阅读说明：本技术 用于降低毒性的共表达嵌合抗原受体和il-6拮抗剂的修饰免疫细胞及其在过继细胞疗法中的用途 (Modified immune cells co-expressing chimeric antigen receptor and IL-6 antagonist for reduced toxicity and their use in adoptive cell therapy ) 是由 胡璧梁 于 2020-01-06 设计创作，主要内容包括：一种免疫细胞群,包括共表达嵌合抗原受体和IL-6信号传导拮抗剂(例如,抗IL6或抗IL-6R抗体)和任选地IL-1信号传导拮抗剂的修饰免疫细胞。本文还提供了制备此类包括修饰免疫细胞的免疫细胞群的方法,和将其用于细胞疗法的方法(例如,以治疗癌症、感染性疾病或免疫疾病)。(An immune cell population comprising modified immune cells that co-express a chimeric antigen receptor and an antagonist of IL-6 signaling (e.g., an anti-IL 6 or anti-IL-6R antibody) and optionally an antagonist of IL-1 signaling. Also provided herein are methods of making such immune cell populations comprising modified immune cells, and methods of using them in cell therapy (e.g., to treat cancer, infectious disease, or immune disease).)

1. A population of immune cells, wherein the immune cells express a chimeric receptor antigen (CAR) and an antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R), wherein the antibody comprises the same heavy chain complementarity determining domain (CDR) and the same light chain CDR as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:1_H) And a light chain variable domain (V) as set forth in SEQ ID NO:2_L) Or (b) V as shown in SEQ ID NO 3_HAnd V as shown in SEQ ID NO. 4_L。

2. The immune cell population of claim 1, wherein the antibody specific for IL-6 or IL-6R comprises the same V as a reference antibody_HAnd the same V_L。

3. The immune cell population of claim 1 or 2, wherein the antibody specific for IL-6 or IL-6R is a single chain antibody fragment (scFv).

4. The immune cell population of claim 3, wherein the scFv comprises the amino acid sequence of SEQ ID NO 13 or SEQ ID NO 14.

5. The immune cell population of any one of claims 1-4, wherein at least 10% of the cells express the CAR and an antibody specific for the IL-6 or IL-6R.

6. The immune cell population of claim 5, wherein about 50 to 70% of the cells express the CAR and the antibody specific for IL-6 or IL-6R.

7. The immune cell population of any one of claims 1-6, wherein the antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R) is a fragment of a bispecific antibody further comprising an antibody specific for granulocyte macrophage colony stimulating factor (GM-CSF).

8. The immune cell population of claim 7, wherein the antibody specific for GM-CSF comprises the same heavy chain complementary determining domains (CDRs) and the same light chain CDRs as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:21_H) And a light chain variable domain (V) as shown in SEQ ID NO:22_L)。

9. The immune cell population of claim 7 or 8, wherein the antibody specific for GM-CSF comprises V as set forth in SEQ ID NO 21_HAnd V as shown in SEQ ID NO. 22_L。

10. The immune cell population of any one of claims 7-9, wherein the antibody specific for GM-CSF is an scFv antibody.

11. The immune cell population of claim 10, wherein the antibody specific for GM-CSF is linked to the antibody specific for IL-6 or IL-6R by a peptide linker.

12. The immune cell population of claim 11, wherein the peptide linker is GSGGSG.

13. The immune cell population of claim 7, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO 28.

14. The immune cell population of any one of claims 1-13, wherein the immune cells express an IL-1 antagonist.

15. The immune cell population of claim 14, wherein the IL-1 antagonist is IL-1 RA.

16. The population of immune cells of any one of claims 1-15, wherein the immune cells comprise a disrupted endogenous IL-2 gene, a disrupted endogenous GM-CSF gene, a disrupted TNFA gene, or a combination thereof.

17. The immune cell population of any one of claims 1-16, wherein the immune cells are T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, precursors thereof, or combinations thereof.

18. The immune cell population of any one of claims 1-17, wherein the immune cells are T cells that do not express an endogenous T cell receptor.

19. The immune cell population of any of claims 1-18, wherein the CAR comprises an extracellular domain specific for a pathological antigen, a transmembrane domain, and a cytoplasmic domain comprising one or more signaling domains.

20. The immune cell population of claim 19, wherein the one or more signaling domains comprise one or more costimulatory domains, a cytoplasmic domain of CD3 ζ, or a combination thereof.

21. The immune cell population of claim 20, wherein the one or more co-stimulatory domains is derived from a co-stimulatory protein selected from the group consisting of CD28, 4-1BB, CD27, OX40 and ICOS.

22. The immune cell population of any of claims 1-21, wherein the CAR binds CD19 or BCMA.

23. The immune cell population of claim 22, wherein the CAR binds CD19 and comprises an extracellular domain that is a single chain antibody fragment comprising the amino acid sequence of SEQ ID No. 52.

24. The immune cell population of claim 22, wherein the CAR binds BCMA and comprises an extracellular domain which is a single chain antibody fragment comprising the amino acid sequence of SEQ ID No. 57.

25. The immune cell population of any one of claims 22-24, wherein the immune cells further express an IL-1 antagonist, optionally the IL-1 antagonist is IL-1 RA.

26. The population of immune cells of any one of claims 22-25, wherein the immune cells have a disrupted endogenous GM-CSF gene and/or a disrupted endogenous TCR gene.

27. The population of immune cells of any one of claims 22-25, wherein the immune cells have wild-type endogenous GM-CSF and/or TCR genes.

28. An immune cell that expresses a chimeric receptor antigen (CAR) and an antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R), wherein the antibody comprises the same heavy chain complementarity determining domain (CDR) and the same light chain CDR as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:1_H) And a light chain variable domain (V) as set forth in SEQ ID NO:2_L) Or (b) V as shown in SEQ ID NO 3_HAnd V as shown in SEQ ID NO. 4_L。

29. The immune cell of claim 28, wherein the antibody specific for IL-6 or IL-6R is according to any one of claims 2 to 4.

30. The immune cell of claim 28 or claim 29, wherein the antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R) is a fragment of a bispecific antibody further comprising an antibody specific for granulocyte macrophage colony-stimulating factor (GM-CSF).

31. The immune cell of claim 30, wherein the antibody specific for GM-CSF is according to any one of claims 8 to 13.

32. The immune cell of any one of claims 28-31, wherein the immune cell expresses an IL-1 antagonist, optionally the IL-1 antagonist is IL-1 RA.

33. The immune cell of any one of claims 28-32, wherein the immune cell comprises a disrupted endogenous IL-2 gene, a disrupted endogenous GM-CSF gene, a disrupted endogenous TNFA gene, or a combination thereof.

34. The immune cell of any one of claims 28-33, wherein the immune cell is a T cell or NK cell, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a mesenchymal stem cell, or a precursor thereof.

35. The immune cell of claim 34, wherein the immune cell is a T cell that does not express an endogenous T cell receptor.

36. The immune cell of any of claims 28-35, wherein the CAR is of any of claims 19-24.

37. The immune cell of claim 36, wherein the CAR is any one of claims 22 to 24, and the immune cell further expresses an IL-1 antagonist, optionally the IL-1 antagonist is IL-1 RA.

38. The immune cell of claim 37, wherein the immune cell has a disrupted endogenous GM-CSF gene and/or a disrupted endogenous TCR gene.

39. The immune cell of claim 37, wherein the immune cell has a wild-type endogenous GM-CSF and/or TCR gene.

40. A method of producing a population of modified immune cells having reduced inflammatory properties, the method comprising:

(i) providing a population of immune cells; and

(ii) introducing into an immune cell a first nucleic acid encoding a Chimeric Antigen Receptor (CAR) of any one of claims 19 to 24 and a second nucleic acid encoding an antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R), wherein the antibody is of any one of claims 1 to 4;

wherein the first nucleic acid and the second nucleic acid are both operably linked to a promoter for expressing the CAR and the antibody in the immune cell.

41. The method of claim 40, wherein the antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R) is a fragment of a bispecific antibody further comprising an antibody specific for granulocyte macrophage colony stimulating factor (GM-CSF).

42. The method of claim 41, wherein the antibody specific for GM-CSF is according to any one of claims 8-13.

43. The method of any one of claims 40-42, further comprising introducing into the immune cell a third nucleic acid encoding an IL-1 antagonist, which antagonist is optionally IL-1 RA.

44. The method of claim 43, wherein the second nucleic acid and the third nucleic acid are on the same vector.

45. The method of any one of claims 40-44, further comprising disrupting an endogenous IL-2 gene, an endogenous GM-CSF gene, an endogenous TNFA, an endogenous T Cell Receptor (TCR) gene, or a combination thereof in the immune cell.

46. The method of any one of claims 40-44, wherein the endogenous GM-CSF gene and/or the TCR gene is not disrupted.

47. The method of claim 45, wherein the endogenous gene is disrupted by a CRISPR gene editing system.

48. The method of any one of claims 40-47, wherein the immune cell is a T cell or NK cell.

49. A method of cell therapy comprising administering to a subject in need thereof a population of immune cells according to any one of claims 1 to 27 or immune cells according to any one of claims 28 to 39.

50. The method of claim 49, wherein the subject is a human patient having cancer, an infectious disease, or an immune disorder.

51. The method of claim 50, wherein the subject is a human patient with cancer, and wherein the human patient has received therapy for the cancer to reduce tumor burden.

52. The method of claim 51, wherein the therapy is chemotherapy, immunotherapy, radiation therapy, or surgery.

53. The method of any one of claims 40-52, wherein the subject is undergoing a lymphodepletion treatment prior to the cell therapy to adapt the subject to the cell therapy.

54. The method of claim 53, wherein the lymphocyte depletion therapy comprises administering fludarabine and/or cyclophosphamide to the subject.

