpha (methamphetamine), levonorgalantamine (2-a), nerolidine (mezolirtisone, mefenadine (sodium), nerolidine (mefenamide, nerolidine (mebendamustine, mebendazole, bexathin-a, bexathin-2, mebendazole, mefena, The compounds of formula (i) include, but are not limited to, the compounds of formula (i) oxpriinterleukin (oprevikins), palifermin (palifermin), pamidronate (pamidronate), pergamase (pegademase), pemetrexed (pegfilgrastim), pemetrexed (pemetrexed) disodium, mithramycin (plicamycin), porfimer sodium (porfimer sodium), quinacrine (quinacrine), labyrine (rasburicase), sargrastim (sargramostim), temozolomide (temozolomide), VM-26, 6-TG, toremifene (toremifene), tretinoin (tretinoin), ATRA, valrubicin (valrubicin), zoledronate (zoledronate), and the pharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone (hydrocortisone), hydrocortisone acetate (hydrocortisone acetate), cortisone acetate (cortisone acetate), hydrocortisone (tixocortol pivalate), triamcinolone acetonide (triamcinolone acetonide), triamcinolone acetonide (triamcinolone alcohol), mometasone (mometasone), amcinolone acetonide (amcinonide), budesonide (budesonide), desonide (desonide), fluocinolone acetonide (fluocinolone acetonide), betamethasone (betamethasone), betamethasone phosphateSodium (betamethasone sodium phosphate), dexamethasone (dexamethosone), sodium dexamethosone phosphate (dexamethosone), sodium dexamethosone sodium phosphate (dexamethosone sodium phosphate), fluocortolone (fluocortolone), hydrocortisone-17-butyrate (hydrocortisone-17-butyrate), hydrocortisone-17-valerate (hydrocortisone-17-valerate), alclomethasone dipropionate (clobetasone dipropionate), prednisone (prednicarbate), clobetasone-17-butyrate (clobetasone-17-butyrate), clobetasol-17-propionate (clobetasol-17-propionate), fluorometholone (fluoxynol acetate), fluoxynisolone (fluocortolone) and fluoxynisolone (flunisolone acetate); immunoselective anti-inflammatory peptides (ImSAID), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN Biotherapeutics, LLC); antirheumatic agents, for example azathioprine, cyclosporin (cyclosporin A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide minocycline (leflunomide irinocycline), sulfasalazine (sulfasalazine), tumour necrosis factor alpha (TNF alpha) blockers, for example etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab (Cimzia), golimumab (Simponi), interleukin 1(Il-1) blockers, for example anakinra (Kineret), T cell co-stimulation blockers, for example, adalimumab (Orencia), interleukin 6(IL-6) blockers, for example, tollizumab Interleukin 13(Il-13) blocking agents such as lerizumab (lebrikizumab); interferon alpha (IFN) blockers, such as rolizumab (rotalizumab); beta 7 integrin blockers, such as rhuMAb beta 7; IgE pathway blockers, such as Anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1/β 2 blockers, such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); miscellaneous research reagents such as sulfur platinum, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyl transferase inhibitors (L-739749,l-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanol, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol,) (ii) a Beta-lapachone (beta-lapachone); lapachol; colchicine; betulinic acid; acetyl camptothecin, scopolectin (scopolectin), and 9-aminocamptothecin); podophyllotoxin; tegafurBexaroteneBisphosphonates, e.g. clodronates (such as Or) EtidronateNE-58095, zoledronic acid/zoledronic acid saltAlendronatePamidronate saltTillofosinate (tirudronate)Or risedronateAnd epidermal growth factor receptor (EGF-R); vaccines, e.g.A vaccine; perhafos (perifosine), COX-2 inhibitors (e.g., celecoxib or etoxib), proteosome inhibitors (e.g., PS 341); CCI-779; tipifarnib (Tipifarnib) (R11577); olaranib (orafenaib), ABT 510; bcl-2 inhibitors, e.g. orlimesen sodiumPixantrone (pixantrone); farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the foregoing, for example CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, an abbreviation for treatment regimen of oxaliplatin (ELOXATINTM) in combination with 5-FU and folinic acid.

Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives (e.g., ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin (oxaprozin), and naproxen), acetic acid derivatives (e.g., indomethacin, sulindac, etodolac, diclofenac), enolic acid derivatives (e.g., piroxicam, meloxicam, tenoxicam, droxicam (droxicam), lornoxicam, and isoxicam), fenamic acid derivatives (e.g., mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid), and COX-2 inhibitors (e.g., celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib (rofecoxib), rofecoxib, and valdecoxib). NSAIDs are useful for alleviating conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthritis, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and symptoms of renal colic.

As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits cell growth in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces proliferation of cells expressing an antigen to which the antibody binds. In another embodiment, the growth inhibitory agent may be one that significantly reduces the percentage of S phase cells. Examples of growth inhibitory agents include agents that block cell cycle progression (at places other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes and topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Those agents that block G1 also spill over into the S phase block, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, methoxyethylamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in chapter 1 of The article "The Molecular Basis of Cancer", edited by Murakami and Israel, entitled "Cell cycle regulations, oncogenes, and anticancer drugs", by Murakami et al (W.B. Saunders, Philadelphia,1995), e.g. page 13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs and are derived from the taxus species. Docetaxel (docetaxel: (b)) Rhone-Poulenc Rorer) is derived from Taxus baccata and is a semi-synthetic analog of paclitaxel: (Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly of tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting mitosis of cells.

"radiation therapy" refers to the use of directed gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to destroy them completely. It will be understood that there are many methods known in the art that can determine the dosage and duration of treatment. Typical treatments are given in one dose, with typical doses ranging from 10 to 200 units per day (Gray).

A "subject" or "individual" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, stadium or pet animals, such as dogs, horses, cats, cattle, etc., preferably, the mammal is a human.

The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies (including but not limited to full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody is a component that has been identified and isolated and/or recovered from its natural environment. Contaminant components of their natural environment are materials that would interfere with antibody research, diagnostic or therapeutic uses, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody (e.g., as determined by the Lowry method), in some embodiments, greater than 99% by weight; (2) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence (e.g., by using a rotary cup sequencer), or (3) homogenization (SDS-PAGE under reducing or non-reducing conditions, using, for example, coomassie blue or silver staining). Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the natural environment of the antibody will not be present. Typically, however, the isolated antibody will be prepared by at least one purification step.

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by a plurality of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of an immunoglobulin, i.e., a variable domain, that comprises an antigen binding site. The constant domains comprise the CH1, CH2, and CH3 domains of the heavy chain (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable part of the antibody and contain the antigen binding site.

The term "variable" refers to the fact that: certain portions of the variable domains vary widely in sequence between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed among the variable domains of the antibody. It is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, predominantly in the beta sheet structure, connected by three HVRs, which form loops connecting and in some cases forming part of the beta sheet structure. The HVRs in each chain are held together tightly by the FR region and, together with the HVRs in the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but have respective effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The "light chains" of antibodies (immunoglobulins) from any mammal can be classified into one of two distinctly different classes, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of its constant regions.

Antibodies (immunoglobulins) can be classified into different classes according to the amino acid sequence of their heavy chain constant domains. Immunoglobulins are largely divided into five classes: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are described, for example, generally in Abbas et al Cellular and mol. The antibody may be part of a larger fusion molecule formed by covalent or non-covalent binding of the antibody to one or more other proteins or peptides.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in substantially intact form, rather than to an antibody fragment as defined below. The term particularly refers to antibodies having a heavy chain comprising an Fc region.

For purposes herein, a "naked antibody" is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, an antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; diabodies (diabodies); a linear antibody; a single chain antibody molecule; multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. The pepsin treatment produced F (ab')2 fragments with two antigen binding sites and still able to cross-link with antigen.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight and non-covalent association. In the single chain Fv (scfv) species, one heavy chain variable region domain and one light chain variable region domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may associate into a "dimer" structure similar to that in a two-chain Fv species. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

The "Fab" fragment contains the heavy and light chain variable domains and also the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab 'fragments differ from Fab fragments in that the Fab' fragment has added to the carboxy terminus of the heavy chain CH1 domain residues that include one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domains carry a free thiol group. F (ab ')2 antibody fragments were originally produced as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, such that the scFv forms the desired antigen binding structure. For reviews of scFv see, for example, Pluckth ü n, The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. (Springer-Verlag, New York,1994), p.269-315.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. The diabody can be a bivalent antibody or a bispecific antibody. Diabodies are more fully described, for example, in: EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations that may be present in minor amounts, such as naturally occurring mutations. Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be to select a unique clone from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to increase affinity for the target, to humanize the target binding sequence, to increase its production in cell culture, to reduce its immunogenicity in vivo, to produce a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use according to the invention can be prepared by a variety of techniques, including, for example: hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14(3):253- Techniques for human or human-like antibodies to loci or genes (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al, Nature 362:255-258 (1993); Bruggemann et al, Yeast in Immunol.7:33 (1993); U.S. pat. Nos.5,545, 807; 5,545, 806; 5,569, 825; 5,625,126; 5,633,425; and 5,661,016; Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al, Nature 368: 856; 859 (1994); Moison 368; Nature 368: 813 (Fishwilk et al, Nature Biotechnology.14: huubl 1996; Nature: 14: 1996; and Nature Bioberr. Reg.93: 1996; Reg et al, Nature 11: 1995); et al, Biotechnol. 11: 1995).

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is bound to a peptide from a particular species or belonging to a particular genusCorresponding sequences in antibodies of an antibody class or subclass are identical or homologous, while the remainder of one or more chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of these antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies includeAn antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing cynomolgus monkeys with an antigen of interest.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an recipient HVR are replaced by residues from an HVR of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not present in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one variable domain, typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), which is typically a human immunoglobulin. For more details see, e.g., Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-E329 (1988); and Presta, curr, Op, Structure, biol.2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.1:105-115 (1998); harris, biochem. Soc. transactions 23: 1035-; hurle and Gross, curr. Op. Biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.

A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and/or an antibody made using any of the techniques disclosed herein for making human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies, including phage display libraries, can be generated using a variety of techniques known in the art. Hoogenboom and Winter, J.mol.biol., 227:381 (1991); marks et al, J.mol.biol., 222:581 (1991). Methods that can also be used to prepare human monoclonal antibodies are described in: cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr Opin Pharmacol.5:368-74 (2001). Human antibodies can be made by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigen challenge, but whose endogenous locus has failed, e.g., immunizing a xenomouse (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XenomousTM technology). See also, e.g., Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006) for human antibodies produced by human B-cell hybridoma technology.

A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for an antigen homolog from a second mammalian species. Typically, a species-dependent antibody "specifically binds" (e.g., has a binding affinity (Kd) value of no more than about 1x 10-7M, preferably no more than about 1x 10-8M, preferably no more than about 1x 10-9M) to a human antigen, but has a binding affinity for an antigen homolog of a second non-human mammalian species that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold weaker than its binding affinity for a human antigen. The species-dependent antibody may be any of the various antibodies as defined above, but is preferably a humanized or human antibody.

The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable domain which is hypervariable in sequence and/or forms structurally defined loops. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 showed the most diversity among six HVRs, and in particular H3 was thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, in Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camelid antibodies consisting of only heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

Many HVR descriptions are used and are included herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk J.mol.biol.196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops and was adopted by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structure. The residues of each of these HVRs are described below.

The HVRs can include the following "extended HVRs": 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL, and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable domain residues are numbered according to the method of Kabat et al, supra.

The HVRs can include the following "extended HVRs": 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL, and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable domain residues are numbered according to the method of Kabat et al, supra.

"framework" or "FR" residues are those variable domain residues other than the HVR residues as defined herein.

The term "numbering of variable domain residues as in Kabat" or "numbering of amino acid positions as in Kabat" and variants thereof refers to the numbering system proposed by Kabat et al above for heavy or light chain variable domains used in antibody assembly. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to a shortening or insertion of the FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat numbering) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat numbering, etc.) after heavy chain FR residue 82. The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequences to regions of homology of "standard" Kabat numbered sequences.

When referring to residues in the variable domain (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al, Sequences of Immunological interest.5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" (e.g., the EU index reported by Kabat et al, supra) is typically used. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

The expression "linear antibody" refers to the antibody described by Zapata et al (1995Protein Eng.8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandemly connected Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with a complementary light chain polypeptide. Linear antibodies can be bispecific or monospecific.

As used herein, the terms "bind," "specific binding," or "specifically for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules (including biological molecules). For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds that target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the antigen, e.g., as measured by Radioimmunoassay (RIA). In certain embodiments, the antibody that specifically binds to the target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

As used herein, the term "sample" refers to a composition obtained or derived from a target subject and/or individual that comprises cellular and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological properties. For example, the phrase "disease sample" and variants thereof refers to any sample obtained from a target subject that is expected or known to comprise cellular and/or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

"tissue sample" or "cell sample" refers to a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue from fresh, frozen and/or preserved organs, tissue samples, biopsies and/or aspirates; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells at any time during pregnancy or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Alternatively, the tissue or cell sample is obtained from a diseased tissue/organ. Tissue samples may contain compounds that do not naturally mix with tissue in the natural environment, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

As used herein, "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased body part (e.g., tissue or cell) of the same subject or individual. For example, healthy and/or non-diseased cells or tissues adjacent to a diseased cell or tissue (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue and/or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased body part (e.g., tissue or cell) of an individual that is not the subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from untreated tissue and/or cells of a body part of an individual that is not the subject or individual.

"effective response" of a patient or "responsiveness" of a patient to drug treatment and similar phrases refer to a clinical or therapeutic benefit that is conferred to a patient having a disease or disorder (such as cancer) or at risk thereof. In one embodiment, such benefits include one or more of the following: extending survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of cancer.

A patient "not responding effectively" to treatment refers to a patient who does not have any of the following: extending survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of cancer.

A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions typically require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using, for example, various assay methods disclosed in the definitions herein.

A "human effector cell" is a leukocyte that expresses one or more fcrs and performs effector functions. Preferably, these cells express at least Fc γ RIII and perform ADCC effector function. Human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells may be isolated from natural sources (e.g., from blood).

A cancer or biological sample "having human effector cells" is a cancer or biological sample in which human effector cells (e.g., infiltrating human effector cells) are present in the sample in a diagnostic test.

A cancer or biological sample "having FcR expressing cells" is one in which FcR expression is present in the sample (e.g., infiltrating FcR expressing cells) in a diagnostic test. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R.

Overview

Provided herein are methods of treating or delaying progression of lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) in an individual comprising administering to the individual an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as astuzumab), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., carboplatin or cisplatin). Also provided herein are methods of enhancing immune function in an individual having lung cancer (e.g., non-small cell lung cancer, e.g., stage IV non-squamous non-small cell lung cancer), the method comprising administering to the individual an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as astuzumab), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., carboplatin or cisplatin). In some embodiments, the treatment extends Progression Free Survival (PFS) and/or Overall Survival (OS) of the individual. In some embodiments, the treatment extends progression-free survival (PFS) and/or Overall Survival (OS) of the individual as compared to a treatment comprising administration of an antimetabolite (e.g., pemetrexed) and a platinum agent (e.g., carboplatin or cisplatin).

In some embodiments, the method comprises treating an individual having stage IV lung cancer (e.g., stage IV non-squamous NSCLC) by administering to the individual a combination of attuzumab, pemetrexed, and carboplatin, wherein the administration comprises an induction phase and a maintenance phase, wherein the induction phase comprises every 21-day cycle of cycles 1 through 4, attuzumab is administered at a dose of 1200mg on day 1, and attuzumab is administered at 500mg/m on day 1²Administering pemetrexed at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1; and wherein the maintenance period comprises each 21-day cycle of each cycle after cycle 4, administering altlizumab at a dose of 1200mg on day 1, and 500mg/m on day 1²Administering pemetrexed at a dose of (a); wherein the individual has not been treated and has stage IV non-squamous non-small cell lung cancer (NSCLC); and wherein the administration extends Progression Free Survival (PFS) of the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, administration extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months.

In some embodiments, the method comprises treating an individual having stage IV lung cancer (e.g., stage IV non-squamous NSCLC) by administering to the individual trastuzumab, pemetrexed, and cisplatin in combination, wherein the administering comprises an induction phase and a maintenance phase, wherein the induction phase comprises each 21-day cycle of cycles 1 through 4, attritzumab administered at a dose of 1200mg on day 1, and 500mg/m on day 1²The dose of (1) administered pemetrexed at 75mg/m on day²Administering cisplatin; and wherein the maintenance period comprises administering atezumab at a dose of 1200mg on day 1 for each 21-day cycle of each cycle after cycle 4 and 500mgm on day 1²Administering pemetrexed at a dose of (a); wherein the individual has not been treated and has stage IV non-squamous non-small cell lung cancer (NSCLC); and wherein the administration extends Progression Free Survival (PFS) of the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, administration extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months.

In some embodiments, the method comprises treating an individual having stage IV lung cancer (e.g., stage IV non-squamous NSCLC) by administering to the individual atuzumab in combination with pemetrexed and carboplatin, wherein the administration comprises an induction period and a maintenance period, wherein the induction period comprises each 21-day period of periods 1 to 6, the attuzumab is administered at a dose of 1200mg on day 1, and the attuzumab is administered at 500mg/m on day 1²Administering pemetrexed at a dose sufficient to achieve an AUC of 6mg/ml/min on day 1; and wherein the maintenance period comprises every 21 day period of each period after the 6 th period, and 12 on day 1Attrituzumab was administered at a dose of 00mg, and 500mg/m on day 1²Administering pemetrexed at a dose of (a); wherein the individual has not been treated and has stage IV non-squamous non-small cell lung cancer (NSCLC); and wherein said administering extends Progression Free Survival (PFS) of said subject. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, administration extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months.

In some embodiments, the method comprises treating an individual having stage IV non-small cell lung cancer (NSCLC) (e.g., stage IV non-squamous NSCLC) by administering to the individual atuzumab in combination with pemetrexed and carboplatin, wherein the administration comprises an induction phase and a maintenance phase, wherein the induction phase comprises each 21-day cycle of cycles 1 through 6, the attuzumab is administered at a dose of 1200mg on day 1, and the attuzumab is administered at 500mg/m on day 1²And on day 1 at 75mg/m²Administering cisplatin; and wherein the maintenance period comprises each 21-day cycle of each cycle after cycle 6, administering altlizumab at a dose of 1200mg on day 1, and 500mg/m on day 1²Administering pemetrexed at a dose of (a); wherein the individual has not been treated and has stage IV non-squamous non-small cell lung cancer (NSCLC); and wherein the administration extends Progression Free Survival (PFS) of the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some cases In embodiments, administration extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months.

PD-1 axis binding antagonists

For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB 2. Alternative names to "PDL 1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL 2" include B7-DC, Btdc, and CD 273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL 2.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PDL1 and/or PDL 2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a particular aspect, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein or an oligopeptide.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).

In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). Navolumab (Bristol-Myers Squibb/Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 andis an anti-PD-1 antibody as described in WO 2006/121168. In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain comprises the following amino acid sequence:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:11), and

(b) the light chain comprises the following amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:12)。

in some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region from SEQ ID NO:11 and SEQ ID NO: 12 (e.g., three heavy chain HVRs from SEQ ID NO:11 and three light chain HVRs from SEQ ID NO: 12). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region from SEQ ID NO:11 and a heavy chain variable domain from SEQ ID NO: 12, a light chain variable domain.

In some embodiments, the anti-PD-1 antibody is Pembrolizumab (Pembrolizumab) (CAS registry number 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck3475, lambrolizumab,And SCH-900475, which is an anti-PD-1 antibody described in WO 2009/114335. In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain comprises the following amino acid sequence:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:13), and

(b) the light chain comprises the following amino acid sequence:

EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:14)。

in some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region from SEQ ID NO:13 and SEQ ID NO: 14 (e.g., three heavy chain HVRs from SEQ ID NO:13 and three light chain HVRs from SEQ ID NO: 14). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region from SEQ ID NO:13 and a heavy chain variable domain from SEQ ID NO: 14, a light chain variable domain.

In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

In some embodiments, the anti-PD-1 antibody is PDR001(CAS registry number 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD 1 antibody that blocks the binding of PDL1 and PDL2 to PD-1.

In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD 1 antibody.

In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).

In some embodiments, the anti-PD-1 antibody is JS-001(Shanghai Junshi). JS-001 is a humanized anti-PD 1 antibody.

In some embodiments, the anti-PD-1 antibody is STI-a1110 (sorento). STI-A1110 is a human anti-PD 1 antibody.

In some embodiments, the anti-PD-1 antibody is incsar-1210 (Incyte). INCSAR-1210 is a human IgG4 anti-PD 1 antibody.

In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).

In some embodiments, the anti-PD-1 antibody is TSR-042 (also known as ANB 011; Tesaro/AnaptysBio).

In some embodiments, the anti-PD-1 antibody is AM0001(ARMO Biosciences).

In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (acoustic biological Holdings). ENUM 244C8 is an anti-PD 1 antibody that inhibits the function of PD-1 without preventing binding of PDL1 to PD-1.

In some embodiments, the anti-PD-1 antibody is ENUM 388D4 (acoustic biological Holdings). ENUM 388D4 is an anti-PD 1 antibody that competitively inhibits binding of PDL1 to PD-1.