55. The method of any one of claims 49 to 54, wherein the immune cells express an anti-CD 19CAR and the subject is a human patient with lymphoblastic leukemia or non-Hodgkin's lymphoma, and wherein optionally the lymphoblastic leukemia is acute lymphoblastic leukemia.

56. The method of any one of claims 49-54, wherein the immune cell expresses an anti-BCMA CAR and the subject is a human patient with multiple myeloma, optionally relapsed or refractory multiple myeloma.

57. The method of claim 55 or claim 56, wherein the immune cells further express an IL-1 antagonist, the IL-1 antagonist optionally being IL-1RA and having a disrupted endogenous GM-CSF gene and/or a disrupted endogenous TCR gene.

58. The method of claim 55 or claim 56, wherein the immune cells further express an IL-1 antagonist, the IL-1 antagonist optionally being IL-1RA and having a wild-type endogenous GM-CSF and/or TCR gene.

59. A bispecific antibody comprising a first antibody fragment specific for IL-6 and a second antibody fragment specific for GM-CSF.

60. The bispecific antibody of claim 59, wherein the antibody fragment specific for IL-6 comprises the same heavy chain complementary determining domain (CDR) and the same light chain CDR as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:1_H) And a light chain variable domain (V) as set forth in SEQ ID NO:2_L) Or (b) V as shown in SEQ ID NO 3_HAnd V as shown in SEQ ID NO. 4_L。

61. The bispecific antibody of claim 60, wherein the antibody fragment specific for IL-6 or IL-6R comprises the same V as the reference antibody_HAnd the same V_L。

62. The bispecific antibody of any one of claims 59 to 61, wherein the antibody fragment specific for IL-6 or IL-6R is a single chain antibody fragment (scFv).

63. The bispecific antibody of claim 62, wherein the scFv comprises the amino acid sequence of SEQ ID NO 13 or SEQ ID NO 14.

64. The bispecific antibody of any one of claims 59 to 63, wherein the antibody fragment specific for GM-CSF comprises the same heavy chain complementary determining domain (CDR) and the same light chain CDR as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:21_H) And a light chain variable domain (V) as shown in SEQ ID NO:22_L)。

65. The bispecific antibody of claim 64, wherein the antibody fragment specific for GM-CSF comprises V as set forth in SEQ ID NO 21_HAnd V as shown in SEQ ID NO. 22_L。

66. The bispecific antibody of claim 64 or claim 65, wherein the antibody specific for GM-CSF is a scFv antibody.

67. The bispecific antibody of any one of claims 59 to 66, wherein the antibody specific for GM-CSF is linked to the antibody specific for IL-6 or IL-6R by a peptide linker.

68. The bispecific antibody of claim 67, wherein the peptide linker is GSGGSG.

69. The bispecific antibody of claim 68, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO 28.

70. A nucleic acid comprising a first nucleotide sequence encoding an antibody fragment specific for IL-6, a second nucleotide sequence encoding a self-cleaving peptide, and a third nucleic acid sequence encoding an IL-1 antagonist.

71. The nucleic acid of claim 70, wherein the first nucleotide sequence encodes a bispecific antibody comprising an antibody fragment specific for IL-6 and an antibody fragment specific for GM-CSF.

Background

Adoptive cell transfer therapy is an immunotherapy that involves ex vivo expansion of autologous or allogeneic immune cells, followed by infusion into a patient. Immune cells can be modified ex vivo to specifically target malignant cells. The prospects for adoptive cell transfer therapy are often limited by toxicity (e.g., cytokine-related toxicity). For example, adoptive cell transfer immunotherapy may trigger a non-physiological increase in cytokine levels (cytokine release syndrome), which may lead to death of the recipient (see, e.g., Morgan et al, Molecular Therapy 18(4):843 851, 2010).

Therefore, it would be of great interest to develop methods to reduce the toxicity associated with adoptive cell transfer immunotherapy while maintaining therapeutic efficacy.

Disclosure of Invention

The present disclosure is based on the following findings: certain antibodies targeting interleukin 6(IL-6) or interleukin 6 receptor (IL-6R) exhibit unexpectedly higher efficacy in inhibiting the IL-6 signaling pathway compared to other IL-6 antagonists. Such anti-IL-6 or anti-IL-6R is expected to be more effective in reducing IL-6 mediated toxicity in CAR-T therapy.

Accordingly, one aspect of the disclosure provides a population of immune cells, wherein the immune cells express a chimeric receptor antigen (CAR) and an antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R), wherein the antibody comprises the same heavy chain complementary determining domains (CDRs) and the same light chain CDRs as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:1_H) And a light chain variable domain (V) as set forth in SEQ ID NO:2_L) Or (b) V as shown in SEQ ID NO 3_HAnd V as shown in SEQ ID NO. 4_L. In some cases, antibodies specific for IL-6 or IL-6R include the same V as described for the reference antibody_HAnd said same V_L. Such antibodies may be single chain antibody fragments (scFv). In a specific example, the scFv can comprise the amino acid sequence of SEQ ID NO 13 or SEQ ID NO 14.

In some embodiments, an immune cell population as described herein can contain at least 10% (e.g., 10% -90% or 50% -70% or more) cells that express the CAR and an antibody specific for the IL-6 or IL-6R.

In some embodiments, the immune cell population described herein can be T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, precursors thereof, or combinations thereof. In some cases, the immune cell is a T cell that does not express an endogenous T cell receptor.

In some embodiments, the population of immune cells expresses a CAR comprising an extracellular domain specific for a pathological antigen, a transmembrane domain, and a cytoplasmic domain comprising one or more signaling domains. The one or more signaling domains can include one or more costimulatory domains, CD3 ζ, or a combination thereof. Exemplary costimulatory domains include, but are not limited to, those from CD28, 4-1BB, CD27, OX40, and ICOS.

In another aspect, provided herein is an immune cell, whichExpressing a chimeric receptor antigen (CAR) and an antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R), wherein the antibody comprises the same heavy chain complementary determining domain (CDR) and the same light chain CDR as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:1_H) And a light chain variable domain (V) as set forth in SEQ ID NO:2_L) Or (b) V as shown in SEQ ID NO 3_HAnd V as shown in SEQ ID NO. 4_L. In some cases, the antibody specific for IL-6 or IL-6R comprises the same V as the reference antibody_HAnd said same V_L. Such antibodies may be single chain antibody fragments (scFv). In particular examples, the scFv can include the amino acid sequence of SEQ ID NO 13 or SEQ ID NO 14.

In any or a population of immune cells or immune cells described herein, the antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R) is a fragment of a bispecific antibody further comprising an antibody specific for granulocyte macrophage colony stimulating factor (GM-CSF). The antibody specific for GM-CSF comprises the same heavy chain complementary determining domain (CDR) and the same light chain CDR as a reference antibody, and wherein the reference antibody comprises (a) a heavy chain variable domain (V) as set forth in SEQ ID NO:21_H) And a light chain variable domain (V) as shown in SEQ ID NO:22_L). In some examples, the antibody specific for GM-CSF comprises V as set forth in SEQ ID NO 21_HAnd V as shown in SEQ ID NO. 22_L。

In some examples, the antibody specific for GM-CSF is a scFv antibody. Such scFv antibodies may be linked to the antibody specific for IL-6 or IL-6R by a peptide linker such as GSGGSG. In a specific example, the bispecific antibody comprises the amino acid sequence of SEQ ID NO 28.

Alternatively or additionally, any immune cell or immune cell population described herein can express an IL-1 antagonist. In some examples, the IL-1 antagonist is IL1RA, which may include the amino acid sequence of SEQ ID No. 29.

In addition, any immune cell or population of immune cells described herein can include a disrupted endogenous IL-2, a disrupted endogenous GM-CSF, a disrupted endogenous TNFA, a disrupted endogenous T Cell Receptor (TCR) gene, or a combination thereof.

In particular examples, the immune cells described herein express an anti-CD 19CAR (e.g., a CAR comprising the extracellular antigen-binding domain of SEQ ID NO:52), an IL-6 antagonist (e.g., a scFv comprising SEQ ID NO:13 or SEQ ID NO:14), an IL-1 antagonist (e.g., IL-1 RA). Such immune cells may have disrupted endogenous GM-CSF and/or TCR genes. In some cases, both endogenous GM-CSF and TCR genes are disrupted in the genetically engineered immune cells disclosed herein. Alternatively, such immune cells may have wild-type endogenous GM-CSF and/or TCR genes. In some cases, the immunized subject may have both wild-type endogenous GM-CSF and wild-type endogenous TCR genes.

In particular examples, the immune cells described herein express an anti-BCMA CAR (e.g., a CAR comprising an extracellular antigen-binding domain comprising SEQ ID NO:57), an IL-6 antagonist (e.g., a scFv comprising SEQ ID NO:13 or SEQ ID NO:14), an IL-1 antagonist (e.g., IL-1 RA). Such immune cells may have disrupted endogenous GM-CSF and/or TCR genes. In some cases, both endogenous GM-CSF and TCR genes are disrupted in the genetically engineered immune cells disclosed herein. Alternatively, such immune cells may have wild-type endogenous GM-CSF and TCR genes. In some cases, the immunized subject may have both wild-type endogenous GM-CSF and wild-type endogenous TCR genes.

Any bispecific antibody capable of binding both IL-6/IL-6R and GM-CSF as described herein is also within the scope of the present disclosure.

In addition, the present disclosure provides a nucleic acid comprising a first nucleotide sequence encoding an antibody fragment specific for IL-6, a second nucleotide sequence encoding a self-cleaving peptide, and a third nucleic acid sequence encoding an IL-1 antagonist. In some cases, the first nucleotide sequence encodes a bispecific antibody comprising the antibody fragment specific for IL-6 and the antibody fragment specific for GM-CSF.

In yet another aspect, the present disclosure provides a method of generating a population of modified immune cells with reduced inflammatory properties, the method comprising: (i) providing a population of immune cells (e.g., those described herein); (ii) introducing into the immune cell a first nucleic acid encoding a Chimeric Antigen Receptor (CAR) as described herein and a second nucleic acid encoding an antibody specific for interleukin-6 (IL-6) or IL-6 receptor (IL-6R) also as described herein. The first nucleic acid and the second nucleic acid are both operably linked to a promoter for expressing the CAR and the antibody in the immune cell. When the immune cell is a T cell, the method may further comprise disrupting an endogenous T cell receptor gene.

Further, the present disclosure provides a method of cell therapy comprising administering to a subject in need thereof a population of immune cells or immune cells as described herein. In some cases, the subject is a human patient with cancer, an infectious disease, or an immune disorder. In some cases, the subject is a human patient with cancer, and wherein the human patient has received therapy for the cancer to reduce tumor burden. Exemplary anti-cancer therapies include, but are not limited to, chemotherapy, immunotherapy, radiation therapy, surgery, or a combination thereof. In some embodiments, the subject may be treated with an conditioning regimen to deplete endogenous lymphocytes following an anti-cancer therapy, thereby subjecting the subject to the cell therapy disclosed herein.

In some cases, the immune cells used in the cell therapy methods disclosed herein express an anti-CD 19CAR (e.g., a CAR comprising an extracellular antigen-binding domain of SEQ ID NO:52), an IL-6 antagonist (e.g., an scFv comprising SEQ ID NO:13 or SEQ ID NO:14), an IL-1 antagonist (e.g., IL-1 RA). Such immune cells may have disrupted endogenous GM-CSF and/or TCR genes. In some cases, both endogenous GM-CSF and TCR genes are disrupted in the genetically engineered immune cells disclosed herein. Alternatively, such immune cells may have wild-type endogenous GM-CSF and/or TCR genes. In some cases, the immunized subject may have both wild-type endogenous GM-CSF and wild-type endogenous TCR genes. Such immune cells are useful for treating human patients with lymphoblastic leukemia (e.g., acute lymphoblastic leukemia) or non-hodgkin's lymphoma.

In some cases, immune cells used in the cell therapy methods disclosed herein express an anti-BCMACAR (e.g., a CAR comprising an extracellular antigen-binding domain comprising SEQ ID NO:57), an IL-6 antagonist (e.g., a scFv comprising SEQ ID NO:13 or SEQ ID NO:14), an IL-1 antagonist (e.g., IL-1 RA). Such immune cells may have disrupted endogenous GM-CSF and/or TCR genes. In some cases, both endogenous GM-CSF and TCR genes are disrupted in the genetically engineered immune cells disclosed herein. Alternatively, such immune cells may have wild-type endogenous GM-CSF and/or TCR genes. In some cases, the immunized subject may have both wild-type endogenous GM-CSF and wild-type endogenous TCR genes. Such immune cells are useful for treating human patients with multiple myeloma, such as relapsed or refractory multiple myeloma.

Also within the scope of the present disclosure are populations of immune cells as described herein for use in the treatment of a disease of interest as also described herein, and the use of such populations of immune cells in the manufacture of a medicament for the treatment of a disease of interest.

The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims.

Drawings

FIG. 1 is a graph showing the effect of various antibodies specific for IL-6 or IL-6R on the inhibition of IL-6 signaling expressed by 293T cells generated by transient transfection of a third generation lentiviral vector encoding a T2A linked anti-CD 19CAR and an antibody.

FIGS. 2A-2B contain graphs showing the combined effect of an IL-6 antagonist and an IL-1 antagonist in inhibiting both IL-1 signaling (2A) and IL-6 signaling (2B).

FIGS. 3A-3C contain graphs showing the effect of anti-IL-6/anti-GM-CSF bispecific antibody in combination with IL1RA on GM-CSF signaling (3A), IL-6 signaling (3B), and IL-1 signaling (3C).

Fig. 4A-4B contain graphs showing proliferation and cytokine expression of T cells with wild-type GM-CSF and T cells with endogenous GM-CSF gene editing by CRISPR.

Fig. 5A-5B contain graphs showing proliferation and cytokine expression of wild-type T cells and T cells with endogenous IL-2 with gene editing by CRISPR.

Fig. 6A-6B contain graphs showing proliferation and cytokine expression of wild-type T cells and T cells with endogenous TNFA by gene editing by CRISPR.

FIGS. 7A-7D contain schematic representations of anti-CD 19/IL6/IL1 TCR-and anti-CD 19/IL6/IL1TCR^-/GM-CSF^-Cells exert IL6(7A) and IL1B (7B) inhibitory effects, secreting similar levels of IFN γ, IL-2 and TNF α, whereas secretion of GM-CSF is significantly reduced (7C) and is capable of inducing cytotoxicity in CD19+ Nalm6 cells (7D).

FIGS. 8A-8D contain a scheme showing anti-BCMA/IL 6/IL1GM-CSF^-The cells exert IL6(8A) and IL1B (8B) inhibitory effects, secreting similar levels of IFN γ and IL-2, whereas GM-CSF is significantly reduced in secretion (8C) and is capable of expressing the protein in BCMA⁺Graph of induced cytotoxicity (8D) in RPMI-8226 cells.

Figures 9A-9H contain graphs showing cytokine secretion levels and other Cytokine Release Syndrome (CRS) characteristics of human cancer patients after the first round of anti-CD 19/IL6/IL1/GM-CSF KO CAR-T cell treatment. FIG. 9A: GM-CSF KO T cells and GM-CSF in wild-type counterpart cells⁺The level of cells. FIG. 9B: GM-CSF levels in human patients at the indicated different time points after T cell infusion. FIG. 9C: IL-6 levels in human patients at various time points indicated after T cell infusion. FIG. 9D: IL1/IL1R blocker levels in human patients at the indicated different time points after T cell infusion. FIG. 9E: CAR vector copy levels in human patients at the indicated different time points after T cell infusion. FIG. 9F: c-reactive protein (CRP) levels in human patients at the indicated different time points after T-cell infusion. FIG. 9G: human patients at different time points indicated after T cell infusionThe interferon gamma (IFN gamma) level of the subject. FIG. 9H shows a graph of the patient's maximum daily temperature (Tmax C.).

Figures 10A-10H contain graphs showing cytokine secretion levels and other Cytokine Release Syndrome (CRS) characteristics of the same human cancer patient after a second round of anti-CD 19/IL6/IL1/CAR-T cell therapy. FIG. 10A: IL-6 levels in human patients at various time points indicated after T cell infusion. FIG. 10B: daily maximum body temperature (c) of human patients at the indicated different time points after T cell infusion. FIG. 10C: IL1/IL1R blocker levels in human patients at the indicated different time points after T cell infusion. FIG. 10D: GM-CSF levels in human patients at the indicated different time points after T cell infusion. FIG. 10E: CRP levels in human patients at the indicated different time points after T cell infusion. FIG. 10F: CAR vector copy levels in human patients at the indicated different time points after T cell infusion. FIG. 10G: CAR-T cell numbers in human patients at the indicated different time points after T cell infusion. FIG. 10H: IFN γ levels in human patients at the indicated different time points after T cell infusion.

Figures 11A-11K contain graphs showing clinical features of refractory multiple myeloma patients treated with anti-BCMA CAR-T cells expressing IL6 and IL-1 antagonists and knocking out GM-CSF and TCR genes. FIG. 11A: efficiency of knockout of GM-CSF by Crispr/Cas9 gene editing during ex vivo expansion of patient T cells. FIG. 11B: variation in IgA concentration before and after CART treatment. FIG. 11C: concentration of IL-6 in peripheral blood during CART treatment. FIG. 11D: daily temperature changes during CART treatment. FIG. 11E: concentration of CRP in peripheral blood during CART treatment. FIG. 11F: concentration of IL1/IL1R blockers in peripheral blood during CART treatment. FIG. 11G: CAR vector copy number per μ g genomic DNA in peripheral blood during CART treatment. FIG. 11H: total number of CAR + T cells in peripheral blood during CART treatment. FIG. 11I: concentration of IFN γ in peripheral blood during CAR-T therapy. FIG. 11J: the concentration of GM-CSF in the peripheral blood during CART treatment. FIG. 11K: comparison of IFN γ and IL6 levels in peripheral blood during CART treatment.

Fig. 12A-12D include graphs showing therapeutic effect of anti-CD 19CAR-T cells expressing IL6 and IL-1 antagonists and knocking out TCR genes. Such T cells either have wild-type GM-CSF or have had GM-CSF knocked out. FIG. 12A: graph showing the change in body weight of mice after treatment. FIG. 12B: graph showing survival of treated mice. FIG. 12C: graph showing the change in leukemia cell levels in blood of treated mice. FIG. 12D: graph showing the change in T cell levels in blood of treated mice.

Figures 13A to 13K contain graphs showing clinical features of non-hodgkin human patients treated with anti-CD 19CAR-T cells expressing IL6 and IL-1 antagonists and knocking out GM-CSF and TCR genes. FIG. 13A: graph showing the efficiency of GM-CSF knock-out by criprpr/Cas 9 gene editing during ex vivo expansion of patient T cells. FIG. 13B: graph showing IL-6 concentration in peripheral blood during CAR-T treatment. FIG. 13C: a graph showing IFNG concentration in peripheral blood during CAR-T treatment. FIG. 13D: graphs comparing IL-6 and IFNG concentrations in peripheral blood of patients during CAR-T treatment. FIG. 13E: graph showing IL1/IL1R blocker concentration in peripheral blood during CART treatment. FIG. 13F: graph showing GM-CSF concentration in peripheral blood during CART treatment. FIG. 13G: graph showing CAR vector copy number per μ g genomic DNA in patient peripheral blood during CAR-T treatment. FIG. 13H: a graph showing the daily maximum body temperature (° c) of human patients at various time points indicated after T cell infusion. FIG. 13I: a graph showing CRP levels in human patients at various time points indicated after T cell infusion.