In some embodiments, the PD-1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and/or a heavy chain variable domain and a light chain variable domain from a PD-1 antibody described in: WO2015/112800 (applicant: Regeneron), WO2015/112805 (applicant: Regeneron), WO2015/112900 (applicant: Novartis), US20150210769 (assigned to Novartis), WO2016/089873 (applicant: Celgene), WO2015/035606 (applicant: Beigene), WO2015/085847 (applicant: Shanghai Hengrui Pharmaceutical/Jiangsu Hengrui medicinal), WO 2015/4835 (applicant: Shanghai Junshi Biosciences/Junmenging Biosciences), WO2012/145493 (Amplimmenmu), US9205148 (assigned to Medmenon), WO2015/119930 (applicant: Pfizer/Merck), WO Pfizer/119923 (applicant: fizer/Merck), WO Pfizer/2016/tyr 25 (WO 2015/2014), WO applicants WO Pfizer/2014/201423 (WO 2014: Sogenrizer/2014) and WO 2014/2014 23 (WO 2014: Biosry).

In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224(CAS registry number: 1422184-00-6; GlaxoSmithKline/MedImmune), also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor as described in WO2010/027827 and WO 2011/066342.

In some embodiments, the PD-1 binding antagonist is a peptide or small molecule compound. In some embodiments, the PD-1 binding antagonist is AUNP-12(Pierre Fabre/Aurigene). See, e.g., WO2012/168944, WO2015/036927, WO2015/044900, WO2015/033303, WO2013/144704, WO2013/132317, and WO 2011/161699.

In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PD-1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL 1. In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits both PDL1 and VISTA. In some embodiments, the PDL1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PDL1 binding antagonist is a small molecule that inhibits PDL1 and TIM 3. In some embodiments, the small molecule is a compound described in WO2015/033301 and WO 2015/033299.

In some embodiments, the PD-1 axis binding antagonist is an anti-PDL 1 antibody. Various anti-PDL 1 antibodies are contemplated and described herein. In any of the embodiments herein, the isolated anti-PDL 1 antibody may bind to human PDL1, e.g., human PDL1 shown in UniProtKB/Swiss-Prot accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the anti-PDL 1 antibody is capable of inhibiting binding between PDL1 and PD-1 and/or between PDL1 and B7-1. In some embodiments, the anti-PDL 1 antibody is a monoclonal antibody. In some embodiments, the anti-PDL 1 antibody is selected from the group consisting of Fab, Fab '-SH, Fv, scFv, and F (ab') ₂An antibody fragment of the fragment. In some embodiments, the anti-PDL 1 antibody is a humanized antibody. In some embodiments, the anti-PDL 1 antibody is a human antibody. Examples of anti-PDL 1 antibodies useful in the methods of the present invention and methods for their preparation are described in PCT patent application WO 2010/077634 a1 and U.S. patent No. 8,217,149, which are incorporated herein by reference.

In some embodiments, the anti-PDL 1 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, the sequences of GFTFSDSWIH (SEQ ID NO: 1), AWISPYGGSTYYADSVKG (SEQ ID NO: 2), and RHWPGGFDY (SEQ ID NO: 3), respectively, and

(b) the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, the sequences of RASQDVSTAVA (SEQ ID NO: 4), SASFLYS (SEQ ID NO: 5), and QQYLYHPAT (SEQ ID NO: 6), respectively.

In some embodiments, the anti-PDL 1 antibody is MPDL3280A, also known as astuzumab and(CAS registry number: 1422185-06-5). In some embodiments, the anti-PDL 1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain variable region sequence comprises the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:7), and

(b) The light chain variable region sequence comprises the amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:8)。

in some embodiments, the anti-PDL 1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain comprises the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:9), and

(b) the light chain comprises the following amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10)。

in some embodiments, the anti-PDL 1 antibody is avilumab (CAS accession No.: 1537032-82-8). Avermelimumab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PDL 1 antibody (Merck KGaA, Pfizer). In some embodiments, the anti-PDL 1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain comprises the following amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:15), and

(b) The light chain comprises the following amino acid sequence:

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:16)。

in some embodiments, the anti-PDL 1 antibody comprises a sequence from SEQ ID NO: 15 and SEQ ID NO: 16 (e.g., three heavy chain HVRs from SEQ ID NO: 15 and three light chain HVRs from SEQ ID NO: 16). In some embodiments, the anti-PDL 1 antibody comprises a sequence from SEQ ID NO: 15 and a heavy chain variable domain from SEQ ID NO: 16, a light chain variable domain.

In some embodiments, the anti-PDL 1 antibody is Devolumab (Durvalumab) (CAS registry number: 1428935-60-7). Devolumab, also known as MEDI4736, is the Fc-optimized human monoclonal IgG1 kappa anti-PDL 1 antibody described in WO2011/066389 and US2013/034559 (MedImmune, AstraZeneca). In some embodiments, the anti-PDL 1 antibody comprises heavy and light chain sequences, wherein:

(a) the heavy chain comprises the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:17), and

(b) The light chain comprises the following amino acid sequence:

EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:18)。

in some embodiments, the anti-PDL 1 antibody comprises a sequence from SEQ ID NO: 17 and SEQ ID NO: 18 (e.g., three heavy chain HVRs from SEQ ID NO: 17 and three light chain HVRs from SEQ ID NO: 18). In some embodiments, the anti-PDL 1 antibody comprises a sequence from SEQ ID NO: 17 and a heavy chain variable domain from SEQ ID NO: 18, a light chain variable domain.

In some embodiments, the anti-PDL 1 antibody is MDX-1105(Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PDL 1 antibody described in WO 2007/005874.

In some embodiments, the anti-PDL 1 antibody is LY3300054(Eli Lilly).

In some embodiments, the anti-PDL 1 antibody is STI-a1014 (sorento). STI-A1014 is a human anti-PDL 1 antibody.

In some embodiments, the anti-PDL 1 antibody is KN035(Suzhou Alphamab). KN035 is a single domain antibody (dAB) generated from a camelid phage display library.

In some embodiments, the anti-PDL 1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by a protease in the tumor microenvironment), activates the antibody antigen binding domain (e.g., by removing the non-binding steric moiety) to cause it to bind its antigen. In some embodiments, the anti-PDL 1 antibody is CX-072(cytomX Therapeutics).

In some embodiments, the PDL1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and/or a heavy chain variable domain and a light chain variable domain from the PDL1 antibody described in: US20160108123 (assigned to Novartis), WO2016/000619 (applicant: Beigene), WO2012/145493 (applicant: Amplimmune), US9205148 (assigned to MedImune), WO2013/181634 (applicant: Sorrento) and WO2016/061142 (applicant: Novartis).

In yet another specific aspect, the antibody further comprises a human or murine constant region. In another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG 4. In yet another specific aspect, the human constant region is IgG 1. In another aspect, the murine constant regions are selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG 3. In another aspect, the murine constant region is IgG 2A.

In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is from a "null effector Fc mutation" or a deglycosylation mutation. In another embodiment, the null effector Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some embodiments, the isolated anti-PDL 1 antibody is deglycosylated. Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used). The glycosylation sites can be conveniently removed from the antibody by altering the amino acid sequence to remove one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Changes can be made by substituting an asparagine, serine, or threonine residue within a glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

In yet another embodiment, the present disclosure provides a composition comprising any of the above anti-PDL 1 antibodies in combination with at least one pharmaceutically acceptable carrier.

In yet another embodiment, the present disclosure provides a composition comprising an anti-PDL 1, an anti-PD-1 or anti-PDL 2 antibody or antigen-binding fragment thereof as provided herein, and at least one pharmaceutically acceptable carrier. In some embodiments, the anti-PDL 1, anti-PD-1, or anti-PDL 2 antibody or antigen-binding fragment thereof administered to the individual is a composition comprising one or more pharmaceutically acceptable carriers. Any pharmaceutically acceptable carrier described herein or known in the art may be used.

Antimetabolites and platinating agents

Antimetabolites

Antimetabolites (e.g., pemetrexed, 5-fluorouracil, 6-mercaptopurine, capecitabine, cytarabine, floxuridine, fludarabine, hydroxyurea, methotrexate, etc.) are widely used antineoplastic agents that interfere with one or more enzymes required for DNA synthesis. Antimetabolites typically act by a variety of mechanisms, including, for example, incorporation into nucleic acids, thereby triggering apoptosis, or, for example, competing for binding sites for enzymes involved in nucleotide synthesis, thereby depleting the supply required for DNA and/or RNA replication and cell proliferation.

Pemetrexed is an exemplary antimetabolite for use in the methods described herein. Pemetrexed is a folic acid analog. The drug pemetrexed disodium heptahydrate has the chemical name L-glutamic acid, N- [4- [2- (2-amino-4, 7-dihydro-4-oxo-1H-pyrrolo [2,3-d ]]Pyrimidin-5 yl) ethyl]Benzoyl radical]-disodium salt heptahydrate with molecular formula C₂₀H₁₉N₅Na₂O₆·7H₂O, molecular weight 597.49.

The pemetrexed heptahydrate disodium salt has the following structure:

pemetrexed inhibits thymine and purineA number of folate dependent enzymes used in the synthesis of pterin, i.e., Thymidylate Synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT) (see Shih et al, (1997) Cancer Res.57: 1116-23). Pemetrexed prevents the formation of DNA and RNA required for the growth and survival of both normal and cancer cells by inhibiting the formation of purine and pyrimidine precursor nucleotides. Pemetrexed may be sold asGIOPEM, PEXATE, PEMANAT, PEMEX, PEMMET, PEXATE, RELISTEXED, TEMERAN, CIAMBRA, etc. are commercially available.

Platinum agents

Platinum agents (e.g., cisplatin, carboplatin, oxaliplatin, and satraplatin) are widely used antineoplastic agents that can cause DNA cross-linking, such as monoadducts, interchain cross-linking, intrachain cross-linking, or DNA protein cross-linking. Platinum assays typically act on the adjacent N-7 position of guanine to form 1, 2 intrachain crosslinks ((Poklar et al, (1996)) Proc. Natl. Acad. Sci. U.S.A.93(15): 7606-11; Rudd et al, (1995). Cancer Chemotherj. Pharmacol.35(4): 323-6). The resulting cross-links inhibit DNA repair and/or DNA synthesis in cancer cells.

Carboplatin is an exemplary platinum coordination compound for use in the methods described herein. The chemical name of carboplatin is platinum, diamine [1, 1-cyclobutane dicarboxy (2-) -O, O' ] -, (SP-4-2), and carboplatin has the following structural formula:

carboplatin is of the formula C₆H₁₂N₂O₄Crystalline powder of Pt, molecular weight 371.25. It is dissolved in water at a rate of about 14mg/mL, and the pH of the 1% solution is between 5 and 7. It is practically insoluble in ethanol, acetone and dimethylacetamide. Carboplatin predominantly produces interchain DNA cross-links, a role that is cell cycle non-specific. Carboplatin can be given the trade nameBIOCARN, BLASTOCARB, BLASTOPLATIN, CARBOKEM, CARBOMAX, CARBOPA, CARBOPLAN, CARBOTEEN, CARBOTINAL, CYTOCARB, DUCARB, KARPLAT, KEMOCARB, NAPROPLAT, NEOPLATIN, NICARBO, ONCOCARBIN, TEVACARB, WOMASTIN, and others are commercially available.

Cisplatin is another exemplary platinum coordination compound used in the methods described herein. The chemical name of cisplatin is diamminedichloroplatinum (diammonilate), which has the following structural formula:

cisplatin is an inorganic water-soluble platinum complex with the molecular formula of Pt (NH)₃)₂Cl₂And the molecular weight is 300.046. After hydrolysis, it reacts with DNA to produce intra-and inter-strand cross-links. These cross-links appear to impair DNA replication and transcription. Cisplatin cytotoxicity has been associated with cell arrest at the G2 phase of the cell cycle. Cisplatin may be referred to by the trade name CDDP, CISPALAN, CISPALAT, PLATKEM, PLATATOCO, PRACTICIS, PLATATICIS, BLASTOLEM, CISMAXMAXAX, CISPALAN, CISLATINUM, CISTERN, DUPLAT, KEMOPLAT, ONCOPLATIN-AQ, PLATINEX, PLATINN, TEVAPLATIN and others are commercially available.

Preparation of antibodies

The antibodies described herein are prepared using techniques available in the art for producing antibodies, exemplary methods of which are described in more detail in the following sections.

The antibody is directed against an antigen of interest (e.g., PD-L1, such as human PD-L1). Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal having a disorder can produce a therapeutic benefit in that mammal.

In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 150nM, less than or equal to 100nM, less than or equal to 50nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM or less than or equal to 0.001 nMM (e.g. 10)^-8M or less, e.g. 10^-8M to 10^{- 13}M, e.g. 10^-9M to 10^-13M)。

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab form of the antibody of interest and its antigen, as described in the assay below. By using the minimum concentration in the presence of a series of unlabeled antigen titrations ( ¹²⁵I) The solution binding affinity of Fab for antigen was measured by equilibration of Fab with labeled antigen and subsequent capture of the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293:865 881 (1999)). To determine the conditions for the assay, capture anti-Fab antibodies (Cappel Labs) were coated with 5. mu.g/mL in 50mM sodium carbonate (pH 9.6)The plate (Thermo Scientific) was used overnight and then blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23 ℃) for two to five hours. In the non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Mixing the antigen with serial dilutions of the Fab of interest. Then incubating the target Fab overnight; however, incubation may be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and used with 0.1% polysorbate 20 in PBSThe plate was washed eight times. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard) and in TOPCOUNT^TMThe gamma counter (Packard) counts the plate for tens of minutes. The concentration of each Fab that gives less than or equal to 20% maximal binding is selected for use in a competitive binding assay.

According to another embodiment, the surface plasmon resonance assay is used at 25 ℃Or(BIAcore, inc., Piscataway, NJ) measured kd (ru) in approximately 10 response units on an immobilized antibody CM5 chip. Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μ g/mL (about 0.2 μ M) with 10mM sodium acetate pH 4.8 before injection at a rate of 5 μ L/min to obtain approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, polysorbate 20 (TWEEN-20) was injected at 0.05% at 25 deg.C at a rate of about 25 μ L/min^TM) Two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS of surfactant (PBST). By fitting both association and dissociation sensorgrams simultaneously, using a simple one-to-one Langmuir binding model: (Evaluation software version 3.2) calculate association rate (kon) and dissociation rate (koff). The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the association rate exceeds 106M-1s-1 as determined by surface plasmon resonance as described above, the association rate can be determined by using a fluorescence quenching technique, e.g., in a spectrometer such as an Aviv Instruments equipped with a flow stopping device or a 8000 series SLM-AMINCO ^TMThe increase or decrease in fluorescence emission intensity (295 nM excitation; 340nM emission, 16nM band pass) of 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 ℃ was measured in the presence of increasing concentrations of antigen in a spectrophotometer (ThermoSpectronic) with a stirred cuvette.

(i) Antigen preparation

Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for the production of antibodies. For transmembrane molecules, for example, receptors, fragments thereof (e.g., extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from a natural source (e.g., cancer cell lines), or may be cells that have been transformed by recombinant techniques to express a transmembrane molecule. Other antigens and forms thereof that can be used to make antibodies will be apparent to those skilled in the art.

(ii) Exemplary antibody-based methods

Preferably, polyclonal antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimide (conjugated via a cysteine residue), N-hydroxysuccinimide (pendant via a lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂Or R¹N ═ C ═ NR (where R and R are¹Is a different alkyl group), it may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin (keyhole limpet hemocyanin), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 μ g or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals were boosted at doses 1/5 to 1/10 of the original amount of peptide or conjugate in freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals were bled and the serum was assayed for antibody titer. Animals were boosted until titers stabilized. Preferably, the animal is augmented with a conjugate of the same antigen (but conjugated to a different protein and/or by a different cross-linking agent). Conjugates can also be prepared in recombinant cell culture as protein fusions. Furthermore, aggregating agents such as alum are suitable for boosting immune responses.

Monoclonal antibodies of the disclosure can be prepared using Hybridoma methods described first in Kohler et al, Nature, 256:495(1975), and further described in, for example, Hongo et al, Hybridoma, 14(3):253-260 (1995); harlow et al, Antibodies A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); hammerling et al, in: Monoclonal Antibodies and T-Cell hybrids 563-681(Elsevier, N.Y., 1981) and Ni, Xiandai Mianyixue, 26(4):265-268(2006) for human-human Hybridomas. Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 relating to the production of monoclonal human native IgM antibodies from hybridoma cell lines. The human hybridoma technique (Trioma technique) is described in Vollmers and Brandlens, Histology and Histopathology,20(3): 927-.

For various other hybridoma techniques, see, e.g., US 2006/258841, US 2006/183887 (fully human antibodies), US 2006/059575, US 2005/287149, US 2005/100546, US 2005/026229, and US patent numbers 7,078,492 and 7,153,507. An exemplary protocol for producing monoclonal antibodies using the hybridoma method is described below. In one embodiment, a mouse or other suitable host animal (e.g., hamster) is immunized to elicit lymphocyte production or is capable of producing antibodies that specifically bind to the protein used for immunization. Antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of the disclosure or a fragment thereof and an adjuvant (e.g., monophosphoryl lipid a (mpl)/Trehalose Distearate (TDM) (Ribi immunochem. The polypeptides (e.g., antigens) of the disclosure or fragments thereof can be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. The serum from the immunized animal is assayed for anti-antigen antibodies and optionally boosted. Lymphocytes are isolated from animals that produce anti-antigen antibodies. Alternatively, lymphocytes may be immunized in vitro.

Following immunization, lymphocytes are isolated, followed by the use of a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells, see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986). Myeloma cells that fuse efficiently can be used, supporting stable high-level antibody production by the selected antibody-producing cells, and being sensitive to a medium such as HAT medium. Exemplary myeloma cells include, but are not limited to, murine myeloma Cell lines (e.g., derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, Calif. USA, and from SP-2 or X63-Ag8-653 cells available from American Type Culture Collection, Rockville, Md. USA). Human myeloma and mouse human heteromyeloma cell lines have also been described for the Production of human Monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and cultured in a suitable medium (e.g., a medium containing one or more agents that inhibit the growth or survival of the unfused parent myeloma cells). For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferably, the serum-free hybridoma cell culture method is used to reduce the use of animal-derived serum, such as fetal bovine serum, described, for example, in Evan et al, Trends in Biotechnology, 24(3), 105-108 (2006).

Oligopeptides as a tool for increasing hybridoma cell culture productivity are described in Franek, Trends in Monoclonal Antibody Research,111-122 (2005). In particular, standard media are rich in certain amino acids (alanine, serine, asparagine, proline) or protein hydrolysate fractions, and synthetic oligopeptides consisting of three to six amino acid residues can significantly inhibit apoptosis. The peptide is present in millimolar or higher concentrations.

Production of monoclonal antibodies that bind to the antibodies of the present disclosure can be determined in the culture medium in which the hybridoma cells are grown. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can be determined by, for example, Scatchard analysis. See, e.g., Munson et al, anal. biochem.107:220 (1980).

Once hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity are identified, the clones can be subcloned by limiting dilution methods and cultured by standard methods. See, e.g., Goding, supra. Suitable media for this purpose include, for example, D MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in the animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods (e.g., protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography). One method of isolating proteins from hybridoma cells is described in US2005/176122 and U.S. Pat. No. 6,919,436. The method involves the use of minimal salts (e.g. lyotropic salts) during the binding process and preferably also small amounts of organic solvents during the elution process.

(iii) Library-derived antibodies

Antibodies of the disclosure can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties, such as the method described in example 3. Other Methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37(O' Brien et al, eds., Human Press, Totowa, NJ,2001), and are further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology 248:161-175(Lo, ed., Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5): 1073-; fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.Methods284(1-2):119-132 (2004).

In some phage display methods, the repertoire of VH and VL genes are individually cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library from which antigen-binding phage can then be selected, as described in Winter et al, Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments as single chain fv (scfv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, all natural components (e.g., all natural components from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self-antigens without any immunization as described by Griffiths et al, EMBO J,12: 725-. Finally, natural libraries can also be made by cloning unrearranged V gene segments from stem cells; and the use of PCR primers containing random sequences to encode highly variable CDR3 regions and to accomplish in vitro rearrangement as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. patent publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

(Iv) chimeric, humanized and human antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.nat' l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (described as "surface remodeling"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer, 83:252-260(2000) (describing the "guided selection" method for FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best fit" approach (see, e.g., Sims et al J.Immunol.151:2296 (1993)); the framework regions of consensus sequences of human antibodies derived from a particular subset of the light or heavy chain variable regions (see, e.g., Carter et al, proc.natl.acad.sci.usa, 89:4285 (1992)); and Presta et al, j.immunol., 151:2623 (1993); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front. biosci.13:1619-1633 (2008)); and the framework regions derived from screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described by van Dijk and van de Winkel, curr. opin. pharmacol.5:368-74(2001) and Lonberg, curr. opin. immunol.20: 450-.