Figures 14A to 14K contain graphs showing clinical features of acute lymphoblastic leukemia human patients treated with anti-CD 19CAR-T cells expressing IL6 and IL-1 antagonists. FIG. 14A: a graph showing IL-6 concentrations in peripheral blood of patients at various time points indicated after T cell infusion. FIG. 14B: a graph showing IFNG concentrations in peripheral blood of patients at various time points indicated after T cell infusion. FIG. 14C: graphs comparing IL-6 and IFNG concentrations in peripheral blood of patients at various time points indicated after T cell infusion. FIG. 14D: a graph showing IL1/IL1R blocker concentrations in peripheral blood of patients at various time points indicated after T cell infusion. FIG. 14E: a graph showing GM-CSF concentrations in peripheral blood of patients at various time points indicated after T cell infusion. FIG. 14F: a graph showing changes in human patient body temperature at various time points indicated after T cell infusion. FIG. 14G: graphs showing CAR vector copy levels in patients at various time points indicated after T cell infusion. FIG. 14H: a graph showing CRP levels in patient peripheral blood at various time points indicated after T cell infusion.

Figures 15A-15P contain graphs showing clinical features of multiple myeloma patients treated with anti-BCMA CAR-T cells expressing IL6 and IL-1 antagonists. FIG. 15A: graph showing patient daily body temperature change at various time points after CAR-T cell infusion. FIG. 15B: graphs showing CRP concentrations in patient peripheral blood at various time points after CAR-T cell infusion. FIG. 15C: graphs showing the concentration of ferritin in the peripheral blood of patients at various time points after CAR-T cell infusion. FIG. 15D: a graph showing IFNG levels in patient peripheral blood at various time points after CAR-T cell infusion. FIG. 15E: graphs showing IL-6 levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15F: graphs comparing IL-6 and IFNG levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15G: graphs showing CAR vector copy number per μ g genomic DNA in patient peripheral blood at various time points post CAR-T cell infusion. FIG. 15H: a graph showing IL1/IL1R blocker levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15I: graphs comparing IL1RA and IL-6 levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15J: graphs showing IL-1B levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15K: graphs showing IL-2 levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15L: graphs showing IL-4 levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15M: graphs showing IL-10 levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15N: graphs showing IL-17A levels in peripheral blood of patients at various time points post CAR-T cell infusion. FIG. 15O: graphs showing TNFA levels in peripheral blood of patients at various time points after CAR-T cell infusion. FIG. 15P: graphs showing GM-CSF levels in patient peripheral blood at various time points post CAR-T cell infusion.

Detailed Description

Adoptive cell transfer immunotherapy relies on immune cell activation and cytokine secretion to eliminate diseased cells. However, systemic overproduction of cytokines can cause safety problems and can sometimes be fatal to the recipient. Morgan et al, Molecular Therapy (Molecular Therapy) 18(4), 843-851, 2010. The present disclosure is directed to overcoming this limitation, in part, by developing immune cells with reduced inflammatory properties. Cytokine Release Syndrome (CRS) is a common type of toxicity associated with CAR-T cell therapy. Toclizumab is an anti-IL 6R antibody commonly used to alleviate CRS in CAR-T therapy. However, high levels of IL-6 still circulate in patients presenting with CRS following CAR-T therapy. Such IL-6 molecules can pass the blood-brain barrier, which may lead to severe neurotoxicity. Furthermore, the time at which CRS occurs in patients receiving CAR-T therapy is unpredictable, making it challenging to decide when tollizumab should be administered to the patient. If a patient presents with CRS and does not immediately receive toslizumab therapy, life threatening.

The present disclosure is based, at least in part, on the identification of specific anti-IL-6 or anti-IL-6R antibodies that exhibit superior effects in inhibiting IL-6 signaling relative to other IL-6 antagonist antibodies. Such anti-IL-6 and anti-IL-6R antibodies are expected to be more effective in reducing the side effects associated with T cell immunotherapy mediated by IL-6 signaling. Without being bound by theory, the methods disclosed herein relate to the automated production of IL-6 antagonists (e.g., those disclosed herein) using CAR-T therapy alone. When CAR-T cells target and kill tumor cells, the CAR-T cells will produce any of the IL-6 antagonists disclosed herein, e.g., scFv antibodies that bind IL-6. At the same time, CAR-T cell mediated tumor cell killing stimulates the host immune system to release IL-6. In time, CAR-T cells produce IL-6 antagonists earlier than the host immune system releases IL-6, and a better neutralization of IL-6 release is expected. The clinical data provided herein have demonstrated successful neutralization of IL-6 storm during CAR-T therapy, making tobramumab treatment unnecessary.

As disclosed herein, genetically engineered immune cells (e.g., T cells) that co-express a CAR targeted to a cancer antigen (e.g., CD19 or BCMA), an IL-6 antagonist, and optionally an IL-1 antagonist, show superior therapeutic efficacy in human patients with various types of cancer, including leukemia, non-hodgkin's lymphoma, and multiple myeloma. Patients receiving CAR-T therapy exhibit reduced CRS severity, reduced or no neurotoxicity, and/or reduced or no other common side effects associated with CAR-T cell therapy (e.g., fever, hypoxia, and/or hypotension), even in the absence of treatment involving anti-CRS drugs such as IL-6 antagonists.

Accordingly, provided herein are modified immune cells expressing a Chimeric Antigen Receptor (CAR) and one or more IL-6 antagonist antibodies, such as those described herein, and therapeutic applications thereof.

I. Modifying immune cells

One aspect of the disclosure provides a modified immune cell having reduced inflammatory properties as compared to a wild-type immune cell of the same type. Wild-type cells refer to those cells without such knock-in and knock-out modifications. Such modified immune cells can include knock-in of one or more IL-6 antagonist antibodies as disclosed herein, and optionally a Chimeric Antigen Receptor (CAR) specific for an antigen of interest (e.g., a cancer antigen). In some cases, modifying the immune cell can further include knocking-in one or more IL-1 antagonists, such as IL-1RA or other IL-1 antagonists known in the art or disclosed herein. In some embodiments, modifying the immune cell can further comprise a knock-out modification of one or more endogenous genes (e.g., GM-CSF and/or TCR). In other embodiments, the modified immune cell can include a wild-type endogenous GM-CSF gene, a wild-type TCR gene, or both.

(i) Antagonistic IL-6 antibodies

IL-6 signals through a complex comprising the membrane glycoprotein gp130 and the IL-6 receptor (IL6R) (see, e.g., Hibi et al, Cell (Cell), 63(6):1149-57, 1990). Binding of IL-6 to IL6R on target cells promotes gp130 homodimerization and subsequent signaling. As used herein, IL6R comprises both a membrane-bound form of IL6R and a soluble form (sIL 6R). When bound to IL-6, soluble IL6R (sIL6R) acts as an agonist and may also promote gp130 dimerization and signaling. Trans-signaling can occur whereby sIL-6R secreted by a particular cell type induces a response to IL-6 in cells expressing only gp130 (see, e.g., Taga et al, Annu Rev Immunol., 15:797-819, 1997; and Rose-John et al, J. Biochem.J.) -300 (Pt2):281-90, 1994). In one example, sIL6R includes the extracellular domain of human IL6R (see, e.g., Peters et al, J Exp Med., 183(4): 1399-.

In some embodiments, the modified immune cells disclosed herein express antagonistic IL-6 antibodies, which can be antibodies that bind IL-6 or bind IL-6 receptor (IL-6R). Such antibodies (antagonist antibodies) can interfere with IL-6/IL-6R binding on immune cells, thereby inhibiting IL-6 mediated cell signaling.

A typical antibody molecule comprises a heavy chain variable region (V)_H) And light chain variable region (V)_L) Which are normally involved in antigen binding. V_HAnd V_LRegions may be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions, known as "framework regions" ("FRs"). Each V_HAnd V_LUsually consisting of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The framework regions and the range of CDRs can be precisely identified using methods known in the art, e.g., by Kabat definition, Chothia definition, AbM definition, and/or contact definition, all of which are well known in the art. See, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest (Sequences of Proteins of Immunological Interest), fifth edition, department of public health and service, American national institutes of health publication No. 91-3242, Chothia et al (1989) Nature (Nature) 342: 877; chothia, C.et Al (1987) J.M.biol.). 196:901-917, Al-lazikani et Al (1997) J.M.M.biol.). 273: 927-948; and Almagro, journal of molecular recognition (J.mol. Recognit.) 17:132-143 (2004). See also hgmp.mrc.ac.uk and bio in.org.uk/abs.

An antibody (used interchangeably with plurals) as used herein is an immunoglobulin molecule capable of specifically binding to a target protein, such as IL-6 or IL-6R, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (such as Fab, Fab ', F (ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of a desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody comprises any class of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody need not belong to any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of the heavy chain of the antibody. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

In some embodiments, an antibody described herein can specifically bind to a target protein or a receptor thereof. Antibodies that "specifically bind" (used interchangeably herein) to a target or epitope are terms well known in the art, and methods of determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or binds to a particular target antigen more frequently, more rapidly, for a longer duration, and/or with greater affinity than it reacts or binds to an alternative target antigen. An antibody "specifically binds" to a target cytokine if it binds with greater affinity, greater avidity, more readily, and/or for a longer duration than it binds to the target cytokine than it does to other substances. For example, an antibody that specifically (or preferentially) binds an IL-6 or IL-6R epitope is one that binds to an IL-6 or IL-6R epitope with greater affinity, greater avidity, greater ease, and/or greater duration than it binds to other IL-6 epitopes, non-IL-6 epitopes, other IL-6R epitopes, or non-IL-6R epitopes. It is also understood by reading this definition that, for example, an antibody that specifically binds a first target antigen may or may not specifically or preferentially bind a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may comprise) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

In some embodiments, an antagonist antibody to a target protein as described herein has suitable binding affinity for the target protein (e.g., human IL-6 or human IL-6R) or an epitope thereof. As used herein, "binding affinity" refers to the apparent binding constant or KA. KA is the reciprocal of the dissociation constant (KD). The antagonistic antibodies described herein can have at least 10 to a target antigen or epitope^-5、10^-6、10^-7、10^-8、10^-9、10^-10M or lower binding affinity (KD). Increased binding affinity corresponds to decreased KD. Higher affinity binding of an antibody to a first antigen relative to a second antigen can be represented by a higher KA (or lower numerical KD) binding to the first antigen than KA (or numerical KD) binding to the second antigen. In such cases, the antibody is specific for the first antigen (e.g., the first protein or mimetic thereof in the first conformation) relative to the second antigen (e.g., the same first protein or mimetic thereof in the second conformation; or the second protein). In some embodiments, the antagonist antibodies described herein have a higher binding affinity (higher KA or less KD) for the mature form of the target protein than for the precursor form of the target protein or another protein, e.g., an inflammatory protein of the same family as the target protein. The difference in binding affinity (e.g., for specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10⁵And (4) doubling.

Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays). An exemplary condition for evaluating binding affinity is in HBS-P buffer (10mM HEPES pH7.4, 150mM NaCl, 0.005% (v/v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of the concentration of the target protein. The concentration of bound protein ([ bound ]) is generally related to the concentration of free target protein ([ free ]), and is expressed as follows:

[ bound ] ([ free ]/(Kd + [ free ])

However, it is not always necessary to determine KA accurately, as it is sometimes sufficient to obtain a quantitative measure of affinity, e.g. determined using methods such as ELISA or FACS analysis, which is proportional to KA, and can therefore be used for comparison, such as to determine whether a higher affinity is e.g. 2-fold higher, to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g. by activity in a functional assay such as an in vitro or in vivo assay.

In some embodiments, an IL-6 antagonist antibody described herein can bind to and inhibit IL-6 signaling by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more). The inhibitory activity of the IL-6 antagonist antibodies described herein can be determined by routine methods known in the art.

The antibodies described herein may be murine, rat, human, or any other source (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., are not produced in animals without human behavior (e.g., such animals are immunized with the desired antigen or fragment thereof).

Any of the antibodies described herein can be monoclonal or polyclonal. "monoclonal antibody" refers to a homogeneous population of antibodies, and "polyclonal antibody" refers to a heterogeneous population of antibodies. These two terms do not limit the source of the antibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to a form of non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or antigen-binding fragment thereof that contains minimal sequences derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies can include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also most preferably comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and/or six) that have been altered relative to the original antibody, also referred to as one or more CDRs "derived" from one or more CDRs from the original antibody. Humanized antibodies may also be involved in affinity maturation.

The heavy chain variable domains (V) of exemplary anti-IL-6 antibodies and anti-IL-6R antibodies are provided below_H) And a light chain variable domain (V)_L) (reference antibodies 1-6) wherein the CDRs are shown in bold (determined according to the rules described in bio in org. uk/abs):

antibody 1 (binding IL-6R):

V_H(SEQ ID NO:1):

V_L(SEQ ID NO:2):

antibody 2 (binding to IL-6):

V_H(SEQ ID NO:3):

V_L(SEQ ID NO:4):

antibody 3 (binding to IL-6):

V_H(SEQ ID NO:5):

V_L(SEQ ID NO:6):

antibody 4 (binding IL-6R):

V_H(SEQ ID NO:7):

V_L(SEQ ID NO:8):

antibody 5 (binding to IL-6):

V_H(SEQ ID NO:9):

V_L(SEQ ID NO:10):

antibody 6 (binding gp 130):

V_H(SEQ ID NO:11):

V_L(SEQ ID NO:12):

in some embodiments, the IL-6 antagonist antibodies described herein bind to the same epitope in an IL-6 antigen (e.g., human IL-6) or IL-6R (e.g., human IL-6R) as one of the reference antibodies provided herein (e.g., antibody 1 or antibody 2) or compete with the reference antibody for binding to an IL-6 or IL-6R antigen. The reference antibodies provided herein comprise antibodies 1-6, the respective structural features and binding activities of which are provided herein. An antibody that binds to the same epitope as a reference antibody described herein can bind to the exact same epitope as the reference antibody or an epitope that substantially overlaps (e.g., contains less than 3 non-overlapping amino acid residues, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue). Whether two antibodies compete with each other for binding to the cognate antigen can be determined by competition assays well known in the art. Such antibodies can be identified as known to those of skill in the art, e.g., those having substantially similar structural features (e.g., complementarity determining regions), and/or those identified by assays known in the art. For example, a competition assay can be performed using one of the reference antibodies to determine whether the candidate antibody binds to the same epitope as the reference antibody or competes for binding to the IL-6 or IL-6R antigen.

In some casesIn some cases, an IL-6 antagonist antibody disclosed herein can include the same heavy chain CDRs and/or the same light chain CDRs as a reference antibody (e.g., antibody 1 or antibody 2) disclosed herein. The heavy and/or light chain CDRs are the regions/residues responsible for antigen binding; such regions/residues may be identified from the amino acid sequences of the heavy/light chain sequences of the reference antibody (as shown above) by methods known in the art. See, for examplewww.bioinf.org.uk/abs(ii) a Almagro, journal of molecular recognition (j.mol. recognit.) 17:132-143 (2004); chothia et al, J.Mol.biol. (J.mol.) 227:799-817(1987), and others known in the art or disclosed herein. Determination of CDR regions in antibodies is within the skill of the art, e.g., the methods disclosed herein, such as the Kabat method (Kabat et Al, Sequences of Proteins of Immunological Interest (Sequences of Immunological Interest), (fifth edition, 1991, national institutes of health, Besseda, Md.) or the Chothia method (Chothia et Al, 1989, Nature (Nature) 342: 877; Al-lazikani et Al (1997) journal of molecular biology (J.Molec.biol.)) 273: 927-948). As used herein, a CDR may refer to a CDR defined by any method known in the art. Two antibodies having the same CDR mean that the two antibodies have the same amino acid sequence of the CDR, determined by the same method.

Also within the scope of the present disclosure are functional variants of any of the exemplary anti-IL-6 or anti-IL-6R antibodies (e.g., antibody 1 or antibody 2) as disclosed herein. The functional variant may be at V relative to a reference antibody_HAnd/or V_LOr one or more amino acid residue variations in one or more HC CDRs and/or one or more LC CDRs while retaining substantially similar binding and biological activity (e.g., substantially similar binding affinity, binding specificity, inhibitory activity, or a combination thereof) as a reference antibody.

In some examples, IL-6 antagonist antibodies disclosed herein include HC CDR1, HC CDR2, and HC CDR3, which collectively contain no more than 10 amino acid variations (e.g., no more than 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared to HC CDR1, HC CDR2, and HC CDR3 of a reference antibody, such as antibody 1 or antibody 2. By "common" is meant that the total number of amino acid variations of all three HC CDRs is within the defined range. Alternatively or additionally, an anti-IL-6 or anti-IL 6R antibody can include LC CDR1, LC CDR2, and LC CDR3 that collectively contain no more than 10 amino acid variations (e.g., no more than 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acid variations), as compared to LC CDR1, LC CDR2, and LC CDR3 of a reference antibody.

In some examples, an IL-6 antagonist antibody disclosed herein can include HC CDR1, HC CDR2, and HC CDR3, at least one of which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variation) as compared to the corresponding HC CDR of a reference antibody, such as antibody 1 or antibody 2. In particular examples, the antibody comprises HC CDR3, which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variation), as compared to HC CDR3 of a reference antibody, such as antibody 1 or antibody 2. Alternatively or additionally, an IL-6 antagonist antibody can include LC CDR1, LC CDR2, and LC CDR3, at least one of which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variation) as compared to a corresponding LC CDR of a reference antibody. In particular examples, the antibody comprises LC CDR3 that contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variations) as compared to LC CDR3 of a reference antibody.

In some cases, the amino acid residue variation may be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein undergoing the amino acid substitution. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods, e.g., "molecular cloning: a Laboratory Manual, J.Sambrook et al, eds., second edition, Cold spring harbor Laboratory Press, Cold spring harbor, New York, 1989, or Current Protocols in Molecular Biology (Current Protocols in Molecular Biology), F.M.Ausubel et al, eds., John Willi father publishing Co., Ltd, New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: ((a) A → G, S; (b) R → K, H; (C) N → Q, H; (D) D → E, N; (E) C → S, A; (F) Q → N; (G) E → D, Q; (H) G → A; (I) H → N, Q; (j) I → L, V; (K) L → I, V; (L) K → R, H; (M → L, I, Y; (N) F → Y, M, L; (o) P → A; (P) S → T; (Q) T → S; (R) W → Y, F; (S) Y → W, F; (T) V → I, L.