Human antibodies can be made by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For an overview of the methods for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech. -112523:1117-1125 (2005). See also, e.g., the description XENOMOUSE^TMU.S. Pat. nos. 6,075,181 and 6,150,584 to technology; description of the inventionU.S. patent numbers 5,770,429 for technology; description of K-M U.S. Pat. No. 7,041,870 to Art, and descriptionU.S. patent application publication No. US 2007/0061900 for technology). The human variable regions from intact antibodies produced by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines have been described for the production of human monoclonal antibodies. (see, e.g., Kozbor J.Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol., 147:86 (1991)), human antibodies produced via human B-cell hybridoma technology are also described by Li et al, Proc.Natl.Acad.Sci.USA, 103: 3557-. Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268(2006) (describing human-human hybridomas). The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlens, Histology and Histopathology,20(3): 927-.

Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

(V) antibody fragment

Antibody fragments may be produced by conventional methods (e.g., enzymatic digestion) or by recombinant techniques. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may improve access to solid tumors. For a review of some antibody fragments, see Hudson et al, (2003) nat. Med.9: 129-.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E.coli, so that large quantities of these fragments can be easily produced. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab '-SH fragments may be recovered directly from E.coli and chemically coupled to form F (ab' )₂Fragments (Carter et al, Bio/Technology 10: 163-. According to another alternative, F (ab')₂And (3) fragment. Fab and F (ab')₂A fragment comprising a salvage receptor binding epitope residue. Other techniques for producing antibody fragments will be apparent to the skilled artisan. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. nos. 5,571,894 and 5,587,458. Fv and scFv are the only species with an intact binding site without constant regions. Thus, they may be suitable for reducing non-specific binding during in vivo use. scFv fusion proteins can be constructed to produce fusion of the effector protein at the amino or carboxy terminus of the scFv. See, Antibody Engineering, ed. borrebaeck, supra. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific antibodies

Multispecific antibodies have binding specificities for at least two different epitopes, wherein the epitopes are typically derived from different antigens. Although such molecules typically bind only two different epitopes (i.e., bispecific antibodies, BsAb), when used herein, the expression includes antibodies with other specificities, such as trispecific antibodies. Bispecific antibodies can be made as full length antibodies or antibody fragments (e.g., F (ab') ₂Bispecific antibodies).

Methods of making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983)). Due to the random diversity of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually done by an affinity chromatography step, is rather cumbersome and the product yield is low. A similar method is disclosed in WO 93/08829 and Trauecker et al, EMBO J., 10:3655-3659 (1991).

One method known in the art for making bispecific antibodies is the "knobs-into-holes" or "protrusion-into-cavity" method (see, e.g., U.S. Pat. No. 5,731,168). In this method, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. The interface of one immunoglobulin polypeptide interacts with the corresponding interface of another immunoglobulin polypeptide, thereby associating the two immunoglobulin polypeptides. These interfaces can be engineered such that a "knob" or "protrusion" (which terms are used interchangeably herein) located on one immunoglobulin polypeptide interface corresponds to a "hole" or "cavity" (which terms are used interchangeably herein) located on another immunoglobulin polypeptide interface. In some embodiments, the hole has the same or similar dimensions as the pestle, and is suitably positioned such that when two interfaces interact, the pestle of one interface can be positioned in the corresponding hole of the other interface. Without wishing to be bound by theory, it is believed that this stabilizes the heteromultimer and favors the formation of the heteromultimer over other species (e.g., homomultimers). In some embodiments, the methods can be used to facilitate heteromultimerization of two different immunoglobulin polypeptides, resulting in a bispecific antibody comprising two immunoglobulin polypeptides having binding specificity for different epitopes.

In some embodiments, the knob may be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, the socket can be constructed by replacing a larger amino acid side chain with a smaller side chain. The pestle or mortar may be present in the original interface or may be synthetically introduced. For example, a knob or hole can be synthetically introduced by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art. The side chain volumes of the various amino acid residues are shown in table 1 below. In some embodiments, the original residue has a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the input residues that form the knob are naturally occurring amino acids and can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and the import residue for forming the socket is a naturally occurring amino acid, and can include alanine, serine, threonine, and valine.

TABLE 1 Properties of amino acid residues

^aThe molecular weight of the amino acid minus the molecular weight of water. From Handbook of Chemistry and Physics,43^rdValues of ed.cleveland, Chemical Rubber Publishing co., 1961.

^bValues from A.A.Zamyytnin, prog.Biophys.mol.biol.24:107-123, 1972.

^cValues from C.Chothia, J.mol.biol.105:1-14, 1975. The accessible surface area is defined in fig. 6-20 of this reference.

In some embodiments, the original residues used to form the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining three-dimensional structures may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these examples, human IgG₁The CH3/CH3 interface of (a) involves sixteen residues on each domain located on four antiparallel beta strands. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel beta strands, so that the structure is dominated by the surrounding solvent and not by the partner CH3The risk of the complementary mortar of domains holding the pestle is minimized. In some embodiments, the mutations that form the corresponding knob and hole in the two immunoglobulin polypeptides correspond to one or more of the pairs provided in table 2.

TABLE 2 exemplary corresponding pestle and mortar Forming mutations^*Group (2)

Mutations are expressed as the original residue, followed by the position of the Kabat numbering system, and then the input residue (all residues are given in the one letter amino acid code). Multiple mutations are separated by colons.

In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain, the CH3 domain comprising one or more amino acid substitutions listed in table 2 above. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of table 2 and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of table 2.

Following DNA mutation as described above, polynucleotides encoding modified immunoglobulin polypeptides having one or more corresponding knob or mortar forming mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Pat. nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, 7,695,936, 8,216,805; U.S. publication No. 2013/0089553; and Spiess et al, Nature Biotechnology 31: 753-. Prokaryotic host cells such as E.coli or eukaryotic host cells such as CHO cells can be used to produce the modified immunoglobulin polypeptides. The corresponding immunoglobulin polypeptides with knob and hole can be expressed in co-cultured host cells and purified together as heteromultimers, or they can be expressed in a single culture, purified separately, and assembled in vitro. In some embodiments, two strains of bacterial host cells (one expressing the immunoglobulin polypeptide with the knob and the other expressing the immunoglobulin polypeptide with the hole) are co-cultured using standard bacterial culture techniques known in the art. In some embodiments, the two strains may be mixed in a specific ratio, for example to achieve equal expression levels in culture. In some embodiments, the two strains may be mixed in a ratio of 50:50, 60:40, or 70: 30. After expression of the polypeptide, the cells can be lysed together and the protein extracted. Standard techniques known in the art that allow measurement of the abundance of homomultimeric versus heteromultimeric species may include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide is expressed separately using standard recombinant techniques, and they can be assembled together in vitro. For example, assembly can be achieved by purifying each modified immunoglobulin polypeptide, mixing them in equal mass and incubating together, reducing the disulfide (e.g., by treatment with dithiothreitol), concentrating, and reoxidizing the polypeptide. The bispecific antibody formed can be purified using standard techniques including cation exchange chromatography and measured using standard techniques including size exclusion chromatography. For a detailed description of these methods, see Speiss et al, Nat Biotechnol 31:753-8, 2013. In some embodiments, the modified immunoglobulin polypeptide may be expressed separately in CHO cells and assembled in vitro using the methods described above.

According to different methods, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. The fusion preferably uses an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, the CH2 and CH3 regions. Typically there is at least one first heavy chain constant region (CH1) present in the fusion that contains the site required for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides in the examples great flexibility to adjust the mutual ratio of the three polypeptide fragments, while the unequal ratios of the three polypeptide chains used in the construction provide the best yield. However, when at least two polypeptide chains are expressed in equal ratios resulting in high yields or when the ratios are of no particular significance, it is possible to insert the coding sequences for two or all three polypeptide chains in one expression vector.

In one embodiment of the method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain combination, since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides a simple way of separation. This process is disclosed in WO 94/04690. For more details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. C with one interface comprising antibody constant domains_H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine), complementary "cavities" of the same or similar size to the larger ones are created at the interface of the second antibody molecule. This provides a mechanism by which the production of heterodimers can be increased over other undesired end products (e.g., homodimers).

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate can be coupled to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). The heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are described in U.S. Pat. No. 4,676,980, as well as in a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science, 229:81(1985) describe a procedure in which intact antibodies are proteolytically cleaved to yield F (ab')₂And (3) fragment. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives was then reconverted to the Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibody produced can be used as an agent for the selective immobilization of enzymes.

Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J.Exp.Med., 175:217-225(1992) describe fully humanized bispecific antibodies F (ab')₂The generation of molecules. Each Fab' fragment was separately secreted from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies.

Various techniques have also been described for the preparation and isolation of bispecific antibody fragments directly from recombinant cell cultures. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al, J.Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. The method can also be used for the production of antibody homodimers. HolliThe "diabody" technique described by nger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-. The fragments comprise a light chain variable domain (V) linked by a linker_H) Heavy chain variable domain of (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V of a segment_HAnd V_LThe domains are forced to complement the V of another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, J.Immunol, 152:5368 (1994).

Antibodies having more than two valencies are contemplated. For example, trispecific antibodies may be prepared. Tuft et al, J.Immunol.147:60 (1991).

(vii) Single domain antibodies

In some embodiments, the antibodies of the present disclosure are single domain antibodies. A single domain antibody is a single polypeptide chain comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516B 1). In one embodiment, the single domain antibody consists of all or part of the heavy chain variable domain of an antibody.

(Viii) antibody variants

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes can be introduced into the amino acid sequence of an antibody of interest when forming the sequence.

(ix) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The target sites for substitution mutations include HVRs and FRs. Conservative substitutions are shown in table 3. Further substantial changes are provided under the heading "exemplary substitutions" in table 1, and are further described below with respect to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

TABLE 3 conservative substitutions

Amino acids can be grouped according to common side chain properties:

a. hydrophobic norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

c. acidity: asp and Glu;

d. alkalinity: his, Lys, Arg;

e. residue of

The chain orientation is Gly and Pro;

f. the aromaticity is Trp, Tyr and Phe.

Non-conservative substitutions will require the exchange of a member of one of these classes for another.

One type of substitution variant involves the replacement of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, one or more of the resulting variants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will have certain biological properties of the parent antibody that are substantially retained. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons that undergo high frequency mutation during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196(2008)) and/or SDR (a-CDR), where the resulting variant VH or VL is subjected to a binding affinity test. Affinity maturation by construction and re-selection from secondary libraries has been described, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37 (edited by O' Brien et al, Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation purposes by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR targeting methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. Such changes may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, or contains no more than one, two, or three amino acid substitutions.

One method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution may be targeted or eliminated. Variants can be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

(X) glycosylation variants

In certain aspects, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Antibody addition or deletion of glycosylation sites can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise bi-antennary oligosaccharides with a branched chain, typically attached through an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present disclosure may be modified to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided comprising an Fc region, wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. In particular, antibodies having reduced fucose relative to the amount of fucose on the same antibody produced in wild-type CHO cells are contemplated herein. That is, they are characterized by a lower amount of fucose than that produced by native CHO cells (e.g., CHO cells that produce a native glycosylation pattern, such as CHO cells containing a native FUT8 gene). In certain embodiments, the antibody is an antibody on which less than about 50%, 40%, 30%, 20%, 10% or 5% of the N-linked glycans comprise fucose. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In certain embodiments, the antibody is an antibody wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely free of fucose, or free of fucose or defucosylated. The amount of fucose is determined by calculating the average amount of fucose at Asn297 in the sugar chain, relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 as determined by MALDI-TOF mass spectrometry, as described in WO 2008/077546. Asn297 refers to the asparagine residue at about position 297 in the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. US 2003/0157108(Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13 CHO cells (Ripka et al, Arch. biochem. Biophys.249:533-545 (1986); U.S. patent application No. US 2003/0157108A1, Presta, L; and WO 2004/056312A 1, Adams et al, inter alia, example 11), and knock-out cell lines, such as alpha-1, 6-fucosyltransferase gene (FUT8), knock-out CHO cells (see, e.g., Yamane-Ohnuki et al, Biotech. biog.87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng.94(4):680-688 (2006); and WO 2003/085107).

Antibodies are also provided with bisected oligosaccharides, for example, where the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al); U.S. Pat. No.6,602,684(Umana et al); US 2005/0123546(Umana et al) and Ferrara et al, Biotechnology and Bioengineering,93(5):851-861 (2006). Also provided are antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).

In certain embodiments, an antibody variant comprising an Fc region described herein is capable of binding to Fc γ RIII. In certain embodiments, an antibody variant comprising an Fc region described herein has ADCC activity in the presence of human effector cells or increased ADCC activity in the presence of human effector cells as compared to an otherwise identical antibody comprising a human wild type IgG1 Fc region.

(xi) Fc region variants

In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In certain embodiments, the disclosure contemplates antibody variants with some, but not all, effector functions, which make them desirable candidates for use where the half-life of the antibody in vivo is important and certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the main cells mediating ADCC, express only Fc (RIII, whereas monocytes express Fc (RI, Fc (RII and Fc (RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravech and Kinet, Annu. Rev. Immunol.9: 457. 492 (1991). non-limiting examples of in vitro assays for assessing ADCC activity of a target molecule are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al, Proc. nat 'l Acad. Sci. USA 83: 7059. 7063(1986)) and Hellstrom, I. et al, Proc. nat' l Acad. Sci. USA 82: 9. clan. 1502 (1985); 5,821,337 (see Bruggemann, M. et al, J.exp.166: 1351)), (see, flow cytometry methods for measuring, e.g., Actic. Acti. Sci. No. 1497- ^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and CytotoxNon-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be assessed in vivo, for example, in an animal model (such as disclosed in Clynes et al, proc. nat' l acad. sci. usa 95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complementActivation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202:163 (1996); Cragg, M.S. et al, Blood101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include those with substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al, J.biol.chem.9(2):6591-6604 (2001))

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In one exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A and K334A.

In some embodiments, alterations are made in the Fc region, resulting in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), as described, for example, in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al, J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al) (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351 for further examples of variants of Fc regions.

Pharmaceutical compositions and formulations

Also provided herein are pharmaceutical compositions and formulations comprising a PD-1 axis binding antagonist (e.g., atelizumab), a platinum agent (e.g., carboplatin or cisplatin), and an antimetabolite (e.g., pemetrexed), for use, e.g., in the treatment of lung cancer (such as non-small cell lung cancer, e.g., stage IV non-squamous non-small cell lung cancer). In some embodiments, the pharmaceutical compositions and formulations further comprise a pharmaceutically acceptable carrier.

In some embodiments, the anti-PDL 1 antibody described herein (e.g., atuzumab) is in a formulation comprising the antibody in an amount of about 60mg/mL, histidine acetate at a concentration of about 20mM, sucrose at a concentration of about 120mM, and a polysorbate (e.g., polysorbate 20) at a concentration of 0.04% (w/v), and the pH of the formulation is about 5.8. In some embodiments, the anti-PDL 1 antibody described herein (e.g., atuzumab) is in a formulation comprising the antibody in an amount of about 125mg/mL, histidine acetate at a concentration of about 20mM, sucrose at a concentration of about 240mM, and a polysorbate (e.g., polysorbate 20) at a concentration of 0.02% (w/v), and the pH of the formulation is about 5.5.

After the antibody of interest is prepared (e.g., techniques for producing antibodies that can be formulated as disclosed herein have been set forth and are known in the art), a pharmaceutical formulation comprising the same is prepared. In certain embodiments, the antibody to be formulated is not pre-lyophilized, and the formulation of interest herein is an aqueous formulation. In certain embodiments, the antibody is a full length antibody. In one embodiment, the antibody in the formulation is an antibody fragment, e.g., F (ab') ₂In this case, a problem that may not occur with a full-length antibody (e.g., splicing of an antibody into Fab) needs to be solved. Present in the formulationA therapeutically effective amount of an antibody is determined, for example, by considering the required dosage volume and mode of administration. About 25mg/mL to about 150mg/mL, or about 30mg/mL to about 140mg/mL, or about 35mg/mL to about 130mg/mL, or about 40mg/mL to about 120mg/mL, or about 50mg/mL to about 130mg/mL, or about 50mg/mL to about 125mg/mL, or about 50mg/mL to about 120mg/mL, or about 50mg/mL to about 110mg/mL, or about 50mg/mL to about 100mg/mL, or about 50mg/mL to about 90mg/mL, or about 50mg/mL to about 80mg/mL, or about 54mg/mL to about 66mg/mL are exemplary antibody concentrations in the formulation.

An aqueous formulation comprising the antibody in a pH buffered solution is prepared. In some embodiments, the pH of the buffer of the present disclosure is in the range of about 5.0 to about 7.0. In certain embodiments, the pH is in the range of about 5.0 to about 6.5, the pH is in the range of about 5.0 to about 6.4, in the range of about 5.0 to about 6.3, the pH is in the range of about 5.0 to about 6.2, the pH is in the range of about 5.0 to about 6.1, the pH is in the range of about 5.5 to about 6.1, the pH is in the range of about 5.0 to about 6.0, the pH is in the range of about 5.0 to about 5.9, the pH is in the range of about 5.0 to about 5.8, the pH is in the range of about 5.1 to about 6.0, the pH is in the range of about 5.2 to about 6.0, the pH is in the range of about 5.3 to about 6.0, the pH is in the range of about 5.4 to about 6.0, the pH is in the range of about 5.5 to about 6.0, the pH is in the range of about 5.5.0 to about 6.0, the pH is in the range of about 5.0, the range of about 6.0, or the pH is in the range of about. In some embodiments, the pH of the formulation is 6.0 or about 6.0. In some embodiments, the pH of the formulation is 5.9 or about 5.9. In some embodiments, the pH of the formulation is 5.8 or about 5.8. In some embodiments, the pH of the formulation is 5.7 or about 5.7. In some embodiments, the pH of the formulation is 5.6 or about 5.6. In some embodiments, the pH of the formulation is 5.5 or about 5.5. In some embodiments, the pH of the formulation is 5.4 or about 5.4. In some embodiments, the pH of the formulation is 5.3 or about 5.3. In some embodiments, the pH of the formulation is 5.2 or about 5.2. Examples of the buffering agent for controlling the pH within this range include histidine (e.g., L-histidine) or sodium acetate. In certain embodiments, the buffer comprises histidine acetate or sodium acetate at a concentration of about 15mM to about 25 mM. In some embodiments, the buffer comprises histidine acetate or sodium acetate at a concentration of about 15mM to about 25mM, about 16mM to about 25mM, about 17mM to about 25mM, about 18mM to about 25mM, about 19mM to about 25mM, about 20mM to about 25mM, about 21mM to about 25mM, about 22mM to about 25mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, or about 25 mM. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.0. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.1. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.3. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.4. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.5. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.6. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.7. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.8. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 5.9. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 6.0. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 6.1. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 6.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 20mM, pH 6.3. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.3. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.4. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.5. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.6. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.7. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.8. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 5.9. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 6.0. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 6.1. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 6.2. In one embodiment, the buffer is histidine acetate or sodium acetate in an amount of about 25mM, pH 6.3.

In some embodiments, the buffer is at a concentration of about 60mM to about 240 mM. In some embodiments, sucrose in the formulation is about 60mM to about 230mM, about 60mM to about 220mM, about 60mM to about 210mM, about 60mM to about 200mM, about 60mM to about 190mM, 60mM to about 180mM, about 60mM to about 170mM, about 60mM to about 160mM, about 60mM to about 150mM, about 60mM to about 140mM, about 80mM to about 240mM, about 90mM to about 240mM, about 100mM to about 240mM, about 110mM to about 240mM, about 120mM to about 240mM, about 130mM to about 240mM, about 140mM to about 240mM, about 150mM to about 240mM, about 160mM to about 240mM, about 170mM to about 240mM, about 180mM to about 240mM, about 190mM to about 240mM, about 200mM to about 240mM, about 80mM to about 160mM, about 100mM to about 140mM, or about 110mM to about 130 mM. In some embodiments, sucrose in the formulation is about 60mM, about 70mM, about 80mM, about 90mM, about 100mM, about 110mM, about 120mM, about 130mM, about 140mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, or about 240 mM.

In some embodiments, the concentration of antibody in the formulation is about 40mg/mL to about 125 mg/mL. In some embodiments, the concentration of antibody in the formulation is about 40mg/mL to about 120mg/mL, about 40mg/mL to about 110mg/mL, about 40mg/mL to about 100mg/mL, about 40mg/mL to about 90mg/mL, about 40mg/mL to about 80mg/mL, about 40mg/mL to about 70mg/mL, about 50mg/mL to about 120mg/mL, about 60mg/mL to about 120mg/mL, about 70mg/mL to about 120mg/mL, about 80mg/mL to about 120mg/mL, about 90mg/mL to about 120mg/mL, or about 100mg/mL to about 120 mg/mL. In some embodiments, the concentration of antibody in the formulation is about 60 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 65 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 70 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 75 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 80 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 85 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 90 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 95 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 100 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 110 mg/ml. In some embodiments, the concentration of antibody in the formulation is about 125 mg/ml.

In some embodiments, a surfactant is added to the antibody preparation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188, etc.). The amount of surfactant added should be such that it reduces aggregation of the formulated antibody and/or minimizes particle formation and/or reduces adsorption in the formulation. For example, the surfactant may be present in the formulation in an amount of about 0.001% to about 0.5% (w/v). In some embodiments, the surfactant (e.g., polysorbate 20) is about 0.005% to about 0.2%, about 0.005% to about 0.1%, about 0.005% to about 0.09%, about 0.005% to about 0.08%, about 0.005% to about 0.07%, about 0.005% to about 0.06%, about 0.005% to about 0.05%, about 0.005% to about 0.04%, about 0.008% to about 0.06%, about 0.01% to about 0.06%, about 0.02% to about 0.06%, about 0.01% to about 0.05%, or about 0.02% to about 0.04%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.005% or about 0.005%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.006% or about 0.006%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.007% or about 0.007%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.008% or about 0.008%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.009%, or about 0.009%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.01% or about 0.01%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.02% or about 0.02%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.03% or about 0.03%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.04% or about 0.04%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.05% or about 0.05%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.06% or about 0.06%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.07% or about 0.07%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.08% or about 0.08%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.1% or about 0.1%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.2% or about 0.2%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.3% or about 0.3%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.4% or about 0.4%. In certain embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.5% or about 0.5%.

In one embodiment, the formulation comprises the above-described agents (e.g., antibodies, buffers, sucrose, and/or surfactants), and is substantially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium chloride. In another embodiment, a preservative may be included in the formulation, particularly when the formulation is a multi-dose formulation. The concentration of the preservative may range from about 0.1% to about 2%, preferably from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers (e.g. as described in Remington's Pharmaceutical sciences)ces 16th edition, Osol, a.ed. (1980) may be included in the formulation provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include: other buffers, co-solvents, antioxidants (including ascorbic acid and methionine), chelating agents (such as EDTA), metal complexes (such as zinc protein complexes), biodegradable polymers (such as polyesters), and/or salt-forming counterions. Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r: (r)) Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

The formulations herein may also comprise more than one protein, preferably those proteins having complementary activities that do not adversely affect other proteins, for the particular indication being treated. For example, when the antibody is anti-PDL 1 (e.g., atelizumab), it can be used in combination with another drug (e.g., a chemotherapeutic agent and an antineoplastic agent).

The pharmaceutical compositions and formulations described herein may be prepared by mixing the active ingredient (e.g., antibody or polypeptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's pharmaceutical Sciences 16th edition, Osol, a.ed. (1980)), in lyophilized formulations or in aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, examples Such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r: (r))Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

Exemplary lyophilized antibody formulations are described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

The formulations and compositions herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively); in colloidal drug delivery systems (e.g., liposomes, albumin, microspheres, microemulsions, nanoparticles, and nanocapsules); or in a coarse emulsion. Such techniques are disclosed in Remington's pharmaceutical Sciences 16 th edition, Osol, A. eds (1980).

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by sterile filtration membranes.

Pharmaceutical formulations of carboplatin, cisplatin and/or pemetrexed are commercially available. For example, carboplatin to includeVarious trade names are known therein (as described elsewhere herein). Cisplatin is prepared byVarious trade names are known therein (as described elsewhere herein). Pemetrexed comprisesVarious trade names (as described elsewhere herein) are known, including giophem, PEXATE and ciambran. In some embodiments, the carboplatin and/or pemetrexed are provided in separate containers. In some embodiments, the cisplatin and/or pemetrexed are provided in separate containers. In some embodiments, carboplatin and/or pemetrexed are used and/or prepared separately for administration to an individual as described in prescription information available for commercial products. In some embodiments, cisplatin and/or pemetrexed are used and/or prepared for administration to an individual, respectively, as described in prescription information available for commercially available products.

Methods of treatment

Provided herein are methods of treating or delaying progression of cancer (e.g., lung cancer, such as non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., carboplatin or cisplatin). In some embodiments, the treatment results in a sustained response in the individual after cessation of treatment. In some embodiments, the treatment extends Progression Free Survival (PFS) in the individual. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, administration extends the Overall Survival (OS) of the individual. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months.

The methods described herein are useful for treating conditions where enhanced immunogenicity is desired, such as increasing tumor immunogenicity for the treatment of cancer. Also provided herein are methods of enhancing immune function in an individual having (e.g., lung cancer, such as non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) comprising administering to the individual an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., carboplatin or cisplatin).

In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is stage IV NSCLC. In some embodiments, the stage IV NSCLC is non-squamous NSCLC. In some embodiments, the NSCLC is stage IV non-squamous non-small cell lung Cancer as defined by or as established by Union International conductor le Cancer/American Joint Committee on Cancer stabilizing system, 7 th edition, histologically or cytologically (see, e.g., Detterbeck et al, (2009) Chest 136: -26071). In some embodiments, stage IV NSCLC has mixed non-small cell histology (e.g., squamous and non-squamous), and the major histological component is or appears to be non-squamous. In some embodiments, the NSCLC is classified as stage IV if the tumor has grown to a nearby structure. In some embodiments, the NSCLC is classified as stage IV if the tumor has grown to nearby structures and/or has reached a proximal lymph node. In some embodiments, the NSCLC is classified as stage IV if the cancer has spread from the initially affected lung to another lung. In some embodiments, NSCLC is classified as stage IV if cancer cells are found in the fluid surrounding the lungs (i.e., malignant pleural effusion). In some embodiments, the NSCLC is classified as stage IV if cancer cells (i.e., malignant pericardial effusion) are found in the fluid surrounding the heart. In some embodiments, NSCLC is classified as stage IV if the cancer has spread as a single tumor outside the chest, e.g., to the distal lymph nodes, liver, bone, and/or brain. In some embodiments, NSCLC is classified as stage IV if the cancer has spread outside the chest as more than one tumor, e.g., to the distal lymph nodes, liver, bone, and/or brain. In some embodiments, stage IV NSCLC is refractory to treatment. More detailed information on the Staging of NSCLC is described in American Joint Committee on Cancer. Lung. in: AJCC Cancer Stable Manual.8 th. New York, NY: Springer; 2017:431-456.

In some embodiments, the subject has a poor prognosis. In some embodiments, the subject is a treatment-naive subject. In some embodiments, the treatment-naive individual is an individual that has not received prior treatment for, e.g., cancer, NSCLC or stage IV non-squamous NSCLC. In some embodiments, the treatment-naive individual is an individual who has not received prior treatment for stage IV non-squamous NSCLC. In some embodiments, the subject is an individual who has not received chemotherapy, e.g., has not received prior chemotherapy to treat, e.g., cancer, NSCLC, and/or stage IV non-squamous NSCLC. In some embodiments, the individual has not received treatment for stage IV non-squamous NSCLC. In some embodiments, the individual has not received prior systemic treatment for stage IV non-squamous NSCLC.

In some embodiments, the individual is asian. In some embodiments, the individual is asian descendant. In some embodiments, the individual is at least 65 years old. In some embodiments, the individual is a never-smoker. In some embodiments, the never-smoker is an adult who never smokes or smokes less than 100 cigarettes in their lifetime. In some embodiments, the individual does not have liver metastases.

In some embodiments, the subject is "PD-L1 high". In some embodiments, a patient is "PD-L1 high" if the total of tumor cells expressing PD-L1 in the pre-treatment sample from the patient is greater than or equal to 50% of the total number of tumor cells in the sample. In some embodiments, expression of PD-L1 on > 50% of tumor cells in the pre-treatment sample is defined/scored as "TC 3". In some embodiments, a patient is "PD-L1 high" if the total of tumor-infiltrating immune cells that express PD-L1 in the pre-treatment sample from the patient is greater than or equal to 10% of the total number of tumor-infiltrating immune cells in the sample. In some embodiments, expression of PD-L1 on ≧ 10% of tumor-infiltrating immune cells in the pre-treatment sample is defined/scored as "IC 3". In some embodiments, the pre-treatment sample is a fresh tumor sample. In some embodiments, the pre-treatment sample is a formalin-fixed paraffin-embedded (FFPE) tumor sample. In some embodiments, the level of PD-L1 expression on tumor cells and/or tumor infiltrating immune cells in the pre-treatment sample is determined by an immunohistochemical assay. In some embodiments, the immunohistochemistry assay is an VENTANA SP142 assay.

In some embodiments, a patient is "PD-L1 low" if the tumor cells expressing PD-L1 in the pre-treatment sample from the patient total 1% to < 5% of the total number of tumor cells in the sample. In some embodiments, PD-L1 expression on 1% to < 5% of tumor cells in the pre-treatment sample is defined/scored as "TC 1". In some embodiments, a patient is "PD-L1 low" if the tumor cells expressing PD-L1 in a pre-treatment sample from the patient total 5% to < 50% of the total number of tumor cells in the sample. In some embodiments, PD-L1 expression on 5% to < 50% of tumor cells in the pre-treatment sample is defined/scored as "TC 2". In some embodiments, a patient is "PD-L1 low" if the tumor-infiltrating immune cells that express PD-L1 in the pre-treatment sample from the patient total 1% to < 5% of the total number of tumor-infiltrating immune cells in the sample. In some embodiments, PD-L1 expression is defined/scored as "IC 1" on 1% to < 5% of tumor immune cells in the pre-treatment sample. In some embodiments, a patient is "PD-L1 low" if the tumor-infiltrating immune cells that express PD-L1 in the pre-treatment sample from the patient total 5% to < 10% of the total number of tumor-infiltrating immune cells in the sample. In some embodiments, PD-L1 expression is defined/scored as "IC 2" on 5% to < 10% of tumor immune cells in the pre-treatment sample. In some embodiments, the pre-treatment sample is a fresh tumor sample. In some embodiments, the pre-treatment sample is a formalin-fixed paraffin-embedded (FFPE) tumor sample. In some embodiments, the level of PD-L1 expression on tumor cells and/or tumor infiltrating immune cells in the pre-treatment sample is determined by an immunohistochemical assay. In some embodiments, the immunohistochemistry assay is an VENTANA SP142 assay.

In some embodiments, the individual is "PD-L1 negative". In some embodiments, a patient is "PD-L1 negative" if the total of tumor cells expressing PD-L1 in a pre-treatment sample from the patient is < 1% of the total number of tumor cells in the sample. In some embodiments, PD-L1 expression on < 1% of tumor cells in a pre-treatment sample is defined as "TC 0". In some embodiments, a patient is "PD-L1 negative" if the total of tumor-infiltrating immune cells that express PD-L1 in the pre-treatment sample from the patient is < 1% of the total number of tumor-infiltrating immune cells in the sample. In some embodiments, PD-L1 expression on < 1% of tumor-infiltrating immune cells in the pre-treatment sample is defined as "IC 0". In some embodiments, the pre-treatment sample is a fresh tumor sample. In some embodiments, the pre-treatment sample is a formalin-fixed paraffin-embedded (FFPE) tumor sample. In some embodiments, the level of PD-L1 expression in tumor cells and/or tumor infiltrating immune cells in the pre-treatment sample is determined by an immunohistochemical assay. In some embodiments, the immunohistochemistry assay is an VENTANA SP142 assay, which is described in further detail elsewhere herein.

In some embodiments, the TC0, TC1, TC2, TC3, IC0, IC1, IC2, and IC3 definitions/scores are summarized in the following table:

exemplary Tumor Cell (TC) and tumor infiltrating Immune Cell (IC) score definitions

TC score highest in the step scheme, followed by IC

Please refer to the evaluation guidelines for VENTANA PD-L1(SP142) IHC assay: and (4) universal non-small cell lung cancer staining training. (see www (dot) rocheplus (dot) es/content/dam/hcp-ports/spain/documents/formalci% C3% B3 n/uph/PD-L1% 20SP 142% 20 Association% 20% 20v2.5(dot) pdf

In some embodiments, The individual has histologically or cytologically confirmed stage IV non-squamous NSCLC (described in Detterbeck et al, (2009) "The new lung cancer staging system." chess.136: 26071-), according to The criteria outlined by The international joint anticancer/american joint committee on staging system 7 th edition). In some embodiments, an individual has NSCLC with mixed non-small cell histology (i.e., squamous and non-squamous), and the individual is considered to have non-squamous NSCLC if the primary histological component is or appears to be non-squamous. In some embodiments, the individual does not have a sensitizing mutation in the EGFR gene. In some embodiments, the individual does not have an ALK fusion oncogene. In some embodiments, the individual is screened for EGFR and ALK status prior to treatment.

In some embodiments, the individual has received prior neoadjuvant chemotherapy, adjuvant chemotherapy, or chemotherapy with the intent to cure non-metastatic disease, and has experienced a no-treatment interval of at least 6 months since the last dose of chemotherapy and/or radiation therapy until the start of treatment. In some embodiments, the individual does not have active or untreated Central Nervous System (CNS) metastases. In some embodiments, the subject has been treated for asymptomatic supratentorial or cerebellar CNS metastasis. In some embodiments, the subject has not metastasized to the midbrain, pons, medulla oblongata, or spinal cord. In some embodiments, the individual has a CNS disease and does not require a corticosteroid for treatment of the CNS disease. In some embodiments, the individual has a new asymptomatic metastasis and has received radiation and/or surgery for CNS metastasis. In some embodiments, an individual with CNS metastasis does not receive stereotactic radiation within 7 days of initiation of treatment. In some embodiments, an individual with a CNS metastasis does not receive whole brain radiation within 14 days of initiation of treatment. In some embodiments, the subject does not have leptomeningeal disease. In some embodiments, the individual is free of uncontrolled tumor pain. In some embodiments, the subject does not have uncontrolled pleural effusion. In some embodiments, the subject is free of uncontrolled pericardial effusion. In some embodiments, the individual has no other malignancy than NSCLC within 5 years prior to the start of treatment.

In some embodiments, the individual has a measurable NSCLC (e.g., stage IV non-squamous NSCLC) as defined by/as RECIST v1.1 criteria (see, e.g., Eisenhauer et al, (2009) "New response evaluation criteria in solid tumors: Revised RECIST peptides (version 1.1)." eur.j. cancer.45: 228-. In some embodiments, the individual is not receiving prior treatment with a CD137 agonist or immune checkpoint blockade therapy, for example, including but not limited to an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the patient has received prior treatment against cytotoxic T lymphocyte-associated antigen 4(CLTA-4), wherein the treatment occurs at least 6 weeks prior to initiation of the treatment described herein.

Any PD-1 axis binding antagonist, antimetabolite, and platinum agent known in the art or described herein can be used in the method. In some embodiments, the PD-1 axis binding antagonist is atelizumab, the antimetabolite is pemetrexed and/or the platinum agent is carboplatin or cisplatin.

In some embodiments, the atezumab is administered at a dose of 1200mg, the carboplatin is administered at a dose sufficient to achieve AUC of 6mg/ml/min, and the pemetrexed is administered at 500mg/m²The dosage of (a).

In some embodiments, the atelizumab is administered at a dose of 1200mg and the cisplatin is at 75mg/m²And pemetrexed at a dose of 500mg/m²The dosage of (a).

In some embodiments, treatment includes an induction phase and a maintenance phase (or "maintenance therapy"). In some embodiments, the induction period comprises administration of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle of cycles 1 through 4, at 500mg/m on day 1²Administering an antimetabolite (e.g., pemetrexed), and administering a platinum agent (e.g., carboplatin) on day 1 at a dose sufficient to achieve an initial target area under the curve (AUC) of 6 mg/mL/min. In some embodiments, the maintenance period includes administering a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle after cycle 4, and 500mg/m on day 1²Administering an antimetabolite (e.g., pemetrexed).

In some embodiments, the induction period includes administration of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle of cycles 1 through 4, at 500mg/m on day 1 ²Administering an antimetabolite (e.g., pemetrexed) at a dose of 75mg/m on day 1²The dose of (a) is administered with a platinum agent (e.g., cisplatin). In some embodiments, the maintenance period includes administering a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle after cycle 4, and 500mg/m on day 1²Administering an antimetabolite (e.g., pemetrexed).

In some embodiments, the induction period includes administration of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle of cycles 1 through 6, at 500mg/m on day 1²Administering an antimetabolite (e.g., pemetrexed), and administering a platinum agent (e.g., carboplatin) on day 1 at a dose sufficient to achieve an initial target area under the curve (AUC) of 6 mg/mL/min. In some embodiments, the maintenance period includes administration of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle after cycle 6, andat 500mg/m on day 1²Administering an antimetabolite (e.g., pemetrexed).

In some embodiments, the induction period includes administration of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle of cycles 1 through 6, at 500mg/m on day 1 ²Administering an antimetabolite (e.g., pemetrexed) at a dose of 75mg/m on day 1²The dose of (a) is administered with a platinum agent (e.g., cisplatin). In some embodiments, the maintenance period includes administering a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atelizumab) at a dose of 1200mg on day 1 for each 21-day cycle after cycle 6 and 500mg/m on day 1²Administering an antimetabolite (e.g., pemetrexed).

Tables 4A and 4B below provide exemplary dosing and administration schedules, including an induction cycle and a maintenance cycle:

table 4A: exemplary dosing and administration schedules

^*Period of 21 days

^□mg/ml/min

Table 4B: exemplary dosing and administration schedules

^*Period of 21 days

^□mg/ml/min

In some embodiments, a 1200mg dose of attrituximab is equivalent to a dose based on average body weight of 15 mg/kg. In some embodiments, the dose of Carboplatin required to achieve an AUC of 6mg/mL/min is calculated according to the Calvert formula (see, e.g., Calvertt et al, (1989) "Carboplatin dose: pro-active evaluation of a single formed pellet on a residual function". J.Clin.Oncol.7: 1748-56; van Warmerdam et al, (1995) J.cancer Res.Clin.Oncol.121(8): 478-486). For more details, see example 1 below.

In some embodiments, progression-free survival (PFS) of an individual is measured according to RECIST v1.1 criteria described in Eisenhauer et al, (2009) "New response evaluation criteria in solid tumors: revived RECIST gummine (Version 1.1)." Eur J cancer.45: 228-47). In some embodiments, PFS is measured as the time period from the start of treatment to the first appearance of disease progression, as determined by RECIST v1.1 criteria. In some embodiments, PFS is measured as the time from the start of treatment to death. In some embodiments, the treatment increases progression-free survival (PFS) in the individual by at least about any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months (including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual by at least about 7.6 months. In some embodiments, the treatment increases PFS in an individual by at least about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 months (including any range between these values) as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) who has received treatment with an antimetabolite (e.g., pemetrexed) and a platinum agent (e.g., carboplatin or cisplatin).

In some embodiments, Overall Survival (OS) is measured as the period of time from the start of treatment to death. In some embodiments, the treatment increases OS of the individual by at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months (including any range between these values). In some embodiments, the treatment increases OS in the individual by at least about 18.1 months. In some embodiments, the treatment increases OS in the individual by at least about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 months (including any range between these values) as compared to an individual having lung cancer (e.g., non-small cell lung cancer, such as stage IV non-squamous non-small cell lung cancer) and receiving treatment with an antimetabolite (e.g., pemetrexed) and a platinum agent (e.g., carboplatin or cisplatin).

In some embodiments, the individual is a human.

In some embodiments, the individual has cancer (that has proven to be resistant) that is resistant to one or more PD-1 axis antagonists. In some embodiments, resistance to a PD-1 axis antagonist comprises relapse of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or new site after treatment. In some embodiments, resistance to a PD-1 axis antagonist comprises progression of cancer during treatment with a PD-1 axis antagonist. In some embodiments, resistance to a PD-1 axis antagonist comprises cancer that is non-responsive to treatment. The cancer may be resistant at the beginning of the treatment, or resistant during the treatment. In some embodiments, the cancer is at an early or late stage.

In another aspect, the individual has a cancer that expresses (e.g., in a diagnostic test, has shown expression of) a PD-L1 biomarker. In some embodiments, the patient's cancer expresses a low PD-L1 biomarker. In some embodiments, the patient's cancer expresses a high PD-L1 biomarker. In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is not present in the sample when it comprises 0% of the sample.

In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is present in the sample when it comprises more than 0% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 1% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 5% of the sample. In some embodiments, the PD-L1 biomarker is present in at least 10% of the sample.

In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof.

In some embodiments of any of the methods, assays, and/or kits, the PD-L1 biomarker is detected in a sample by protein expression. In some embodiments, protein expression is determined by Immunohistochemistry (IHC). In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 antibody. In some embodiments, the PD-L1 biomarker is detected by IHC as weak staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC as moderate staining intensity. In some embodiments, the PD-L1 biomarker is detected by IHC as a strong staining intensity. In some embodiments, the PD-L1 biomarker is detected on tumor cells, tumor infiltrating immune cells, stromal cells, and any combination thereof. In some embodiments, the staining is membrane staining, cytoplasmic staining, or a combination thereof.

In some embodiments, the PD-L1 biomarker is detected using an anti-PD-L1 rabbit monoclonal primary antibody. In some embodiments, PD-L1 is detected in a formalin fixed paraffin embedded sample. In some embodiments, the anti-PD-L1 rabbit monoclonal primary antibody is detected with a secondary antibody comprising a detectable label. In some embodiments, the assay used to detect PD-L1 is the VENTANA PD-L1(SP142) assay (available from Commercially available) which are described in more detail in the examples.

In some embodiments of any of the methods, assays, and/or kits, the absence of the PD-L1 biomarker is detected as the absence of staining or no staining in the sample. In some embodiments of any of the methods, assays, and/or kits, the presence of the PD-L1 biomarker is detected as any staining in the sample.

The PD-1 axis binding antagonist (e.g., atuzumab), antimetabolite (e.g., pemetrexed), and platinating agent (e.g., carboplatin or cisplatin) can be administered in any order. For example, a PD-1 axis binding antagonist (e.g., atelizumab), an antimetabolite (e.g., pemetrexed), and a platinating agent (e.g., carboplatin or cisplatin) can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PD-1 axis binding antagonist (e.g., atelizumab), the antimetabolite (e.g., pemetrexed), and the platinum agent (e.g., carboplatin or cisplatin) are in separate compositions. In some embodiments, one or more (or all three) of a PD-1 axis binding antagonist (e.g., altuzumab), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., carboplatin or cisplatin) are in the same composition.