In some embodiments, an IL-6 antagonist antibody disclosed herein can include heavy chain CDRs that share at least 80% (e.g., 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs of a reference antibody, such as antibody 1 or antibody 2. Alternatively or additionally, the antibody can include light chain CDRs that share at least 80% (e.g., 85%, 90%, 95%, or 98%) identity with light chain CDRs of a reference antibody. In some embodiments, an IL-6 antagonist antibody can include a heavy chain variable region having at least 80% (e.g., 85%, 90%, 95%, or 98%) identity to a heavy chain variable region of a reference antibody, such as antibody 1 or antibody 2, and/or a light chain variable region having at least 80% (e.g., 85%, 90%, 95%, or 98%) identity to a light chain variable region of a reference antibody.

The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc.Natl.Acad.Sci.USA 87: 2264-. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al, J.mol.biol. (215: 403-) -10, 1990. BLAST protein searches using the XBLAST program can be performed with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the target protein molecule. In the case of gaps between two sequences, gap BLAST can be utilized as described in Altschul et al, Nucleic Acids reviews (Nucleic Acids Res.) 25(17):3389-3402, 1997. When BLAST and gapped BLAST programs are used, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used.

The present disclosure also provides germline variants of any of the reference IL-6 antagonist antibodies disclosed herein. Germline variants contain one or more mutations in the framework regions relative to their parent antibody to the corresponding germline sequence. To make germline variants, the heavy or light chain variable region sequences (e.g., framework sequences) of a parent antibody or portion thereof can be used as a query against an antibody germline sequence database (e.g., biolnfo. org. uk/abs/, www.vbase2.org, or imgt. org) to identify the corresponding germline sequences used by the parent antibody and the amino acid residue variations in one or more framework regions between the germline sequences and the parent antibody. One or more amino acid substitutions can then be introduced into the parent antibody based on the germline sequence to produce a germline variant.

In some examples, an antagonist antibody described herein is a human antibody or a humanized antibody. Alternatively or additionally, the antagonistic antibody is a single chain antibody (scFv). Exemplary scFv antibodies are provided below.

IL6/IL6R scFv 1(SEQ ID NO:13):

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSS

IL6/IL6R scFv 2(SEQ ID NO:14):

EIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQKPGQAPRLLIYDMSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPYTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLWGYYALDIWGQGTTVTVSS

IL6/IL6R scFv 3(SEQ ID NO:15):

QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGKLLKPGGSLKLSCAASGFTFSSFAMSWFRQSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCARGLWGYYALDYWGQGTSVTVSS

IL6/IL6R scFv 4(SEQ ID NO:16):

QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTMLRDTSKNQFSLRLSSVTAADTAVYYCARSLARTTAMDYWGQGSLVTVSSGGGGSGGRASGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIK

(ii) Antibodies specific for GM-CSF

Granulocyte, macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2(CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. GM-CSF acts as a cytokine and stimulates stem cells to produce granulocytes (e.g., neutrophils, eosinophils, and/or basophils) and monocytes. GM-CSF triggers an immune/inflammatory cascade whereby activation of a small number of macrophages can rapidly lead to an increase in their numbers, a key process for fighting infections. GM-CSF may also have an effect on mature cells of the immune system, e.g., inhibiting neutrophil migration and causing alterations in receptors expressed on the cell surface.

In some embodiments, the modified immune cells disclosed herein express an antibody specific for GM-CSF, alone or in combination with any other inhibitor disclosed herein, e.g., an antagonist IL-6 antibody, which may be an antibody that binds IL-6 or binds an IL-6 receptor (IL-6R). Such antibodies (antagonistic antibodies) can inhibit GM-CSF-mediated cell signaling, thereby down-regulating the immune response triggered by GM-CSF.

The anti-GM-CSF antibodies described herein can have any form and/or be from any suitable species, e.g., intact (i.e., full length), antigen-binding fragments (such as Fab, Fab ', F (ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins including antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. In some embodiments, an anti-GM-CSF antibody described herein can specifically bind to GM-CSF of a particular species, e.g., human GM-CSF.

In some embodiments, the anti-GM-CSF antibodies described herein can have suitable binding affinity for a target protein or an epitope thereofForce. For example, an anti-GM-CSF antibody can have at least 10 for a target antigen or epitope (e.g., human GM-CSF or an epitope thereof)^-5、10^-6、10^-7、10^-8、10^-9、10^-10M or lower binding affinity (KD). In some embodiments, an anti-GM-CSF antibody described herein can bind to and inhibit GM-CSF signaling by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more). The inhibitory activity of the anti-GM-CSF antibodies described herein can be determined by conventional methods known in the art.

The heavy chain variable domains (V) of exemplary anti-GM-CSF antibodies are provided below_H) And a light chain variable domain (V)_L) (reference antibodies 7 to 9) wherein the CDRs are shown in bold (determined according to the rules described in bio in org. uk/abs):

reference antibody 7:

V_H(SEQ ID NO:17):

V_L(SEQ ID NO:18):

reference antibody 8:

V_H(SEQ ID NO:19):

V_L(SEQ ID NO:20):

reference antibody 9:

V_H(SEQ ID NO:21):

V_L(SEQ ID NO:22):

in some embodiments, an anti-GM-CSF antibody described herein binds to the same epitope in a GM-CSF antigen as one of the reference antibodies provided herein, such as antibody 9, or competes with the reference antibody for binding to the GM-CSF antigen. The reference antibodies provided herein comprise antibodies 7 to 9, the respective structural features and binding activities of which are provided herein. Such antibodies can be identified as known to those of skill in the art, e.g., those having substantially similar structural features (e.g., complementarity determining regions), and/or those identified by assays known in the art. For example, a competition assay may be performed using one of the reference antibodies to determine whether the candidate antibody binds to the same epitope as the reference antibody or competes for binding to the GM-CSF antigen.

In some cases, an anti-GM-CSF antibody disclosed herein can include the same heavy chain CDRs and/or the same light chain CDRs as a reference antibody disclosed herein (e.g., antibody 9). The heavy and/or light chain CDRs are the regions/residues responsible for antigen binding; such regions/residues may be identified from the amino acid sequences of the heavy/light chain sequences of the reference antibody (as shown above) by methods known in the art. See also the description above.

Also within the scope of the present disclosure are functional variants of any exemplary anti-GM-CSF antibody (e.g., antibody 9) as disclosed herein. The functional variant may be at V relative to a reference antibody_HAnd/or V_LOr one or more amino acid residue variations in one or more HC CDRs and/or one or more LC CDRs while retaining substantially similar binding and biological activity (e.g., substantially similar binding affinity, binding specificity, inhibitory activity, or a combination thereof) as a reference antibody.

In some examples, an anti-GM-CSF antibody disclosed herein includes HC CDR1, HC CDR2, and HC CDR3, which collectively contain no more than 10 amino acid variations (e.g., no more than 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared to the HC CDR1, HC CDR2, and HC CDR3 of a reference antibody, such as antibody 9. Alternatively or additionally, an anti-GM-CSF antibody may include LC CDR1, LC CDR2, and LC CDR3 that collectively contain no more than 10 amino acid variations (e.g., no more than 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared to LC CDR1, LC CDR2, and LC CDR3 of a reference antibody.

In some examples, an anti-GM-CSF antibody disclosed herein can include HC CDR1, HC CDR2, and HC CDR3, at least one of which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variations) as compared to the corresponding HC CDR of a reference antibody, such as antibody 9. In particular examples, the antibody includes an HC CDR3 that contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variations) as compared to the HC CDR3 of a reference antibody, such as antibody 9. Alternatively or additionally, an anti-GM-CSF antibody described herein may include LC CDR1, LC CDR2, and LC CDR3, at least one of which contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variations) as compared to the corresponding LC CDR of a reference antibody. In particular examples, the antibody comprises LC CDR3 that contains no more than 5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acid variations) as compared to LC CDR3 of a reference antibody. In some cases, the amino acid residue variation may be a conservative amino acid residue substitution.

In some embodiments, an anti-GM-CSF antibody disclosed herein can include heavy chain CDRs that share at least 80% (e.g., 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs of a reference antibody, such as antibody 9. Alternatively or additionally, the antibody can include light chain CDRs that share at least 80% (e.g., 85%, 90%, 95%, or 98%) identity with light chain CDRs of a reference antibody. In some embodiments, an anti-GM-CSF antibody may include a heavy chain variable region having at least 80% (e.g., 85%, 90%, 95%, or 98%) identity to a heavy chain variable region of a reference antibody, such as antibody 9, and/or a light chain variable region having at least 80% (e.g., 85%, 90%, 95%, or 98%) identity to a light chain variable region of a reference antibody. In some embodiments, the anti-GM-CSF antibodies described herein can be germline variants of any of the exemplary anti-GM-CSF antibodies described herein, e.g., antibody 9.

In some examples, an anti-GM-CSF antibody described herein is a human or humanized antibody. Alternatively or additionally, the antagonistic antibody is a single chain antibody (scFv). Exemplary scFv antibodies are provided below.

GM-CSF scFv1(SEQ ID NO:23):

GM-CSF scFv2(SEQ ID NO:24):

GM-CSF scFv3(SEQ ID NO:25):

(iii) Bispecific antibodies specific for IL-6 and GM-CSF

Also provided herein are bispecific antibodies to both IL-6 and GM-CSF. Bispecific antibodies include two binding moieties, one specific for IL-6/IL-6R and the other specific for GM-CSF. Bispecific antibodies can have any form known in the art or disclosed herein. In some embodiments, the IL-6/IL-6R with specificity of the binding part can be derived from any of the example IL-6 antagonist antibodies (e.g., antibody 1 or antibody 2) or also as described herein its functional variants. Alternatively or additionally, the binding moiety specific for GM-CSF may be derived from any of the exemplary anti-GM-CSF antibodies described herein (e.g., antibody 9) or functional variants thereof as also described herein.