The PD-1 axis binding antagonist (e.g., atuzumab), the antimetabolite (e.g., pemetrexed), and the platinum agent (e.g., carboplatin or cisplatin) can be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the antimetabolite (e.g., pemetrexed) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the platinum agent (e.g., carboplatin or cisplatin) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the PD-1 axis binding antagonist (e.g., atelizumab), the antimetabolite (e.g., pemetrexed), and the platinum agent (e.g., carboplatin or cisplatin) are administered by intravenous infusion. An effective amount of a PD-1 axis binding antagonist (e.g., atuzumab), an antimetabolite (e.g., pemetrexed), and a platinum agent (e.g., carboplatin or cisplatin) can be administered to prevent or treat the disease.

In some embodiments, there is provided a method of treating lung cancer (e.g., stage IV non-squamous non-small cell lung cancer (NSCLC)) in an individual (e.g., an individual not treated for stage IV non-squamous non-small cell lung cancer (NSCLC)), comprising administering to the individual an effective amount of atezumab, pemetrexed, and carboplatin, wherein the administration comprises an induction period and a maintenance period, wherein the induction period comprises each 21-day period of periods 1 through 4, the atezumab is administered at a dose of 1200mg on day 1, and the atezumab is administered at a dose of 500mg/m on day 1²Administering pemetrexed at a dose sufficient to achieve an initial target curve of 6mg/mL/minCarboplatin was administered at the dose of Area Under (AUC). In some embodiments, the maintenance period comprises administering atelizumab at a dose of 1200mg on day 1 for each 21-day cycle after cycle 4, and 500mg/m on day 1²The dosage of pemetrexed is administered.

In some embodiments, there is provided a method of treating lung cancer (e.g., stage IV non-squamous non-small cell lung cancer (NSCLC)) in an individual (e.g., an individual not treated for stage IV non-squamous non-small cell lung cancer (NSCLC)), comprising administering to the individual an effective amount of atezumab, pemetrexed, and cisplatin, wherein the administration comprises an induction phase and a maintenance phase, wherein the induction phase comprises each 21-day cycle of cycles 1 through 4, the atezumab administered at a dose of 1200mg on day 1, and the atezumab administered at 500mg/m on day 1 ²And on day 1 at 75mg/m²Administering cisplatin. In some embodiments, the maintenance period comprises administering atelizumab at a dose of 1200mg on day 1 for each 21-day cycle after cycle 4, and 500mg/m on day 1²The dosage of pemetrexed is administered.

In some embodiments, there is provided a method of treating lung cancer (e.g., stage IV non-squamous non-small cell lung cancer (NSCLC)) in an individual (e.g., an individual not treated for stage IV non-squamous non-small cell lung cancer (NSCLC)), comprising administering to the individual an effective amount of atezumab, pemetrexed, and carboplatin, wherein the administration comprises an induction period and a maintenance period, wherein the induction period comprises each 21-day period of periods 1 through 6, the atezumab administered at a dose of 1200mg on day 1, and the atezumab administered at a dose of 500mg/m on day 1²And administering on day 1 carboplatin at a dose sufficient to achieve an initial target area under the curve (AUC) of 6 mg/mL/min. In some embodiments, the maintenance period comprises administering atelizumab at a dose of 1200mg on day 1 for each 21-day cycle after cycle 6, and 500mg/m on day 1²The dosage of pemetrexed is administered.

In some embodiments, there is provided a method of treating lung cancer (e.g., stage IV non-squamous non-small cell lung cancer (NSCLC)) in an individual (e.g., an individual not treated for stage IV non-squamous non-small cell lung cancer (NSCLC))Cell Lung Cancer (NSCLC)) comprising administering to an individual an effective amount of atuzumab, pemetrexed, and cisplatin, wherein the administration comprises an induction phase and a maintenance phase, wherein the induction phase comprises each 21-day cycle of cycles 1 to 6, the attuzumab is administered at a dose of 1200mg on day 1, and 500mg/m on day 1²And on day 1 at 75mg/m²Administering cisplatin. In some embodiments, the maintenance period comprises administering atelizumab at a dose of 1200mg on day 1 for each 21-day cycle after cycle 6, and 500mg/m on day 1²The dosage of pemetrexed is administered.

In some embodiments, the method extends the subject's PFS (e.g., at least any one of 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 months, including any range between these values) and/or the subject's OS (e.g., at least any one of about 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 months, including any range between these values). In some embodiments, the treatment increases Progression Free Survival (PFS) of the individual for at least about 7.6 months, and/or increases OS of the individual for at least about 18.1 months. In some embodiments, the method extends PFS (e.g., at least about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 months, including any range therebetween) and/or Os (e.g., at least about any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4.5, 5, 5.5, 6, 6.5, or 7 months, including any range therebetween) in the individual as compared to an individual having lung cancer (e.g., non-small cell lung cancer, e.g., stage IV non-squamous non-small cell lung cancer) who has received treatment with a platinum agent (e.g., carboplatin or cisplatin) and an antimetabolite (e.g., pemetrexed).

In some embodiments, e.g., as described in table 4A above, on day 1 of each 21-day cycle of cycles 1 through 4, pemetrexed is administered followed by altuzumab, and carboplatin or cisplatin is administered following pemetrexed. In some embodiments, e.g., as described in table 4B above, on day 1 of each 21-day cycle of cycles 1 through 6, pemetrexed is administered followed by altuzumab, and carboplatin or cisplatin is administered following pemetrexed.

In some embodiments, on day 1 of each 21-day cycle of cycles 1 through 4, atuzumab is administered intravenously over 60(± 15) minutes, pemetrexed is administered intravenously over about 10 minutes, and carboplatin is administered intravenously over about 30-60 minutes. In some embodiments, on day 1 of cycle 1, alemtuzumab is administered intravenously over 60(± 15) minutes, pemetrexed is administered intravenously over about 10 minutes, and carboplatin is administered intravenously over about 30-60 minutes; on day 1 of cycles 2 through 4, alemtuzumab is administered intravenously over 30(± 10) minutes, pemetrexed is administered intravenously over about 10 minutes, and carboplatin is administered intravenously over about 30-60 minutes. In some embodiments, on day 1 of each 21-day cycle after cycle 4, atuzumab is administered intravenously over 60(± 15) minutes and pemetrexed is administered intravenously over about 10 minutes. In some embodiments, on day 1 of each 21-day cycle after cycle 4, atuzumab is administered intravenously within 30(± 10) minutes and pemetrexed is administered intravenously within about 10 minutes.

In some embodiments, the atuzumab is administered intravenously over 60(± 15) minutes on day 1, pemetrexed over about 10 minutes on day 1, and cisplatin is administered intravenously over about 1-2 hours on day 1 for each 21-day cycle of cycles 1 through 4. In some embodiments, on cycle 1, atezumab is administered intravenously over 60(± 15) minutes on day 1, pemetrexed is administered intravenously over about 10 minutes on day 1, and cisplatin is administered intravenously over about 1-2 hours on day 1; on cycles 2 through 4, atezumab was administered intravenously over 30(± 10) minutes on day 1, pemetrexed over about 10 minutes on day 1, and intravenously over about 30-60 minutes on day 1. In some embodiments, the atuzumab is administered intravenously over 60(± 15) minutes on day 1, and the pemetrexed is administered intravenously over about 10 minutes on day 1, every 21-day cycle after cycle 4. In some embodiments, the atuzumab is administered intravenously within 30(± 10) minutes on day 1, and the pemetrexed is administered intravenously within about 10 minutes on day 1, every 21-day cycle after cycle 4.

In some embodiments, the atuzumab is administered intravenously over 60(± 15) minutes on day 1, the pemetrexed is administered intravenously over about 10 minutes on day 1, and the carboplatin is administered intravenously over about 30-60 minutes on day 1, for each 21-day cycle of cycles 1 through 6. In some embodiments, on cycle 1, atezumab is administered intravenously over 60(± 15) minutes on day 1, pemetrexed is administered intravenously over about 10 minutes on day 1, and carboplatin is administered intravenously over about 30-60 minutes on day 1; on cycles 2 through 6, atezumab was administered intravenously over 30(± 10) minutes on day 1, pemetrexed over about 10 minutes on day 1, and carboplatin was administered intravenously over about 30-60 minutes on day 1. In some embodiments, attrituximab is administered intravenously over 60(± 15) minutes on day 1, and pemetrexed is administered intravenously over about 10 minutes on day 1, every 21-day cycle after cycle 6. In some embodiments, attrituximab is administered intravenously over 30(± 10) minutes on day 1, and pemetrexed is administered intravenously over about 10 minutes on day 1, every 21-day cycle after cycle 6.

In some embodiments, the atuzumab is administered intravenously over 60(± 15) minutes on day 1, pemetrexed over about 10 minutes on day 1, and cisplatin is administered intravenously over about 1-2 hours on day 1 for each 21-day cycle of cycles 1 through 6. In some embodiments, on cycle 1, atezumab is administered intravenously over 60(± 15) minutes on day 1, pemetrexed is administered intravenously over about 10 minutes on day 1, and cisplatin is administered intravenously over about 1-2 hours on day 1; in cycles 2 through 6, atezumab was administered intravenously on day 1 over 30(± 10) minutes, pemetrexed was administered intravenously over about 10 minutes on day 1, and cisplatin was administered intravenously over about 30-60 minutes on day 1. In some embodiments, attrituximab is administered intravenously over 60(± 15) minutes on day 1, and pemetrexed is administered intravenously over about 10 minutes on day 1, every 21-day cycle after cycle 6. In some embodiments, attrituximab is administered intravenously over 30(± 10) minutes on day 1, and pemetrexed is administered intravenously over about 10 minutes on day 1, every 21-day cycle after cycle 6.

As a general proposition, a therapeutically effective amount of the antibody administered to a human will be in the range of about 0.01 to about 50mg/kg of patient body weight, whether by one or more administrations. In some embodiments, the antibody used is administered daily, for example, at about 0.01 to about 45mg/kg, about 0.01 to about 40mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15mg/kg, about 0.01 to about 10mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosage regimens may be useful. In one embodiment, the anti-PDL 1 antibody described herein is administered to a human at a dose of about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, or about 1400mg on day 1 of a 21-day cycle. The dose may be administered in a single dose or in multiple doses (e.g., 2 or 3 doses), such as an infusion. The dose of antibody administered in the combination therapy can be reduced compared to monotherapy. The progress of the therapy can be readily monitored by conventional techniques.

In some embodiments, the method may further comprise additional therapies. The additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nano-therapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side-effect limiting agent (e.g., an agent intended to reduce the occurrence and/or severity of a therapeutic side-effect, such as an anti-nausea agent, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation.

In some embodiments, the adjunctive therapy includes CT-011 (also known as Pidilizumab (Pidilizumab) or MDV 9300; CAS registry number 1036730-42-3; CureTech/Medvation). CT-011, also known as hBAT or hBAT-1, is an antibody described in WO 2009/101611. In some embodiments, the additional therapeutic agent comprises an antibody comprising heavy and light chain sequences, wherein:

(a) The heavy chain comprises the following amino acid sequence:

QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:19), and

(b) the light chain comprises the following amino acid sequence:

EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:20)。

in some embodiments, the additional therapeutic antibody comprises a heavy chain variable region from SEQ ID NO:19 and SEQ ID NO: 20 (e.g., three heavy chain HVRs from SEQ ID NO:19 and three light chain HVRs from SEQ ID NO: 20). In some embodiments, the additional therapeutic antibody comprises a heavy chain variable region from SEQ ID NO:19 and a heavy chain variable domain from SEQ ID NO: 20, a light chain variable domain.

Other additional therapeutic antibodies contemplated for use herein include, but are not limited to: alemtuzumab (Campath), bevacizumab (bGenentech); cetuximab (Imclone); panitumumab (A) Amgen), rituximab (Genentech/Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (Genentech), tositumomab (Bexxar, Corixia), antibody drug conjugate gemtuzumab ozolomicin (Wyeth), aprezumab, aselizumab, atilizumab, piplizumab, mabbivatuzumab, mocantotuzumab, sidaclizumab, polyethylene glycol-conjugated certuzumab, cidfusituzumab, cidtuzumab, darlizumab, eculizumab, efavirenzumab, epratuzumab, erilizumab, universal lizumab, aryltuzumab, gemtuzumab ozolomide, oxidutuzumab ozotacin, ipizumab, labuzumab, lintuzumab, matuzumab, mevizumab, motavizumab, natalizumab, nimotuzumab, nolovizumab, nuvivuzumab, adolizumab, omalizumab, olizumab, pertuzumab, rituzumab, ritulizumab, pertuzumab, ritulizumab, and the like, tacatuzumab tetraxetan, taclizumab, talilizumab, tefralizumab, tollizumab, simon interleukin mab, tucusituzumab, umuzumab, ultlizumab, tussilizumab, and anti-leukin mab Interleukin 12(ABT-874/J695, Wyeth Research and Abbott Laboratories).

In some embodiments, the additional therapy is a therapy targeting the PI3K/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemoattractant. In some embodiments, the additional therapy is CTLA-4 (also known as CD152), e.g., a blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or) Tremellimumab (also known as ticilimumab or CP-675,206), antagonists against B7-H3 (also known as CD276) (e.g., blocking antibody MGA271), antagonists against TGF β (e.g., metelilimumab (also known as CAT-192), tresolimumab (also known as GC1008), or LY2157299), including adoptive therapy of T cells (e.g., cytotoxic T cells or CTLs) expressing Chimeric Antigen Receptors (CARs), including adoptive therapy of T cells containing dominant inhibitory TGF β receptors (e.g., dominant inhibitory TGF β type II receptors); treatment comprising a HERCREEM regimen (see, e.g., clinical trials. gov identification NCT00889954), agonists against CD137 (also known as TNFRSF9, 4-1BB or ILA) (e.g., activated antibody, urelumab (also known as BMS-663513)), agonists against CD40 (e.g., activated antibody, CP-870893), agonists against OX40 (also known as CD134) (e.g., activated antibody, administered in combination with a different anti-OX 40 antibody (e.g., AgonOX)), agonists against CD27 (e.g., activated antibody, CDX-1127, indoleamine-2, 3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-dnmt)), antibody-drug conjugates (in some embodiments, comprising cysteine or monad aurine (MMAE), naae 2 b-dnpi conjugate (also known as MMAE 0600A) or MMAE 75rg 3599), antibody-drug conjugate (also known as MMAE A) or MMAE 75rg 3599) Trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine or Genentech), DMUC5754A, antibody-drug conjugates targeting endothelin B receptor (EDNBR) (e.g., an antibody conjugated to MMAE against EDNBR), antibodies to VEGF (e.g., VEGF-a), bevacizumab (also known as VEGF-a)Genentech), an antibody against angiopoietin 2 (also known as Ang2), MEDI 3617; antineoplastic agents, drugs targeting CSF-1R (also known as M-CSFR or CD115), anti-CSF-1R (also known as IMC-CS 4); interferons, such as interferon alpha or interferon gamma, Roferon-A, GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargrastim or) IL-2 (also known as aldesleukin or) IL-12, antibodies targeting CD20 (in certain embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or GA 101)) Or rituximab), an antibody targeting GITR (in certain embodiments, the antibody targeting GITR is TRX 518); in combination with a Cancer vaccine (in some embodiments, the Cancer vaccine is a peptide Cancer vaccine, in some embodiments a personalized peptide vaccine; in some embodiments, the peptide Cancer vaccine is a multivalent long peptide, polypeptide, peptide mixture, hybrid peptide, or peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104:14-21, (2013)); in combination with an adjuvant (a TLR agonist such as Poly-ICLC (also known as a TLR agonist) (also known as a peptide vaccine) ) LPS, MPL or CpG ODN, Tumor Necrosis Factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, HVEM antagonists, ICOS agonists (e.g., by administration of ICOS-L or agonist antibodies to ICOS), CX3CL1 targeted therapies, CXCL10 targeted therapies, CCL5 targeted therapies, LFA-1 or ICAM1 agonists, selectin agonists, targeted therapies, B-Raf inhibitors, vemurafenib (also known as CCL 5)dabrafenib (also known as dabrafenib)) Erlotinib (also known as erlotinib)) MEK inhibitors such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2), cobimetinib (also known as GDC-0973 or Xl-518), trametinib (also known as trametinib)) K-Ras inhibitors, c-Met inhibitors, onartuzumab (also known as MetMAb), ALK inhibitors, AF802 (also known as CH5424802 or Alletinib), inhibitors of phosphatidylinositol 3-kinase (PI3K), BKM120, idelalisib (also known as GS-1101 or CAL-101), periplosine (also known as KRX-0401), Akt, MK2206, GSK690693, GDC-0941, inhibitors of mTOR, sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or Teclinolimus (also known as CCI-779 or Alletinib (Alletinib)), inhibitors of phosphatidylinositol 3-kinase (PI3K), BKM120, idelalisib (also known as GS-1101 or CAL-101), periplosine) Everolimus (also known as RAD001), ridaforolimus (also known as AP-23573, MK-8669 or deforolimus), OSI-027, AZD8055, INK128, PI3K/mTOR dual inhibitor, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, PF-05212384 (also known as PKI-587). The additional therapy may be one or more chemotherapeutic agents described herein.

IX. detection and diagnostic method

In some embodiments, the sample is obtained prior to treatment with a PD-1 axis binding antagonist (e.g., atelizumab), a platinum agent (e.g., carboplatin or cisplatin), and an antimetabolite (e.g., pemetrexed). In some embodiments, the tissue sample is a formalin-fixed and paraffin-embedded specimen, fresh, or frozen.

In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells, and any combination thereof.

The presence and/or expression level/amount of a biomarker (e.g., PD-L1) may be determined qualitatively and/or quantitatively based on any suitable criteria known in the art, including but not limited to DNA, mRNA, cDNA, protein fragments, and/or gene copy number. In certain embodiments, the presence and/or expression level/amount of the biomarker in the first sample is increased or elevated as compared to the presence/absence and/or expression level/amount in the second sample. In certain embodiments, the presence/absence and/or expression level/amount of the biomarker in the first sample is reduced or decreased compared to the presence and/or expression level/amount in the second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosures for determining the presence/absence and/or expression level/amount of a gene are described herein.

In some embodiments of any of the methods, increased expression refers to an overall increase in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) of any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected using methods known in the standard art (e.g., the methods described herein). In certain embodiments, increased expression refers to an increase in the expression level/amount of a biomarker in a sample that is at least about any one of 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 25X, 50X, 75X, or 100X of the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).

In some embodiments of any of these methods, reduced expression refers to an overall reduction in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected, for example, using methods known in the standard art described herein. In certain embodiments, reduced expression refers to a reduction in the expression level/amount of the biomarker in the sample by at least about any one of 0.9X, 0.8X, 0.7X, 0.6X, 0.5X, 0.4X, 0.3X, 0.2X, 0.1X, 0.05X, or 0.01X of the expression level/amount of the biomarker in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

The presence and/or expression levels/amounts of various biomarkers in a sample can be analyzed by a variety of methods, many of which are known in the art and understood by the skilled artisan, including but not limited to: immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assay, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assay, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") including quantitative real-time PCR ("qRT-PCR") and other amplification type detection methods, such as branched DNA, ba, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or serial analysis of gene expression ("sise"), and any of a variety of analyses that can be performed by protein, gene, and/or tissue array analysis. Typical Protocols for assessing the status of genes and gene products can be found, for example, In Ausubel et al, 1995, Current Protocols In Molecular Biology, Units 2(Northern Blotting), 4(Southern Blotting), 15(Immunoblotting) and 18(PCR Analysis). Multiplex immunoassays may also be used, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD").

In some embodiments, the presence and/or expression level/amount of a biomarker is determined using the following method: (a) performing gene expression profiling, PCR (e.g., rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (e.g., a cancer sample); and b) determining the presence and/or expression level/amount of the biomarker in the sample. In some embodiments, microarray methods include the use of microarray chips having one or more nucleic acid molecules that hybridize under stringent conditions to nucleic acid molecules encoding the genes described above, or having one or more polypeptides (e.g., peptides or antibodies) that bind to one or more proteins encoded by the genes described above. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

Methods for evaluating mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using labeled ribonucleoproteins specific for one or more genes, Northern blotting, and related techniques) and various nucleic acid amplification assays (e.g., RT-PCR using complementary primers specific for one or more genes, as well as other amplification type detection methods, such as branched DNA, SISBA, TMA, and the like).

mRNA measurements can be conveniently performed on mammalian samples using Northern blot, dot blot or PCR analysis. In addition, such methods can include one or more steps that allow the method to determine the level of a target mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence for a "housekeeping" gene (e.g., an actin family member)). Alternatively, the sequence of the amplified target cDNA may be determined.

Alternative methods include protocols for examining or detecting mRNA (e.g., target mRNA) in a tissue or cell sample by microarray technology. Test and control mRNA samples from the test and control tissue samples were reverse transcribed and labeled using a nucleic acid microarray to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the order and location of each component of the array is known. For example, genes whose expression correlates with an increase or decrease in clinical benefit of anti-angiogenic therapy can be selected for arrangement on a solid support. Hybridization of a labeled probe to a particular array member indicates that the sample from which the probe was derived expresses the gene.

According to some embodiments, the presence and/or expression level/amount of the aforementioned gene is measured by observing its protein expression level. In certain embodiments, the method comprises contacting the biological sample with an antibody (e.g., an anti-PD-L1 antibody) directed against a biomarker described herein under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo. In one embodiment, the antibody is used to select a subject (e.g., a biomarker selected for an individual) that is suitable for treatment with a PD-L1 axis binding antagonist.

In certain embodiments, the sample is examined for the presence and/or expression level/amount of a biomarker protein using IHC and staining protocols. IHC staining of tissue sections has proven to be a reliable method for determining or detecting the presence of proteins in a sample. In some embodiments of any of these methods, assays, and/or kits, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 is detected by immunohistochemistry. In some embodiments, the increased expression of the PD-L1 biomarker in the sample from the individual is increased expression of a protein, and in further embodiments, an IHC assay is used. In one embodiment, the expression level of the biomarker is determined using the following method: (a) IHC analysis of a sample (e.g., a cancer sample) with an antibody; and b) determining the expression level of the biomarker in the sample. In some embodiments, IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line stained sample or tissue sample from a non-cancer patient).

IHC may be used in conjunction with other techniques, such as morphological staining and/or fluorescence in situ hybridization. There are two general IHC methods available: direct and indirect assays. According to the first assay, the binding of the antibody to the target antigen is determined directly. This direct assay is visualized without further antibody interaction using a labeled reagent, such as a fluorescent label or an enzyme-labeled primary antibody. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. When the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.

The primary and/or secondary antibodies used in IHC will typically be labeled with a detectable moiety. Many tags are available, which are generally divided into the following categories: (a) radioisotopes, e.g.³⁵S、¹⁴C、¹²⁵I、³H and¹³¹i; (b) colloidal gold particles; (c) fluorescent labels including, but not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores (e.g., SPECTRUM ORANGE7 and SPECTRUM GREEN7) and/or derivatives of any one or more of the above; (d) various enzyme-substrate labels are available, and U.S. Pat. No. 4,275,149 provides a review of some of them. Examples of enzyme labels include luciferases (e.g., luciferases and bacterial luciferases; U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinones (2, 3-dihydrophthalazinones), malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, lactoperoxidase, microperoxidase, and the like.

Examples of enzyme-substrate combinations include, for example: horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate; alkaline Phosphatase (AP) with p-nitrophenyl phosphate as chromogenic substrate; and β -D-galactosidase (β -D-Gal) having a chromogenic substrate (e.g., p-nitrophenyl- β -D-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl- β -D-galactosidase). For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

In some embodiments of any of the methods, PD-L1 is detected by immunohistochemistry using an anti-PD-L1 diagnostic antibody (i.e., primary antibody). In some embodiments, the PD-L1 diagnostic antibody specifically binds to human PD-L1. In some embodiments, the PD-L1 diagnostic antibody is a non-human antibody. In some embodiments, the PD-L1 diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the PD-L1 diagnostic antibody is a monoclonal antibody. In some embodiments, the PD-L1 diagnostic antibody is directly labeled.

The specimen thus prepared can be mounted and covered with a cover slip. Slide evaluation is then determined, for example, using a microscope, and staining intensity criteria routinely used in the art can be employed. In one embodiment, it is understood that when cells and/or tissue from a tumor are examined using IHC, staining is typically determined or assessed in the tumor cells and/or tissue (as opposed to stroma or surrounding tissue that may be present in the sample). In some embodiments, it is understood that when IHC is used to examine cells and/or tissues from a tumor, staining includes determining or evaluating tumor infiltrating immune cells, including immune cells within or surrounding the tumor.

In some embodiments, PDL1 expression is assessed on a tumor or tumor sample. As used herein, a tumor or tumor sample may encompass a portion or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample can further encompass the area of the tumor occupied by tumor-associated intratumoral cells and/or tumor-associated stroma (e.g., hyperplastic stroma around an associated tumor). The tumor-associated intratumoral cells and/or tumor-associated matrix can include an immune-infiltrating region (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent to and/or associated with the main tumor mass. In some embodiments, PDL1 expression is assessed on tumor cells. In some embodiments, PDL1 expression is assessed on immune cells within the tumor region (e.g., tumor infiltrating immune cells) as described above.

In an alternative method, the sample may be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and the complex detected. The presence of biomarkers can be detected in a variety of ways, such as by Western blotting and ELISA procedures for assaying a variety of tissues and samples, including plasma or serum. A variety of immunoassay techniques are available using this assay format, see, e.g., U.S. Pat. nos. 4,016,043, 4,424,279 and 4,018,653. These include non-competitive types of single-and two-site or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarkers.

The presence and/or expression level/amount of a selected biomarker in a tissue or cell sample can also be examined by function-based or activity-based assays. For example, if the biomarker is an enzyme, assays known in the art can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

In certain embodiments, the samples are normalized for differences in the amount of biomarker determined and differences in the quality of the samples used and differences between assay runs. Such normalization can be achieved by detecting and incorporating the expression of certain normalization biomarkers, including well-known housekeeping genes. Alternatively, normalization may be based on the mean or median signal of all genes being measured or a large subset thereof (global normalization approach). The normalized amount of subject tumor mRNA or protein measured is compared to the amount found in the reference set on a gene-by-gene basis. The normalized expression level of each mRNA or protein in each tested tumor of each subject can be expressed as a percentage of the expression level measured in the reference set. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall within a certain percentile of this range, which can be determined by methods well known in the art.

In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsies are commonly used to obtain representative tumor tissue masses. Alternatively, tumor cells may be obtained indirectly in the form of tissues or body fluids known or believed to contain the tumor cells of interest. For example, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushing, or from sputum, thoracic fluid, or blood. Genes or gene products can be detected from cancer or tumor tissue or other body samples (e.g., urine, sputum, serum, or plasma). The same techniques discussed above for detecting a target gene or gene product in a cancerous sample can be applied to other body samples. Cancer cells may be shed from cancerous lesions and appear in such body samples. By screening such body samples, a simple early diagnosis of these cancers can be made. In addition, by testing for target genes or gene products in such body samples, the progress of the treatment can be more easily monitored.

In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual that are obtained at one or more different time points than the test sample. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at an earlier time point than when the test sample was obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell or control tissue may be useful if the reference sample is obtained during a preliminary diagnosis of cancer and the test sample is obtained later on at the time of metastasis of the cancer.

In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a plurality of samples from a combination of one or more healthy individuals that are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a plurality of samples from a combination of one or more individuals having a disease or disorder (e.g., cancer) that is not the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue or pooled plasma or serum samples from one or more individuals other than the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue or pooled plasma or serum samples of one or more individuals with a disease or disorder (e.g., cancer) that is not the subject or individual.

In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., biopsy). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and/or bone/bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (e.g., glioblastoma, neuroblastoma, etc.).

In some embodiments, a tumor tissue sample (the terms "tumor sample" are used interchangeably herein) may encompass a portion or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample can further encompass the area of the tumor occupied by tumor-associated intratumoral cells and/or tumor-associated stroma (e.g., hyperplastic stroma around an associated tumor). The tumor-associated intratumoral cells and/or tumor-associated matrix can include an immune-infiltrating region (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent to and/or associated with the main tumor mass.

In some embodiments, the PD-L1 biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot hybridization, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof. In some embodiments, the PD-L1 biomarker is detected using FACS analysis. In some embodiments, the PD-L1 biomarker is PD-L1. In some embodiments, PD-L1 expression is detected in a blood sample. In some embodiments, PD-L1 expression is detected on circulating immune cells in the blood sample. In some embodiments, the circulating immune cells are CD3+/CD8+ T cells. In some embodiments, the immune cells are isolated from the blood sample prior to analysis. Any suitable method may be used to isolate/enrich for such cell populations, including but not limited to cell sorting. In some embodiments, PD-L1 expression is elevated in a sample from an individual who is responsive to treatment with an inhibitor of the PD-L1/PD-1 axis pathway, such as an anti-PD-L1 antibody. In some embodiments, PD-L1 expression is elevated on circulating immune cells, such as CD3+/CD8+ T cells, in the blood sample.

In some embodiments, the anti-PD-L1 rabbit monoclonal primary antibody is detected with a secondary antibody comprising a detectable label. In some embodiments, the assay used to detect PD-L1 is the VENTANA PD-L1(SP142) assay (available fromCommercially available) which are described in more detail in the examplesThe above-mentioned processes are described.

Certain aspects of the present disclosure relate to the measurement of the expression level of one or more genes or one or more proteins in a sample. In some embodiments, the sample may comprise white blood cells. In some embodiments, the sample can be a peripheral blood sample (e.g., from a patient having a tumor). In some embodiments, the sample is a tumor sample. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basement membrane, and any other cell type associated with a tumor. In some embodiments, the sample is a tumor tissue sample containing tumor infiltrating leukocytes. In some embodiments, the sample can be processed to separate or isolate one or more cell types (e.g., leukocytes). In some embodiments, the sample may be used without separating or differentiating cell types.

Tumor samples may be obtained from a subject by any method known in the art, including but not limited to biopsy, endoscopy, or surgery. In some embodiments, the tumor sample can be prepared by methods such as freezing, fixing (e.g., by using formalin or similar fixative), and/or embedding in paraffin. In some embodiments, a tumor sample may be sectioned. In some embodiments, a fresh tumor sample (i.e., a sample that has not been prepared by the methods described above) may be used. In some embodiments, tumor samples may be prepared by incubation in solution to preserve mRNA and/or protein integrity.

In some embodiments, the sample may be a peripheral blood sample. Peripheral blood samples may include leukocytes, PBMCs, and the like. Any technique known in the art for isolating leukocytes from a peripheral blood sample can be used. For example, a blood sample may be drawn, red blood cells may be lysed, and a white blood cell pellet may be isolated and used for the sample. In another example, density gradient separation can be used to separate white blood cells (e.g., PBMCs) from red blood cells. In some embodiments, a fresh peripheral blood sample (i.e., a sample that has not been prepared by the methods described above) may be used. In some embodiments, peripheral blood samples may be prepared by incubation in solution to preserve mRNA and/or protein integrity.

In some embodiments, responsiveness to treatment may refer to any one or more of: extending survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of cancer. In some embodiments, response may refer to improvement in one or more factors according to the RECIST guideline publication for determining tumor status (i.e., response, stability, or progression) in a cancer patient. For a more detailed discussion of these guidelines, see Eisenhauer et al, Eur J Cancer 2009; 45: 228-47; topalian et al, N Engl J Med 2012; 366: 2443-54; wolchok et al, Clin Can Res 2009; 15: 7412-20; and therase, P., et al J.Natl.cancer Inst.92:205-16 (2000). A responsive subject may refer to a subject whose cancer shows improvement, e.g., based on one or more factors based on RECIST criteria. Non-responsive subjects may refer to subjects whose cancer does not show improvement, e.g., based on one or more factors based on RECIST criteria.

Conventional response criteria may not be sufficient to characterize the anti-tumor activity of immunotherapeutics, which may result in a delayed response that may begin with an initial apparent radiological progression, including the appearance of new lesions. Thus, modified response criteria have been developed that take into account the possible appearance of new lesions and allow confirmation of radiological progress in subsequent evaluations. Thus, in some embodiments, reactivity may refer to improvement in one or more factors according to immune-related response criteria 2 (irRC). See, e.g., Wolchok et al, Clin Can Res 2009; 15:7412-20. In some embodiments, new lesions are added to a defined tumor burden and then used, for example, for subsequent radiologic progression assessment. In some embodiments, the presence of a non-target lesion is included in the assessment of a complete response, not in the assessment of radiologic progression. In some embodiments, radiologic progression may be determined based solely on measurable disease and/or may be determined by continuous assessment ≧ 4 weeks from the date of first recording.

In some embodiments, responsiveness may include immune activation. In some embodiments, responsiveness may include therapeutic efficacy. In some embodiments, responsiveness may include immune activation and therapeutic efficacy.

X. preparation or kit

In another embodiment of the invention, an article of manufacture or kit is provided comprising a PD-1 axis binding antagonist (e.g., atelizumab) and/or a platinating agent (e.g., carboplatin or cisplatin) and/or an antimetabolite (e.g., pemetrexed). In some embodiments, the article of manufacture or kit further comprises a package insert comprising instructions for administering the PD-1 axis binding antagonist in combination with a platinum agent (e.g., carboplatin or cisplatin) and an antimetabolite (e.g., pemetrexed) to treat or delay progression of cancer (e.g., lung cancer, such as non-small cell lung cancer (NSCLC) including stage IV non-squamous NSCLC) or to enhance immune function in an individual having cancer (e.g., lung cancer, such as NSCLC, including stage IV non-squamous NSCLC). Any PD-1 axis binding antagonist, platinum agent known in the art may be included in the article of manufacture or kit. In some embodiments, the kit comprises atezumab, carboplatin or cisplatin, and pemetrexed.

In some embodiments, the PD-1 axis binding antagonist (e.g., atelizumab), the platinum agent (e.g., carboplatin or cisplatin), and the antimetabolite (e.g., pemetrexed) are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials, for example glass, plastic (such as polyvinyl chloride or polyolefin) or metal alloys (such as stainless steel or hastelloy). In some embodiments, the container contains the formulation, and a label on or associated with the container can indicate instructions for use. The article of manufacture or kit may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further comprises one or more other agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes.

This description is to be construed as sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples of the invention

The present disclosure will be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: phase III open randomized study of atelizumab in combination with carboplatin + pemetrexed or cisplatin pemetrexed + versus carboplatin-pemetrexed or cisplatin pemetrexed in patients who have not received chemotherapy and have stage IV non-squamous non-small cell lung carcinoma + (NSCLC) +

This study was conducted to evaluate the efficacy, safety and pharmacokinetic comparison of altuzumab in combination with carboplatin + pemetrexed + or cisplatin + pemetrexed versus carboplatin + pemetrexed or cisplatin-pemetrexed in patients who had not received chemotherapy and had stage IV non-squamous non-small cell lung cancer (NSCLC). The following summary outlines the specific goals and corresponding endpoints of the study.

Object of study

The common primary efficacy goals of this study are as follows:

efficacy of alemtuzumab + carboplatin + pemetrexed versus carboplatin + pemetrexed and of alemtuzumab + cisplatin + pemetrexed versus cisplatin + pemetrexed was evaluated in a treatment (ITT) intended population with Progression Free Survival (PFS) assessed by the investigator according to RECIST v1.1 (see, e.g., Eisenhauer et al, (2009) "Newresponse evaluation criteria in solid tumors: Revised recistguline (Version 1.1)." Eur J cancer.45:228-47) or death due to any cause (whichever occurred first).

The efficacy of atuzumab + carboplatin + pemetrexed, and of atuzumab + cisplatin + pemetrexed, versus cisplatin + pemetrexed was evaluated as Overall Survival (OS) (defined as the time from random grouping to death due to any cause).

Secondary efficacy goals for this study were as follows:

the efficacy of astuzumab + carboplatin + pemetrexed and of astuzumab + cisplatin + pemetrexed versus cisplatin + pemetrexed was evaluated in terms of Objective Response Rate (ORR) (defined as Partial Response (PR) or Complete Response (CR)) according to RECIST v 1.1.

The efficacy of astuzumab + carboplatin + pemetrexed versus carboplatin + pemetrexed, and of astuzumab + cisplatin + pemetrexed versus cisplatin + pemetrexed, was evaluated with the investigator-assessed duration of response (DOR) according to RECIST v 1.1.

Assessment of OS rates for 1 and 2 years.

The effect of astuzumab was determined by measurement of patient-reported changes from baseline in lung cancer symptoms of cough, dyspnea, chest pain, or arm/shoulder pain using the european cancer research and treatment organization (EORTC) quality of life questionnaire-core 30(QLQ-C30) and supplementary lung cancer module (QLQ-LC 13).

TTD (time to exacerbation) of lung cancer symptoms in patients, defined as the time from random grouping to exacerbation (10 point change) in EORTC QLQ-30 and EORTC QLQ-LC 13.

Using the lung cancer Symptom (SILC) scale symptom severity score, the effect of attentizumab was determined by the change from baseline in patient-reported lung cancer symptom (chest pain, dyspnea and cough) scores.

The safety goals for this study were as follows:

national cancer institute general terminology for adverse events standard (NCI CTCAE) v 4.0, the incidence, nature and severity of adverse events were used to assess the safety and tolerability of atuzumab + carboplatin + pemetrexed or atuzumab + cisplatin + pemetrexed.

Assessment of changes in vital signs, physical findings and clinical laboratory results during and after study treatment administration. The safety and tolerability of altuzumab as a maintenance therapy in combination with carboplatin + pemetrexed, or in combination with cisplatin + pemetrexed, or with pemetrexed alone, was evaluated.

Evaluation of the incidence and titer of therapeutic antibodies (ATA) against astuzumab and investigation of the potential relationship between immunogenic response and pharmacokinetics, safety and efficacy. The pharmacokinetic objectives of this study were:

characterization of the pharmacokinetics of altuzumab with carboplatin + pemetrexed, cisplatin + pemetrexed, or pemetrexed alone.

Characterization of the pharmacokinetics of carboplatin in combination with altuzumab + pemetrexed.

Characterization of the pharmacokinetics of cisplatin in combination with atuzumab + pemetrexed.

Characterization of the pharmacokinetics of pemetrexed in combination with atuzumab + carboplatin or in combination with atuzumab + cisplatin

Determine the maximum serum atuzumab concentration (Cmax) observed after infusion (group a).

Determination of the minimum serum alemtuzumab concentration (Cmin) observed 120 days (+ -30 days) before infusion, discontinuation of treatment and the last dose of alemtuzumab (group a) in the selected cycle.

Determination of plasma concentrations of carboplatin or cisplatin (group a).

Determination of plasma concentration of Pemetrexed (group A)

The exploratory goals of this study were:

assessment of Progression Free Survival (PFS) rate at 6 months and 1 year marker time points.

Assess the Overall Survival (OS) rate at 3 years for each treatment group.

Efficacy of atelizumab in subgroups based on demographic and baseline characteristics was evaluated by OS and investigator-assessed PFS according to RECIST v 1.1.

Efficacy of atelizumab was assessed by milestone survival.

The relationship between biomarkers (including but not limited to programmed death-ligand 1(PD-L1), programmed death 1(PD-1), somatic mutations, etc.) in tumors and blood is evaluated according to the definition of Immunohistochemistry (IHC), quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), next generation sequencing, and/or other methods and measures of efficacy.

Assessment of predictive, prognostic and pharmacodynamic exploratory biomarkers in specimens and/or fresh tumor tissue and blood and their association with disease state, resistance mechanisms and/or response to the study treatment.

Assessment of the status of PD-L1-, immune and NSCLC-associated and other exploratory biomarkers, their association with disease status and/or response to alemtuzumab combination chemotherapy in specimens and/or fresh tumor tissues and blood (or blood derivatives) collected before, during or after or in progress with treatment with alemtuzumab.

Assess and compare the health status of patients according to the EuroQoL 5 dimensional level 5 (EQ-5D-5L) questionnaire to generate utility scores for compensating the economic model.

The effect of attentimab in each treatment comparison was determined by changes from baseline in Patient Reported Outcome (PRO) for health-related quality of life, lung cancer-related symptoms and health status as assessed by european cancer research and treatment organization quality of life questionnaires EORTC QLQ-C30 and QLQ-LC 13.

Design of research

Detailed information for a randomized, phase III, multicenter, open study aimed at assessing the safety and efficacy of (a) attritumab + carboplatin + pemetrexed versus carboplatin + pemetrexed therapy and (b) attritumab + cisplatin + pemetrexed versus cisplatin + pemetrexed therapy on patients who have not received chemotherapy and who have stage IV non-squamous NSCLC is described below. The following scheme 1 illustrates the study design:

scheme 1

In scheme 1 above, ECOG PS refers to "eastern cooperative tumor group expression status", NSCLC refers to "non-small cell lung cancer", and RECIST v1.1 refers to "response assessment criteria for solid tumors, version 1.1".

Approximately 568 patients were recruited in all sites of the global recruitment phase for this study. Patients were randomized by gender (male vs. female), smoking status (never vs. present and/or previous), ECOG (i.e. eastern cooperative tumor group) performance status (0vs.1), and chemotherapy regimen (carboplatin vs. cisplatin), and then grouped by 1:1 to receive one of the following treatment regimens as shown in table 5 below. Further details regarding ECOG expression status are provided in Oken et al, (1982) Am J Clin Oncol.5: 649-.

Table 5: treatment group

The induction period was four or six cycles with 21 days as one cycle. The number of cycles (i.e., four or six) of induction treatment was determined by the investigator and determined and recorded prior to randomization. Induction therapy was administered on a 21 day cycle until the following (whichever occurred first) occurred: 1) four or six cycles of administration, 2) unacceptable toxicity or 3) documented disease progression.

After the induction period, patients who did not experience progression or unacceptable toxicity continued to maintenance treatment with altuzumab + pemetrexed (group a) or pemetrexed alone (group B). Patients randomized to group a or group B continued treatment with attrituzumab + pemetrexed maintenance or pemetrexed maintenance until disease progression, unacceptable toxicity or death. Patients randomized to group a continued treatment with atelizumab for progressive disease over RECIST v1.1 during the induction or maintenance phase, provided they experienced clinical benefit assessed by the investigator as follows:

for treatment group a: during treatment (induction or maintenance), patients showing evidence of clinical benefit are allowed to continue to use atlizumab after RECIST v1.1 for progressive disease is met if all of the following conditions are met:

Evidence of clinical benefit assessed by the investigator.