In some embodiments, bispecific antibodies described herein can be configured as a single fusion polypeptide comprising a first scFv fragment specific for IL-6/IL-6R and a second scFv fragment specific for GM-CSF. The two scFv fragments may be linked by a peptide linker. Exemplary bispecific antibodies are provided below:

bispecific Ab1 (bold and italic peptide linker) (SEQ ID NO: 26):

bispecific Ab2 (bold and italic peptide linker) (SEQ ID NO: 27):

bispecific Ab3 (bold and italic peptide linker) (SEQ ID NO: 28):

(iv) IL-1 antagonists

Interleukin-1 is a cytokine known in the art and comprises two isoforms, IL-1 α and IL-1 β. IL-1 plays an important role in the up-and down-regulation of acute inflammation and other biological pathways.

In some embodiments, the IL-1 antagonist expressed in the modified immune cells disclosed herein can be an interleukin-1 receptor antagonist (IL-1 RA). IL-1RA is a naturally occurring polypeptide that is secreted by a variety of cells, such as immune cells, epithelial cells, and adipocytes. It binds to the cell surface IL-1R receptor, thereby preventing cell signaling triggered by IL-1/IL-1R interaction. Human IL-1RA is encoded by the IL1RN gene. The following is an exemplary amino acid sequence of human IL-1RA (SEQ ID NO: 29):

the bold and italicized N-terminal fragments refer to the signal peptide in native IL-1 RA. IL-1RA for use in the present application may include an amino acid sequence (excluding the signal peptide) corresponding to the mature polypeptide of human IL-1RA described above. The following is an exemplary amino acid sequence of mature human IL-1RA (SEQ ID NO: 58):

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTS FESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

in some cases, the signal peptide may be replaced by a different signal sequence, for example, MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 59). The resulting IL-1RA will have the complete sequence (SEQ ID NO: 60):

other IL-1 antagonists include, but are not limited to, anti-IL-1 α or anti-IL-1 β antibodies.

(v) Chimeric Antigen Receptor (CAR)

The modified immune cells disclosed herein can further express a chimeric antigen receptor, which is an artificial cell surface receptor that redirects the binding specificity of the immune cell (e.g., T cell) to the pathological antigen to which the CAR binds, thereby eliminating the target diseased cell by, for example, effector activity of the immune cell. The CAR constructs typically include an extracellular antigen-binding domain fused to at least an intracellular signaling domain. Cartellieri et al, J Biomed Biotechnol (J Biomed Biotechnol) 2010:956304,2010. The extracellular antigen-binding domain, which may be a single chain antibody fragment (scFv), is specific for an antigen of interest (e.g., a pathological antibody, such as a cancer antigen), and the intracellular signaling domain may mediate cell signaling leading to immune cell activation. Thus, an immune cell expressing a CAR construct specific for an antigen of interest can bind to a diseased cell (e.g., tumor cell) expressing the antigen, resulting in activation of the immune cell and elimination of the diseased cell. In some embodiments, the extracellular antigen-binding domain targets a tumor antigen, such as CD19 or BCMA. In a specific example, the extracellular antigen-binding domain is a single chain antibody fragment that binds CD19, such as SEQ ID NO: 52. In other examples, the extracellular antigen-binding domain is a single chain antibody fragment that binds to BCMA, e.g., SEQ ID NO: 57.

The CAR constructs disclosed herein can include one or more intracellular signaling domains. In some examples, the CAR includes an intracellular signaling domain comprising an Immunoreceptor Tyrosine Activation Motif (ITAM). Such intracellular signaling domains may be from CD3 ζ, CD3 δ/epsilon, CD3 γ/epsilon, or from MHC class I molecules or suitable receptors, such as TNF receptors, immunoglobulin-like receptors, Fc receptors, cytokine receptors, activated NK cell receptors, BTLAs, integrins, or Toll ligand receptors. Furthermore, the CAR construct may further comprise one or more co-stimulatory signaling domains, which may be derived from a co-stimulatory receptor, e.g., Signaling Lymphocyte Activating Molecule (SLAM), OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CDlla/CD18),4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp 1, NKp 1, NKp 1, CD1, CD1, CD1 alpha, CD1 beta, IL 21 gamma, IL7 alpha, ITGA 72, VLAl, CD1, VLITGA 1, CD1, CD1, GAMMA 1, GAITGB 72, GAITGB 1, GAITC 1, GAITGB 72, GAITGA 1, GAITGB 72, GAITC 1, GAITGB 72, GAITC 1, GAITB 1, GAITC 1, GAITX 1, GAITB 1, GAITX 1, GAITC 1, GAITX 366957, GAITX 1, GAITX 366955, GAITX 1, GAITX 366957, GAITX 1, GAITX 366955, GAITX 1, GAIT, CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a or a ligand that specifically binds to CD 83.

The CAR constructs disclosed herein may further comprise a transmembrane hinge domain, which may be obtained from a suitable cell surface receptor such as CD28 or CD 8. In some cases, the CAR construct can further include a hinge domain, which can be located between the transmembrane domain and the intracellular signaling domain.

(vi) Knock-in modified immune cells with IL-6 antagonist antibodies, anti-GM-CSF antibodies and/or IL-1 antagonists

Also provided herein are populations of immune cells comprising knock-in modified cells having an IL-6 antagonist antibody (e.g., scFv1 or scFv2), an anti-GM-CSF antibody, an IL-1 antagonist, or a combination thereof, as described herein, and optionally a CAR construct. In some cases, the IL-6 antagonist antibody and the anti-GM-CSF antibody may be in the form of a bispecific antibody, such as those described herein.

In some embodiments, the genetically modified immune cells can include knock-in modifications of an IL-6 antagonist (e.g., an anti-IL-6 antagonist antibody, such as scFv1 or scFv2) and an IL-1 antagonist, such as IL-1 RA. Such immune cells may have an endogenous GM-CSF gene and optionally also an endogenous TCR gene. Alternatively, the immune cell may have an endogenous GM-CSF gene and a disrupted endogenous TCR gene.

The modified immune cells disclosed herein include knock-in modifications to express antagonistic IL-6 antibodies, anti-GM-CSF antibodies, bispecific antibodies that bind IL-6/IL-6R and GM-CSF, IL-1 antagonists disclosed herein, or a combination thereof. Knock-in modifications can include delivery of one or more exogenous nucleic acids encoding an IL-6 antagonist antibody, an anti-GM-CSF antibody, a bispecific antibody, an IL-1 antagonist disclosed herein, or a combination thereof to a host cell (e.g., an immune cell as described herein). The exogenous nucleic acid is operably linked to a suitable promoter such that the encoded protein (e.g., cytokine antagonist and/or immunosuppressive cytokine) can be expressed in the host cell. In some cases, the exogenous nucleic acid encoding the IL-6 antagonist antibody can be integrated into the genome of the host cell. In other cases, the exogenous nucleic acid may remain extrachromosomal (not integrated into the genome).

In some cases, any modified immune cell can include further knock-in modifications to express the CAR constructs disclosed herein.

The modified immune cells comprising one or more knock-in modifications can comprise one or more exogenous nucleic acids (e.g., an exogenous expression cassette) for expressing immunosuppressive cytokines and/or antagonists of one or more inflammatory proteins of interest as described herein. For the purposes of this disclosure, it is to be expressly understood that the term "antagonist" encompasses all previously identified terms, names and functional states and characteristics whereby the target protein itself, the biological activity or the result of the biological activity of the target protein is substantially abolished, reduced or neutralized to any meaningful degree, for example, by at least 20%, 50%, 70%, 85%, 90% or more.

Modifying immune cells disclosed herein can also include knocking out one or more inflammatory proteins (e.g., an inflammatory cytokine or soluble receptor thereof, an inflammatory growth factor, or a cytotoxic molecule), knocking in one or more inflammatory protein antagonists, or immunosuppressive cytokines, or a combination thereof.

Exemplary inflammatory cytokines or soluble receptors thereof include interleukin 1 alpha (IL1 alpha), interleukin 1 beta (IL1 beta), interleukin 2(IL-2), interleukin 5(IL-5), interleukin 6(IL-6), interleukin 7(IL-7), interleukin 8(IL-8), interleukin 9(IL-9), interleukin (IL-12), interleukin 15(IL-15), interleukin 17(IL-17), interleukin 18(IL-18), interleukin 21(IL-21), interleukin 23(IL-23), sIL-1RI, sIL-2 Ra, soluble IL-6 receptor (sIL6R), interferon alpha (IFN alpha), interferon beta (IFN beta), interferon gamma (IFN gamma), macrophage inflammatory proteins (e.g., MIP alpha and MIP beta), macrophage colony stimulating factor 1(CSF1), Leukemia Inhibitory Factor (LIF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), C-X-C motif chemokine ligand 10(CXCL10), chemokine (C-C motif) ligand 5(CCL5), eotaxin, Tumor Necrosis Factor (TNF), monocyte chemotactic protein 1(MCP1), gamma interferon-induced monocyte factor (MIG), advanced glycosylation end product Receptor (RAGE), C-reactive protein (CRP), angiopoietin-2, and Von Willebrand Factor (VWF).

Examples of inflammatory proteins of interest include, but are not limited to, inflammatory cytokines or their soluble receptors (e.g., IL2, IL1 α, IL1 β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2R α, sIL6R, IFN α, IFN β, IFN γ, MIP α, MIP β, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, and VWF), inflammatory growth factors (e.g., TGF α, VEGF, HGF, EGF, and FGF), and cytotoxic molecules (e.g., perforin, granzyme, and ferritin).

The immune cell population as described herein can be further modified to express an exogenous cytokine, a chimeric synNotch receptor, a chimeric immunoreceptor, a chimeric costimulatory receptor, a chimeric killer cell immunoglobulin-like receptor (KIR), and/or an exogenous T cell receptor. This may be done before, after or simultaneously with the knock-in and/or knock-out modification. Such receptors can be cloned and integrated into any suitable expression vector using conventional recombinant techniques. Considerations for designing chimeric antigen receptors are also known in the art. See, e.g., Sadelain et al, Cancer discovery (Cancer Discov.), 3(4):388-98, 2013.