Absence of symptoms and signs (including laboratory value exacerbations [ e.g., new or worsening hypercalcemia ]), indicates a clear progression of the disease.

There was no decline in ECOG performance status attributable to disease progression.

No tumor progression at critical anatomical sites (e.g. leptomeningeal disease) that the protocol allows for medical intervention to fail to address.

Patients must provide written consent to acknowledge deferral of other treatment options in support of continuing study treatment at initial progression.

Chemotherapy treatment was discontinued in all patients who showed no evidence of progressive disease for RECIST v1.1 (groups a and B).

Tables 6A and 6B below provide dosing and administration schedules for the treatment regimens in table 5:

table 6A: dosing and administration schedules for 4 cycle induction phase therapy

Period of 21 days

□mg/ml/min

Table 6B: dosing and administration schedules for 6 cycle induction phase therapy

^*Period of 21 days

^□mg/ml/min

Regardless of dose delay, patients were evaluated for tumors at baseline and every 6 weeks (+ -7 days) at the first 48 weeks after cycle 1 day. Regardless of the delay in therapeutic dose, tumor assessments are required every 9 weeks (+ -7 days) after completion of the tumor assessment at week 48. Tumor assessments were performed on patients until radiographic disease progression according to RECIST v1.1 or loss of clinical benefit (for patients receiving only atzumab-therapy who continued treatment after radiographic disease progression according to RECIST v 1.1), withdrawal of consent, termination of the study by the sponsor, or death (whichever occurred first). Patients who discontinued treatment for reasons other than radiographic disease progression (e.g., toxicity) continued to undergo planned tumor assessment until radiographic disease progression according to RECIST v1.1 or lost clinical benefit (for patients receiving alemtuzumab-treatment who continued treatment after radiographic disease progression according to RECIST v 1.1), withdrawal consent, termination of the study by the investigator, or death (whichever occurred first), regardless of whether the patient began a new anti-cancer treatment.

If clinically feasible, patients are advised to take tumor biopsy samples for collection as the radiographic disease progresses. These data were used to investigate whether radiographic findings were consistent with the presence of tumors. In addition, these data were analyzed to assess the correlation between tumor tissue changes and clinical outcome and to further understand the underlying mechanisms of progression and resistance to atuzumab compared to this mechanism following chemotherapy alone. The exploratory biomarker assessment was not used for any treatment-related decisions.

Patients who continue treatment following radiographic progression of RECIST v1.1, continue tumor assessment every 6 weeks (+ -7 days) or more if a worsening of symptoms occurs. For these patients, tumor assessments were performed every 6 weeks (+ -7 days) regardless of study time until study treatment was discontinued.

Patients who discontinued treatment for reasons other than radiographic disease progression according to RECIST v1.1 (e.g., toxicity, worsening of symptoms) continue to undergo planned tumor assessments at the same frequency as if the patient continued to receive study treatment (i.e., every 6 weeks (+ 7 days) 48 weeks after cycle 1 day, then every 9 weeks (+ 7 days), regardless of whether treatment dose was delayed, until radiographic progression according to RECIST v1.1, withdrawal consent, initiation of the study termination, or death (whichever comes first).

Patients who started a new anti-cancer therapy without radiographic disease progression according to RECIST v1.1 continued to undergo planned tumor assessment until radiographic disease progression according to RECIST v1.1 (or loss of clinical benefit, for patients receiving alemtuzumab treatment who continued alemtuzumab treatment after radiographic disease progression according to RECIST v 1.1), withdrawal consent, death, or termination of the study by the sponsor (whichever was first).

Despite evidence of radiographic progress, tumor assessments continued on patients receiving atuzumab treatment and continuing to have clinical benefit according to the above schedule.

Patient's health

Patients were eligible for this study if they had not received chemotherapy-and had stage IV non-squamous NSCLC.

Inclusion criteria

The main inclusion criteria included: 18 years old or older; ECOG performance status is 0 or 1; histologically or cytologically confirmed stage IV non-squamous NSCLC (according to The International Union anti-cancer/United states Commission on cancer staging System 7 th edition; Detterback et al (2009) "The new lung cancer staging system" Chest 136: 260-71); patients with mixed non-small cell histology (i.e., squamous and non-squamous) tumors qualify if the major histology component appears to be non-squamous; no prior treatment for stage IV non-squamous NSCLC; patients who have previously received neoadjuvant therapy, adjuvant chemotherapy, radiation therapy, or chemo-radiation therapy intended to cure non-metastatic disease must experience a no-treatment interval of at least 6 months from random groupings since the last dose of chemotherapy and/or radiation therapy; patients with a history of asymptomatic CNS metastases qualify only if: (a) metastasis is supratentorial and/or cerebellar metastasis (i.e., no midbrain, pons, medulla oblongata, or spinal cord metastasis); (b) patients do not need to continuously receive corticosteroid hormones for treatment of CNS disorders; (c) patients had no stereotactic radiation within 7 days or no whole brain radiation within 14 days prior to randomization; (d) between the completion of CNS-directed treatment and radiation influencer screening, the patient had no evidence of intermediate progression; patients who find a new asymptomatic CNS metastasis in a screening scan must undergo radiation therapy and/or surgery for CNS metastasis. Patients who find a new asymptomatic CNS metastasis in a screening scan must undergo radiation therapy and/or surgery for CNS metastasis. After treatment, these patients may be eligible if all other criteria are met, without the need for additional brain scans prior to randomization. Eligible patients meeting RECIST v1.1 definition should exhibit measurable disease (previously irradiated lesions are considered measurable disease only if disease progression after irradiation of their site is explicitly recorded, since previously irradiated lesions are not the only disease site); adequate hematology and end organ function as defined by the following laboratory test results obtained within 14 days prior to randomization:

Omicron ANC is more than or equal to 1500 cells/mu L, and is supported by granulocyte colony-stimulating factor.

The lymphocyte count is more than or equal to 500/mu L.

The platelet count is more than or equal to 100,000/mu L when not transfused.

O hemoglobin is more than or equal to 9.0 g/dL. (patients were allowed to transfuse blood to meet this standard.)

Omicron or aPTT is less than or equal to 1.5 times the upper normal limit (ULN). (this applies only to patients who do not receive anticoagulation therapy; patients who receive anticoagulation therapy must be maintained at a stable dose.)

Omicron AST, ALT and alkaline phosphatase ≦ 2.5 × ULN, except:

patients with liver metastases recorded: AST and/or ALT is less than or equal to 5 multiplied by ULN.

Patients with liver or bone metastases documented: alkaline phosphatase ≤ 5 × ULN.

O serum bilirubin is less than or equal to 1.25 × ULN. (patients with known Gilbert's disease and serum bilirubin levels ≦ 3 × ULN are included.)

The calculated creatinine clearance (CRCL) is > 45mL/min, or if cisplatin is used, the calculated CRCL must be > 60 mL/min.

Patients were encouraged to submit tumor tissue samples (if any) prior to treatment. If there is no tumor tissue available (e.g., due to exhaustion of prior diagnostic tests), the patient is still eligible. If there is tumor tissue available, it is preferred to use formalin-fixed paraffin-embedded (FFPE) tumor specimens in representative paraffin blocks, or unstained freshly cut serial sections (preferably at least 10) taken from FFPE tumor specimens. If there are no 10 slices, fewer slices may be committed. If no FFPE specimen is available as described above, any type of specimen, including fine needle aspiration, cell pellet (e.g., from pleural effusion), and lavage fluid specimens, is acceptable. The specimens were accompanied by relevant pathological reports. Any available tumor tissue samples need to be submitted before or within 4 weeks after enrollment.

Exclusion criteria

Key exclusion criteria included: patients with sensitizing mutations in the EGFR gene or ALK fusion oncogene; treatment with any other study agent with therapeutic intent within 28 days prior to randomization; assessing determined activity or untreated CNS metastasis by Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) during screening and prior radiographic assessment; spinal cord compression that has not been specifically treated by surgery and/or radiation, or spinal cord compression that has been previously diagnosed and treated but no evidence that the disease is clinically stable for > 2 weeks prior to randomization; leptomeningeal disease; tumor-related uncontrolled pain (patients requiring analgesics must receive a stable treatment regimen when entering the study; symptomatic lesions requiring palliative radiation therapy (e.g., bone metastases or metastases causing nerve impact) should be treated prior to randomization (patients should recover from the effects of radiation without the required minimum recovery time.) for asymptomatic metastatic lesions, further growth of the lesion may be possible in patients with asymptomatic metastatic lesionsWhich can lead to functional defects or intractable pain (e.g., epidural metastases that are currently not associated with spinal compression), the patient is considered for local area treatment, if appropriate, before being randomized. Exclusion criteria also included: uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage (monthly or more frequently, but with indwelling catheters (e.g., for example) ) Patient unlimited drainage frequency); uncontrolled or symptomatic hypercalcemia (> 1.5mmol/L ionized calcium or calcium > 12mg/dL or corrected serum calcium > ULN; patients receiving denosumab before randomization, if eligible, need to be discontinued and replaced with bisphosphonates in the study); other malignancies except SCLC within 5 years prior to randomization are grouped, but those with negligible risk of metastasis or death (e.g., expected-5 years OS > 90%) have expected curative results (e.g., adequately treated cervical in situ cancer, basal or squamous cell skin cancer, surgically treated local prostate cancer with curative intent, surgically treated ductal carcinoma in situ with curative intent); known tumor PD-L1 expression status as determined by IHC analysis of other clinical studies (e.g., patients with PD-L1 expression status were identified, but not qualified, when screened into studies with anti-PD-1 or anti-PD-L1 antibodies); women who were pregnant, lactating, or intended to be pregnant during the study; a history of autoimmune diseases, including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granuloma, and, Syndrome, Guillain-Barre syndrome, multiple sclerosis, vasculitis, or glomerulonephritis (patients with a history of autoimmune-related hypothyroidism and eligible to receive thyroid replacement hormone treatment; type I diabetes-controlled patients eligible to receive insulin treatment); history of idiopathic pulmonary fibrosis,Histological pneumonia (e.g., bronchiolitis obliterans), drug pneumonia, idiopathic pneumonia, or breast CT scan screening for evidence of active pneumonia. (history of permissible radiation pneumonitis in the radiation area (fibrosis)); HIV detection result is positive; with active hepatitis B (chronic or acute; defined as hepatitis B surface antigen [ HBsAg ] at screening]Positive test results) or Hepatitis C Virus (HCV); active tuberculosis; severe infections, including but not limited to complications from hospitalization, bacteremia or severe pneumonia, occurred within 4 weeks prior to randomization; patients receiving therapeutic oral or intravenous antibiotics within 2 weeks prior to randomization (patients receiving prophylactic antibiotic therapy (e.g., for preventing urinary tract infection or preventing exacerbations of chronic obstructive pulmonary disease), who are eligible for significant cardiovascular disease, such as New York Heart Association heart disease (grade II or higher), myocardial infarction, or cerebrovascular accident, unstable arrhythmia, or unstable angina within 3 months prior to randomization) (patients with known coronary artery disease, congestive heart failure failing the above criteria, or patients with left ventricular ejection fraction < 50% should have a stable treatment regimen that should be optimized according to the treatment physician's opinion and consulted with the cardiologist as appropriate; major surgery other than diagnosis performed within 28 days prior to randomization or anticipated need of major surgery during the course of the study; prior allogeneic bone marrow transplantation or solid organ transplantation; any other disease, a treatment regimen, Metabolic dysfunction, physical examination findings, or clinical laboratory findings that may reasonably suspect a disease or condition that prohibits the use of study drugs or may affect interpretation of outcomes or put a patient at high risk for treatment complications; patients with a disease or condition that affects their ability to understand, follow, and/or comply with the present study procedure; treatment with other study drugs with curative intent within 28 days prior to randomization; administering an attenuated live vaccine within 4 weeks before a randomized cohort or expected study period would require such an attenuated live vaccine; prior treatment with EGFREGFRALK inhibitors or ALK inhibitors; any approved anti-cancer therapy, including hormone therapy, was performed within 21 days prior to initiation of study treatment; in that Treatment was first with a CD137 agonist or immune checkpoint blockade therapy, anti-PD-1 and anti-PD-L1 therapeutic antibodies. (patients who had previously received anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) treatment were eligible for cohort, provided that the last dose of anti-CTLA-4 was used at least 6 weeks prior to randomization and that the patient did not have a history of severe immune-related adverse effects by CTLA-4(NCI CTCAE grade 3 and 4)). Treatment with any other study agent with curative intent within 28 days prior to randomization, treatment with systemic immune stimulators (including but not limited to interferon and interleukin 2) within 4 weeks prior to randomization or within 5 half-lives of the drug (whichever is longer) (allowing prior treatment with cancer vaccine); systemic immunosuppressive drugs (including but not limited to corticosteroids, cyclophosphamide, azathioprine, methotrexate, thalidomide and anti-tumor necrosis factor [ anti-TNF ] were administered within 2 weeks prior to randomization]Drug) is administered. Patients who had received an acute, low dose (≦ 10mg oral prednisone or equivalent) of systemic immunosuppressant were eligible to participate in the study. In addition, corticosteroids (< 10mg oral prednisone or equivalent) are allowed for chronic obstructive pulmonary disease, mineralocorticoids (e.g., fludrocortisone) for orthostatic hypotension patients, and small-dose supplementation of corticosteroids for adrenal insufficiency. Patients were excluded if: a history of severe allergic, sensitive or other hypersensitivity reactions to the chimeric or humanized antibody or fusion protein; known to be hypersensitive or allergic to any component of biopharmaceuticals produced by chinese hamster ovary cells or altlizumab preparations; a history of anaphylaxis to carboplatin, cisplatin, or other platinum-containing compounds; patient hearing impairment (cisplatin); grade 2 peripheral neuropathy defined by NCI CTCAE v4.0 (cisplatin); creatinine clearance < 60mL/min (for cisplatin) or < 45mL/min (for carboplatin).

Method of treatment

568 patients were randomized (1:1) to receive treatment with altuzumab + carboplatin + pemetrexed or altuzumab + cisplatin + pemetrexed (group a) or carboplatin + pemetrexed or cisplatin + pemetrexed (group B). (details of groups A and B are shown in Table 5 above).

During the induction period, the chemotherapy cycle counts to a predetermined number (4 or 6) of induction chemotherapy cycles, so long as at least one chemotherapeutic component is administered at least once during a 21 day cycle. Cycles without chemotherapeutic components administered are not counted in the total number of induced chemotherapy cycles.

Patients who experienced no further clinical benefit (for patients who were enrolled in group a), or disease progression (for patients who were enrolled in group B) at any time during the induction period discontinued all study treatments. Without the above criteria, following an induction period of 4 or 6 cycles, patients began maintenance therapy (group A Atlizumab + pemetrexed or group B pemetrexed)

During treatment (induction or maintenance), group a patients with evidence of clinical benefit were allowed to continue to receive atlizumab treatment after RECIST v1.1 for disease progression was met. However, chemotherapy treatment was discontinued.

Patients received antiemetic drugs and intravenous water for platinum-pemetrexed treatment according to local standards of care and manufacturer's instructions. However, due to their immunomodulatory effects, pre-operative administration of steroids is limited when clinically feasible. In addition, topical use of steroids as a one-line treatment is recommended when clinically feasible in the event of pemetrexed-related rashes. Table 7 below lists the pre-operative administration of pemetrexed. Table 8 below lists the infusion times for therapeutic administration of pemetrexed + platinum during the induction and maintenance phases.

Table 7: preoperative drug for pemetrexed

IM in muscle

PO ═ oral administration

Q9w every 9 weeks.

TABLE 8 treatment regimen based on Pemetrexed + platinum chemotherapy

AUC under the concentration-time curve is area

IV is intravenous

Progressive disease of PD

Q3w every 3 weeks.

578 patients (1:1) were randomized to receive treatment with atuzumab + pemetrexed + carboplatin or cisplatin (group a) or pemetrexed + carboplatin or cisplatin (group B). (details of treatment groups A and B are shown above in tables 5, 6A and 6B). Tables 9A and 9B below show the patient demographics and baseline characteristics.

Table 9A: patient demographics and baseline characteristics

Prevalence is calculated in biomarker evaluable populations

Table 9B: baseline characteristics

ECOG, eastern cooperative group of tumors; PS, presentation State

^aIndian or alaska indigenous ethnicity (n-2), black or african americans (n-6) and unknown ethnicity (n-38) are not included in the table.^B2 patients had baseline ECOG PS depletion.^CThe PD-L1 status was available in 60% of patients. PD-L1-high (TC3/IC 3): PD-L1 is expressed in more than or equal to 50% of tumor cells or more than or equal to 10% of tumor infiltrating immune cells; PD-L1-Low (TC12/IC 12): PD-L1 is more than or equal to 1 percent<50% of tumor cells or more than or equal to 1% of <10% of patients expressed in tumor-infiltrating immune cells; and PD-L1-negative (TC0/IC 0): PD-L1 in<1% of tumor cells and<1% tumor infiltration immunityA patient expressed in a germ cell.

Patients received the first dose of study medication on the day of randomization, if available. If not, the first dose is administered within 5 days after the random grouping. Atezumab was provided by the sponsor. Carboplatin, cisplatin and pemetrexed are background treatments and are considered non-research medical products (NIMPs). Carboplatin, cisplatin and pemetrexed were used in the commercial formulations. The attrituzumab drug product is provided in a sterile liquid form in a 20-mL glass vial. The vial is designed to deliver 20mL (1200mg) of the atuzumab solution, but may contain a volume in excess of the specified volume, so that the full 20-mL volume can be delivered.

The induction phase of the study consisted of four or six cycles of altemamab/placebo plus chemotherapy, each cycle lasting 21 days. The number of cycles (four or six) of induction treatment was determined by the investigator and recorded prior to randomization. See above protocol. On day 1 of each cycle, all eligible patients were administered study medication in the following order:

Group A: abiralizumab → Pemetrexed → Carboplatin or cisplatin

Group B: pemetrexed → carboplatin or cisplatin

During the induction period, study treatment was administered on day 1 in the following manner:

1. abiralizumab (1200mg, equivalent to a dose of 15mg/kg based on mean body weight) was administered intravenously over 60(± 15) minutes (which is the first infusion, and may be shortened to 30(± 10) minutes for subsequent infusions), followed by

2. Permetrexed (500 mg/m) was administered intravenously over about 10 minutes²) Then, then

3. Administering carboplatin intravenously over 30-60 minutes to achieve an initial target concentration-time area under the curve (AUC) of 6mg/mL/min (Calvert formula dosing);

or

At 75mg/m within 1-2 hours²The dose of (a) cisplatin is administered intravenously.

Calvert formula was used to calculate the carboplatin dose for AUC 6 (Calvert et al, (1989) J Clin Oncol 7: 1748-56):

calvert formula:

total dose (mg) ═ target AUC (x) (glomerular filtration rate GFR ] +25)

The GFR used in the Calvert's equation to calculate AUC-based dosing should not exceed 125 mL/min. For the purposes of this protocol, GFR is considered equivalent to creatinine clearance (CRCL). The CRCL is calculated according to institutional guidelines or the method described in Cockcroft and Gault (1976) Nephron 16:31-41 using the following formula:

Wherein: CRCL ═ creatinine clearance (mL/min)

Age (age) of the patient

Wt is the weight (kg) of the patient

Serum creatinine (mg/dL)

For patients with abnormally low serum creatinine levels, the GFR can be estimated by using a minimum creatinine level of 0.8mg/dL or by limiting the estimated GFR to 125 mL/min. Physicians were advised to limit the dose of carboplatin to the desired exposure (AUC) to avoid potential toxicity from overdosing. The maximum dose was calculated according to Calvert formula described in carboplatin labeling as follows:

carboplatin maximum dose (mg) ═ target AUC (mg × min/mL) × (GFR +25mL/min)

For patients with normal renal function, the maximum dose is limited to 125mL/min based on an estimate of GFR. Higher estimated GFR values are not used. For the target AUC 6, the maximum dose was 6 × 150 to 900 mg. For the target AUC 5, the maximum dose is 5 × 150 to 750 mg. For the target AUC 4, the maximum dose was 4 × 150 to 600 mg. For additional details regarding carboplatin administration, see: www (dot) fda (dot) gov/aboutfda/centeroffis/offis/second product and bacterium/cd/ucm 228974.htm

During the induction period, the chemotherapy cycle counts to a predetermined number (4 or 6) of induction chemotherapy cycles, so long as at least one chemotherapeutic component is administered at least once during a 21 day cycle. Is prepared from Cycles of chemotherapy administration were not counted in the total number of cycles of induced chemotherapy. Following the induction period, patients began using atezumab (i.e., 1200mg, intravenous infusion, as described above) and pemetrexed (i.e., 500 mg/m) on day 1 of each 21-day cycle following the induction period²Intravenous infusion as described above) for maintenance therapy. See figure 1 and the above study protocol). Dose modification of alemtuzumab was not allowed.

Allowed therapy

For any infusion of atuzumab after cycle 1, antihistamines can be used as pre-operative medication. The patient should continue the following treatments during the study:

oral contraceptives

Hormone replacement therapy

Prophylactic or therapeutic anticoagulant therapy (e.g. low molecular weight heparin or warfarin at a stable dosage level)

Palliative radiotherapy (e.g., treatment of known bone metastases) provided it does not interfere with the assessment of the tumor target lesion (e.g., the irradiated lesion is not the only disease site, as this would render the patient's tumor response unevaluble according to RECIST v 1.1)

No retention of Abutilizumab was required during palliative radiotherapy.

Inactivated influenza vaccination

Megestrol as an appetite stimulant

Inhalation of glucocorticoids for chronic obstructive pulmonary disease

Mineralocorticoids (e.g. fludrocortisone)

Low doses of corticosteroids for patients with orthostatic hypotension or adrenocortical insufficiency

Generally, supportive care is clinically adopted for patient care in accordance with local standards. Patients experiencing infusion-related symptoms may have received acetaminophen, ibuprofen, diphenhydramine and/or famotidine or another H as per standard practice₂The symptomatic treatment of receptor antagonists. Severe infusion-related events are manifested by dyspnea, hypotension, wheezing, bronchospasm, tachycardia, and blood oxygen saturationReduced degree of neutralization or respiratory distress, and treatment with supportive therapy (e.g., supplemental oxygen and beta) according to clinical indications₂Adrenergic agonists).

Prophylactic treatment of patients receiving treatment with atelizumab

It is well known that systemic corticosteroids and TNF-alpha inhibitors attenuate the potentially beneficial immunological effects of treatment with atelizumab. Thus, in the case of conventional administration of systemic corticosteroids or TNF- α inhibitors, the attending physician first considers alternative drugs including antihistamines. If a replacement drug is not feasible, the systemic corticosteroid and TNF- α inhibitor should be administered by the attending physician as appropriate, except in patients for which CT scanning of the contrast agent is contraindicated (i.e., patients with contrast agent allergy or impaired renal clearance). Systemic corticosteroid hormones are recommended and are cautiously considered by the attending physician to treat certain adverse events associated with the treatment with astuzumab.

Forbidden therapy

Any concomitant therapy intended to treat cancer (whether health authorities-approved or experimental) is contraindicated at various time periods prior to the initiation of study treatment (depending on the anti-cancer drug), and during study treatment until disease progression is noted and the patient has discontinued study treatment. Forbidden concomitant therapies include, but are not limited to, chemotherapy, hormonal therapy, immunotherapy, radiation therapy, research medications, or herbal therapies (unless otherwise specified).

Unless otherwise stated, patients prohibited the use of the following drugs during the study:

dinoteumab; patients receiving denosumab prior to enrollment must be willing and eligible to receive bisphosphonate therapy.

Any attenuated live vaccine (e.g.,) Within 4 weeks prior to randomization or during treatment or within 5 months after the last attritumab dose (for patients randomized to attritumab).

Pre-operative use of steroids in patients contraindicated for CT scanning of contrast agents (i.e. patients with contrast agent allergy or impaired renal clearance); in such patients, a contrast-free CT scan of the chest, and a contrast-free CT scan or MRI of the abdomen and pelvis are performed.

Concurrent herbal therapies are not recommended because their pharmacokinetics, safety and potential drug-drug interactions are generally unknown. However, the investigator can decide to use it for the patients in the study as appropriate, as long as there is no known interaction with any study therapeutic. As mentioned above, herbal therapies intended for cancer treatment are prohibited.

Tumor and response assessment

Patient safety and tolerability were closely monitored throughout the study and toxicity was assessed prior to each dose.

The medical history of each patient includes clinically significant disease, surgery, cancer history (including prior cancer treatment and surgery), reproductive status, smoking history, and screening for all medications (e.g., prescription, over-the-counter, herbal or homeopathic, nutritional supplements) used by the patient within 7 days prior to visit.

The history of NSCLC cancer includes prior cancer treatments, surgery, and assessment of the status of tumor mutations (e.g., sensitized EGFR mutations, ALK fusion status). For patients who have not previously been tested for a tumor mutational status, testing at the time of screening is required. For these patients, either local testing or centralized evaluation is performed during screening. If EGFR mutation or ALK status tests are not performed locally, additional tumor sections are needed to focus the assessment of the mutation status of these genes. Demographic data include age, gender, and self-reported race/ethnicity.

A complete physical examination includes an assessment of the head, eyes, ears, nose and throat, as well as the cardiovascular, dermatological, musculoskeletal, respiratory, gastrointestinal, genitourinary and nervous systems. Any abnormalities found at baseline are recorded.

In subsequent visits (or clinical indications), limited physical examinations for symptoms were performed. Abnormalities in the change from baseline were noted in the patient notes. New or worsening clinically significant abnormalities are recorded as adverse events.

Vital signs include measuring temperature, pulse rate, respiratory rate, and systolic and diastolic pressures while the patient is sitting down.

Tumor and response assessment

Screening evaluations included Computed Tomography (CT) scans of the chest and abdomen (oral/intravenous contrast unless contraindicated) or Magnetic Resonance Images (MRI). Screening requires CT or MRI scans of the pelvis under clinical instructions or in subsequent response assessment according to local-care criteria. If possible, a chest helical CT scan can be performed, but this is not required.

Screening requires either CT (with contrast agents if not contraindicated) or MRI on the head to assess CNS metastasis in all patients. If the scan is ambiguous, an MRI scan of the brain is required to confirm or deny the diagnosis of baseline CNS metastases. Patients with active or untreated CNS metastases did not qualify for the study (see exclusion criteria).

If a CT scan for tumor assessment is performed in a Positron Emission Tomography (PET)/CT scanner, a CT acquisition is required to meet the criteria of a full contrast diagnostic CT scan.

If clinically indicated, bone and CT scans of the neck may also be performed. Other methods of assessing measurable disease according to RECIST v1.1 were used at the discretion of the investigator.

Tumor assessments were allowed to be used as a standard of care-before obtaining informed consent and within 28 days of cycle 1 day 1, rather than repeat testing. All known disease sites need to be documented at the time of screening and the records re-evaluated at each subsequent tumor evaluation. Patients with a history of irradiated brain metastases at screening need not receive an imaging brain scan for subsequent tumor assessment unless clinically clear scan indications are available. The same radiographic imaging procedure (e.g., the same imaging protocol for CT scans) as used to assess the disease site at screening was used throughout the study. The response was evaluated by researchers using RECIST v1.1 (see Eisenhauer et al, (2009) New response evaluation criteria in solid tumors: Revised RECIST guideline (Version 1.1) Eur J cancer.45:228-47) and modified RECIST criteria. Modified RECIST criteria are derived from RECIST v1.1(Eisenhauer et al; Topalian et al, (2012) N Engl J Med.366: 2443-54; and Wolchok et al, (2009) Clin Can Res 15:7412-20) and immune-related response criteria (Wolchok et al; Nishino et al, (2014) J Immunother Can.2: 17; and Nishino et al, (2013) Clin Can Res.19: 3936-43). Evaluation is performed by the same evaluator, if possible, to ensure internal consistency of visits. The results should be reviewed by the investigator before the next cycle of dosing.

Patients were evaluated for tumors every 6 weeks (+ -7 days) at 48 weeks after cycle 1 day, after completion of tumor evaluation at 48 weeks, then every 9 weeks (+ -7 days) regardless of treatment delay until radiographic progression according to RECIST v1.1 (loss of clinical benefit, only for patients receiving alemtuzumab treatment who continued treatment after radiographic disease progression according to RECIST v 1.1), withdrawal consent, death, or termination of the study by the sponsor (whichever occurred earlier).

Patients who discontinued treatment for reasons other than radiographic disease progression according to RECIST v1.1 (e.g., toxicity, worsening of symptoms) continued to undergo planned tumor assessment until radiographic disease progression according to RECIST v1.1 (or lost clinical benefit, for patients receiving alemtuzumab treatment who continued alemtuzumab treatment after radiographic disease progression according to RECIST v 1.1), withdrawal consent, death, or termination of the study by the sponsor (whichever came first).

Patients who started a new anti-cancer therapy without radiographic disease progression according to RECIST v1.1 continued to undergo planned tumor assessment until radiographic disease progression according to RECIST v1.1 (or loss of clinical benefit, for patients receiving alemtuzumab treatment who continued alemtuzumab treatment after radiographic disease progression according to RECIST v 1.1), withdrawal consent, death, or termination of the study by the sponsor (whichever was first).

Despite evidence of radiographic progress, tumor assessments continued on patients receiving atuzumab treatment and continuing to have clinical benefit according to the above schedule.

Exploratory analysis of progression-free survival

Progression-free survival at milestone-meaningful time points: PFS rates were estimated for each treatment group using the Kaplan-Meier method, defined as the probability of patient survival without disease progression after randomized cohort (e.g., at 6 months and 1 year), and 95% CI was estimated using the Greenwood's equation. The 95% CI of the difference in PFS rates between treatment groups was estimated using normal approximation and the standard error was calculated using the Greenwood method.

Non-protocol-specified anti-cancer therapies: the number of patients receiving non-protocol-specific anti-cancer therapy prior to the occurrence of a PFS event, the effect of the non-protocol-specific anti-cancer therapy on PFS was assessed. If > 5% of patients in any treatment group received non-protocol-specified anti-cancer therapy prior to the occurrence of a PFS event, a sensitivity analysis comparison is made between treatment groups of patients receiving non-protocol-specified anti-cancer therapy before examining the PFS event for the last tumor assessment date prior to receiving non-protocol-specified anti-cancer therapy.

Subgroup analysis: to assess the concordance of the study results in subgroups defined by demographics (e.g., age, gender, and race/ethnicity), baseline prognostic characteristics (e.g., ECOG performance status, smoking status, and type of chemotherapy), PFS duration checks were performed on these subgroups. A summary of PFS was generated separately for each level of categorical variable used for comparisons between treatment groups, including Kaplan-Meier estimates of unfractionated HR and median PFS estimated according to the Cox proportional hazards model.

And (3) sensitivity analysis: sensitivity analysis was performed to assess the potential impact of lack of planned tumor assessments on the primary analysis of PFS, as determined by researchers using PFS event attribution rules. The following two attribution rules are considered: (1) if a patient misses two or more planned tumor assessments before the day of a PFS event according to RECIST v1.1, the patient is examined in the last tumor assessment before the first misses for these visits. (2) If a patient misses two or more planned tumor assessments before the day of a PFS event according to RECIST v1.1, the patient is considered to have progressed on the date that these assessments were first missed. The attribution rules apply to patients in both treatment groups.

Visit-the effect on OS will be assessed according to the number of patients who were visited. If > 5% of patients in any treatment group were missed due to OS, a sensitivity analysis comparison between treatment groups will be performed, wherein missed patients will be considered dead on the last day of known life.

Exploratory analysis of overall survival

Loss-visit: the effect of missed-on OS was assessed by the number of patients who were missed. If > 5% of patients in any treatment group were lost of OS, a sensitivity analysis comparison between treatment groups was performed, where the lost patients were considered dead on the last day of known life.

Subgroup analysis: to assess the consistency of the study results in subgroups defined by demographics (e.g. age, gender and race/ethnicity), baseline prognostic characteristics (e.g. ECOG performance status, smoking status and chemotherapy type, presence of baseline liver metastases), these subgroups were subjected to an OS duration check. A summary of survival was generated separately for each level of categorical variables used for comparisons between treatment groups, including the non-stratified HR and Kaplan-Meier estimates of median survival time estimated according to the Cox proportional hazards model.

Overall survival of 3-year milestone: the OS rate at 3 years was estimated for each treatment group using the Kaplan-Meier method and 95% CI was calculated using the standard error from the Greenwood equation. The 95% CI for OS rate differences between the two treatment groups was estimated using a normal approximation method.

Milestone overall life analysis: to assess the impact of long-term survival and delayed clinical outcome, a milestone OS analysis (Chen) was performed(2015)J Natl Cancer Inst.107: djv 156). The milestone OS is the OS endpoint that performs cross-section assessment at a pre-specified point in time. Performing a milestone OS analysis using the same method as the method specified by the primary OS analysis

Non-protocol-specified anti-cancer therapies: the effect of non-protocol specified anti-cancer therapy on OS is assessed on the basis of the number of patients receiving such therapy. For example, the duration from the start of a non-protocol specified anti-cancer therapy to the death or check-up date may have been reduced by a range of possible effects on the OS of subsequent non-protocol specified anti-cancer therapies (e.g., 10%, 20%, 30%).

Exploratory biomarker analysis: exploratory biomarker analyses were performed to understand the relationship of these markers to study drug responses, including efficacy and/or adverse events. Tumor biomarkers include, but are not limited to, PD-L1 and CD8 as defined by IHC, qRT-PCR or other methods. Other pharmacodynamic analyses were performed as appropriate.

The status of programmed death-ligand 1(PD-L1) IHC was determined using the Ventana anti-PD-L1 (SP142) rabbit monoclonal primary antibody Immunohistochemistry (IHC) assay.

Description of the apparatus: the Ventana anti-PD-L1 (SP142) rabbit monoclonal primary antibody was intended for semi-quantitative immunohistochemical evaluation of PD-L1 protein in formalin-fixed paraffin-embedded non-small cell lung cancer (NSCLC) tissues stained on a Ventana BenchMark ULTRA automatic slide staining machine. It may help to select NSCLC cancer patients with locally advanced or metastatic disease who may benefit from treatment with atelizumab.

The Ventana anti-PD-L1 (SP142) rabbit monoclonal primary antibody is a pre-diluted ready-to-use antibody product optimized for use on the Ventana Medical Systems automated BenchMark ULTRA platform with the Ventana Medical Systems OptiView DAB IHC detection kit and OptiView amplification kit. A5-mL dispenser of anti-PD-L1 (SP142) rabbit monoclonal primary antibody contains about 36 μ g of rabbit monoclonal antibody against PD-L1 protein and reagents sufficient for 50 tests. Reagents and IHC procedures were optimized for use on a BenchMark ULTRA automatic slide staining machine using Ventana System Software (VSS).

The scoring system comprises: staining of PD-L1 by anti-PD-L1 (SP142) rabbit monoclonal primary antibody in NSCLC was observed in both tumor cells and tumor-infiltrating immune cells using Ventana anti-PD-L1 (SP142) rabbit monoclonal primary antibody.

Results

The results of the study are listed in table 10 below:

table 10: summary of the major efficacy endpoints

Table 10 shows that this study demonstrates that the investigator-assessed progression-free survival (PFS) of the ITT population has a statistically significant and clinically significant improvement. In addition, studies have shown an improvement in Overall Survival (OS).

Patients treated with altuzumab + pemetrexed + carboplatin or cisplatin exhibited an increased progression-free survival compared to patients treated with pemetrexed + carboplatin or cisplatin. See fig. 2. Patients receiving altuzumab + pemetrexed + carboplatin or cisplatin had a 6-month PFS of 59.14% compared to 40.93% for patients receiving pemetrexed + carboplatin or cisplatin. The 12-month PFS was 33.71% for patients receiving altuzumab + pemetrexed + carboplatin or cisplatin versus 16.97% for patients receiving pemetrexed + carboplatin or cisplatin. The overall survival of patients treated with altuzumab + pemetrexed + carboplatin or cisplatin is numerically improved compared to patients treated with pemetrexed + carboplatin or cisplatin. See fig. 3(NE — FIG not evaluated). The 6-month OS was 59.61% for patients receiving altuzumab + pemetrexed + carboplatin or cisplatin versus 55.39% for patients receiving placebo + pemetrexed + carboplatin or cisplatin. The 12-month OS was 59.6% for patients receiving attrituzumab + pemetrexed + carboplatin or cisplatin compared to 55.4% for patients receiving placebo + pemetrexed + carboplatin or cisplatin.

In addition, the confirmed Overall Response Rate (ORR) for patients receiving treatment with attrituximab + pemetrexed + carboplatin or cisplatin was 47%, while the confirmed ORR for patients receiving treatment with pemetrexed + carboplatin or cisplatin was 32% (CR: group A1.7% vs. group B0.7%; CR/PR: group A46.9% vs. group B32.2%). See fig. 4. Unidentified ORR was also improved in group a (CR ═ complete remission; CR/PR ═ complete remission/partial remission; SD ═ stable disease; PD ═ progressive disease). As shown in table 11 below, the median duration of confirmed response (DOR) was 10.1 months for patients receiving altuzumab + pemetrexed + carboplatin or cisplatin (i.e., group a), and 7.2 months for patients receiving pemetrexed + carboplatin or cisplatin (i.e., group B). DOR was evaluated according to RECIST v1.1 criteria. The unidentified DOR was also improved in group a. 42% of patients in group A showed a sustained response, whereas 30% of patients in group B showed a sustained response.

Table 11: median response duration confirmed in treatment groups a and B

The benefits of PFS were observed in all subgroups analyzed. See fig. 5A. An increase in OS values was also observed. See fig. 6. Consistent results were shown in each clinical subgroup.

The safety of alemtuzumab + pemetrexed + carboplatin or cisplatin is consistent with the known risks of individual therapeutic ingredients. No new security signal is found. The key safety parameters were consistent with the results of other first-line NSCLC studies involving atelizumab in combination with platinum chemotherapy.

This study shows that initial (first line) treatment with the combination of atuzumab + pemetrexed + carboplatin or cisplatin reduces the risk of disease progression or death (PFS) compared to chemotherapy alone (i.e., pemetrexed + carboplatin or cisplatin). There was also a numerical improvement in the overall survival of patients treated with altuzumab + pemetrexed + carboplatin or cisplatin compared to patients receiving chemotherapy alone (i.e., pemetrexed + carboplatin or cisplatin).

Example 2: efficacy of astuzumab in combination with carboplatin pemetrexed or cisplatin pemetrexed as first-line treatment in a critical subgroup of + IV + stage non-squamous non-small cell lung cancer (NSCLC) patients

Exploratory efficacy analyses were performed for PFS and metaphase OS in clinically relevant patient subgroups (e.g. race, age, smoking history and baseline liver metastases) based on the results described in example 1.

578 patients were enrolled. Median follow-up time was 14.8 months. Baseline characteristics were substantially balanced between treatment groups. See table 12 and fig. 5B, which are PFS and mid-OS data in the key subgroups.

Table 12: PFS and mid OS data in critical subgroups

Addition of atuzumab to carboplatin or cisplatin + pemetrexed increased PFS and OS values for most critical clinical subgroups. Survival benefits appear to be more pronounced in asian patients, elderly patients and never-smokers.

Example 3: exploratory analysis: progression-free survival, patient's PD-L1 status evaluable by the biomarker in example 1

PD-L1 expression levels on tumor infiltrating Immune Cells (IC) and tumor-cells (TC) in baseline tissue samples obtained from patients evaluable for biomarkers (i.e., from example 1) were analyzed. Tumor cells were scored as TC0, TC1, TC2 or TC3, and tumor-infiltrating immune cells were scored as IC0, IC1, IC2 and IC 3.

Assay scores for each therapeutic component were TC3 or IC3 (i.e., "PD-L1 high"); TC1, TC2, IC1, or IC2 (or "PD-L1 low"); and Overall Response Rate (ORR) and progression free survival rate (PFS) in patients with TC0 or IC0 (i.e., "PD-L1 negative"). The results of these analyses are shown in fig. 7A, 7B and 7C.

As shown in FIG. 7A, the ORR of "PD-L1 high" patients receiving Attuzumab + pemetrexed + carboplatin or cisplatin was 72%. In contrast, the ORR was 55% in "PD-L1 high" patients receiving pemetrexed + carboplatin treatment. The median PFS was 10.8 months for "PD-L1 high" patients receiving attrituzumab + pemetrexed + carboplatin or cisplatin, and 6.5 months for "PD-L1 high" patients in the control group. The 12-month PFS in the treatment group "PD-L1 high" patients was 46%, while the 12-month PFS in the control group "PD-L1 high" patients was 25%.

There was no significant difference in ORR or median PFS in the treatment group of "PD-L1 low" patients compared to the control group. The 12-month PFS was 27% in the treatment group "PD-L1 low" patients and 20% in the control group "PD-L1 low" patients. See fig. 7B.

FIG. 7C shows that the ORR of "PD-L1 negative" patients receiving Attuzumab + pemetrexed + carboplatin or cisplatin is 44%. In contrast, the ORR was 27% in "PD-L1 negative" patients receiving pemetrexed + carboplatin treatment. The median PFS for "PD-L1 negative" patients receiving attrituzumab + pemetrexed + carboplatin or cisplatin was 8.5 months, while the median PFS for the control "PD-L1 negative" patients was 4.9 months. The 12-month PFS of the treatment group "PD-L1 negative" patients was 35%, while the 12-month PFS of the control group "PD-L1 negative" patients was 8%. Median duration of response (DOR) was 10.1 months for the treatment group of "PD-L1 negative" patients, while 4.2 months for the control group of "PD-L1 negative" patients. Although the present disclosure has been described in considerable detail previously for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

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Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 11

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 12

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 13

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 14

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 15

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 15

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 16

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 16

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 17

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 18

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 19

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Ser Gly Glu Ser Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Thr Ser Leu Thr Ala Glu Asp Thr Gly Met Tyr Phe Cys

85 90 95

Val Arg Val Gly Tyr Asp Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 20

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Arg Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Cys Leu Thr Ile Asn Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Phe Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210