The immune cell disclosed herein can be a T cell, NK cell, dendritic cell, macrophage, B cell, neutrophil, eosinophil, basophil, mast cell, myeloid-derived suppressor cell, mesenchymal stem cell, precursor thereof, or a combination thereof.

Methods of making modified immune cells

Any knock-in and knock-out modifications can be introduced into a suitable immune cell by conventional methods and/or routes described herein. Typically, such methods will involve delivering the genetic material into an appropriate immune cell to down-regulate the expression of the endogenous inflammatory protein of interest, to express the cytokine antagonist of interest, or to express the immunosuppressive cytokine of interest.

(A) Knock-in embellishment

To generate knock-ins of one or more cytokine antagonists described herein, the coding sequence for any of the antagonists and/or immunosuppressive cytokines described herein can be cloned into a suitable expression vector (e.g., including, but not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors, PiggyBac transposon vectors, and sleepangbeauty transposon vectors) and introduced into a host immune cell using conventional recombinant techniques. Sambrook et al, molecular cloning: a Laboratory Manual, third edition, Cold spring harbor Laboratory Press. Thus, a modified immune cell of the present disclosure may include one or more exogenous nucleic acids encoding at least one cytokine antagonist or at least one immunosuppressive cytokine. In some cases, the coding sequence for one or more antagonists and/or one or more immunosuppressive cytokines is integrated into the genome of the cell. In some cases, the coding sequence for the one or more antagonists is not integrated into the genome of the cell.

The exogenous nucleic acid comprising the coding sequence for the cytokine antagonist or immunosuppressive cytokine of interest may further comprise a suitable promoter, which may be operably linked to the coding sequence. As used herein, a promoter refers to a nucleotide sequence (site) on a nucleic acid to which RNA polymerase can bind to initiate transcription of the encoding DNA (e.g., for cytokine antagonists) into mRNA, which is then translated into the corresponding protein (i.e., expression of the gene). A promoter is said to be "operably linked" to a coding sequence when it is in the correct functional position and orientation relative to the coding sequence to control ("drive") the initiation of transcription and expression of that coding sequence (to produce the corresponding protein molecule). In some cases, the promoters described herein may be constitutive, which initiates transcription independently of other regulatory factors. In some cases, the promoters described herein may be inducible, depending on the regulatory factor of transcription. Exemplary promoters include, but are not limited to, ubiquitin, RSV, CMV, EF 1a, and PGK 1. In one example, one or more nucleic acids encoding one or more antagonists of one or more inflammatory cytokines such as those described herein, operably linked to one or more suitable promoters, can be introduced into an immune cell by conventional methods to drive expression of the one or more antagonists.

In addition, the exogenous nucleic acids described herein may further contain, for example, some or all of: selectable marker genes, such as the neomycin gene used to select stable or transient transfectants in mammalian cells; enhancer/promoter sequences for high level transcription from the human CMV immediate early gene; transcriptional termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma viral origin of replication and ColE1 for correct episomal replication; a multifunctional multiple cloning site; and the T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable methods for producing a vector containing a transgene are well known and available in the art. Sambrook et al, molecular cloning: a Laboratory Manual, third edition, Cold spring harbor Laboratory Press.

In some cases, multiple cytokine antagonists as described herein can be constructed in a polycistronic fashion in one expression cassette, such that the multiple cytokine antagonists act as separate polypeptides. In some instances, an internal ribosome entry site can be inserted between two coding sequences to achieve this goal. Alternatively, a nucleotide sequence encoding a self-cleaving peptide (e.g., T2A or P2A) may be inserted between the two coding sequences. Exemplary designs of such polycistronic expression cassettes are provided in the examples below.

(B) Knock-out modifications

Any method known in the art for down-regulating the expression of an endogenous gene in a host cell may be used to reduce the level of production of the endogenous cytokine/protein of interest as described herein. Gene editing methods may involve the use of an endonuclease capable of cleaving a target region in an endogenous allele. Non-homologous end joining in the absence of template nucleic acid can repair double-strand breaks in the genome and introduce mutations (e.g., insertions, deletions, and/or frameshifts) into the target site. Gene editing methods are generally classified according to the type of endonuclease involved in generating a double-strand break in the target nucleic acid. Examples include, but are not limited to, regularly interspaced clustered short palindromic repeats (CRISPR)/endonuclease systems, transcription activator-like effector nucleases (TALENs), Zinc Finger Nucleases (ZFNs), endonucleases (e.g., ARC homing endonucleases), meganucleases (e.g., mega-TAL), or combinations thereof.

Various gene editing systems using meganucleases have been described in the art, including modified meganucleases; see, e.g., Steentoft et al, reviews in Glycobiology (Glycobiology) 24(8), 663-80, 2014; belfort and Bonocora, Methods of molecular biology (Methods Mol Biol.) 1123:1-26 (2014); hafez and Hausner, Genome 55 (Genome), 553-69 (2012); and the references cited therein. In some examples, a knock-out event can be combined with a knock-in event — an exogenous nucleic acid encoding a desired molecule, such as those described herein, can be inserted by gene editing into the locus of a target endogenous gene of interest.

In some cases, endogenous genes can be knocked out using CRISPR technology. Exemplary target endogenous genes include IL-2, GM-CSF, TNFA T-cell receptor, β 2M, and the like. Exemplary gRNAs for knock-out of target endogenous genes IL-2, GM-CSF and TNFA are provided in the examples below.

Alternatively, any knock-out modification can be achieved by methods known in the art using antisense oligonucleotides (e.g., interfering RNAs, such as shRNA or siRNA) or ribozymes. Antisense oligonucleotides specific for a target cytokine/protein refer to oligonucleotides complementary or partially complementary to a target region of the endogenous gene of the cytokine or to the mRNA encoding them. Such antisense oligonucleotides can be delivered into target cells by conventional methods. Alternatively, such antisense oligonucleotides may be expressed using an expression vector such as a lentiviral vector or an equivalent thereof.

(C) Preparation of immune cell populations comprising modified immune cells

A population of immune cells comprising any of the modified immune cells described herein or a combination thereof can be prepared by introducing one or more knock-in modifications, one or more knock-out modifications, or a combination thereof into a population of host immune cells. Knock-in and knock-out modifications can be introduced into the host cell in any order.

In some cases, one or more modifications are introduced into a host cell in a sequential manner without isolating and/or enriching the modified cells after a previous modification event and before a next modification event. In that case, the resulting immune cell population may be heterogeneous, including cells with different modifications or different combinations of modifications. Such immune cell populations may also include unmodified immune cells. The level of each modification event occurring in the immune cell population can be controlled by the amount of genetic material inducing such modification relative to the total number of host immune cells. See also the discussion above.

In other cases, the modified immune cells can be isolated and enriched after the first modification event and before the second modification event. This approach will result in the generation of a substantially homogeneous population of immune cells with knock-in and/or knock-out modifications of all the introduced cells.

In some examples, the knock-in modification and the knock-out modification are introduced separately into the host immune cell. For example, knock-out modifications are made by gene editing to knock-out the endogenous gene of the target cytokine, and knock-in modifications are made by delivering a separate exogenous expression cassette into the host immune cell to produce one or more cytokine antagonists. In some cases, the knock-in and knock-out events can occur simultaneously, e.g., a knock-in cassette can be inserted into the locus of the target gene to be knocked out.

Therapeutic applications

Any immune cell population comprising modified immune cells as described herein can be used in adoptive immune cell therapy to treat a target disease, such as leukemia or lymphoma. Due to the introduction of knock-in and knock-out modifications of immune cells, particularly knock-in IL-6 antagonist antibodies, anti-GM-CSF antibodies (possibly fragments of bispecific antibodies that also contain fragments that bind to IL-6 or IL-6R), IL-1 antagonists described herein, or combinations thereof, it is expected that their therapeutic use will reduce cytotoxicity associated with conventional adoptive immune cell therapy (reducing inflammatory cytokines produced by both immune cells used in adoptive immune cell therapy and endogenous immune cells of the recipient, which can be activated by infused immune cells), while achieving the same or better therapeutic effect.

To practice the methods of treatment described herein, an effective amount of an immune cell population comprising any of the modified immune cells described herein can be administered to a subject in need of treatment by a suitable route (e.g., intravenous infusion). It is also within the scope of the present disclosure that the immune cell population can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition prior to administration. The immune cells can be autologous to the subject, i.e., the immune cells are obtained from a subject in need of treatment, modified to reduce expression of one or more target cytokines/proteins, such as those described herein, to express one or more cytokine antagonists described herein, to express the CAR construct and/or exogenous TCR, or a combination thereof. The resulting modified immune cells can then be administered to the same subject. Administration of autologous cells to a subject results in a reduction in rejection of immune cells as compared to administration of non-autologous cells. Alternatively, the immune cells may be allogeneic, i.e., the cells are obtained from a first subject modified as described herein and administered to a second subject that is different from the first subject but is of the same species. For example, the allogeneic immune cells may be derived from a human donor and administered to a human recipient that is different from the donor.

The subject to be treated can be a mammal (e.g., a human, mouse, pig, cow, rat, dog, guinea pig, rabbit, hamster, cat, goat, sheep, or monkey). The subject may have cancer, an infectious disease, or an immune disorder. Exemplary cancers include, but are not limited to, hematological malignancies (e.g., B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma). Exemplary infectious diseases include, but are not limited to, Human Immunodeficiency Virus (HIV) infection, epstein-barr virus (EBV) infection, Human Papilloma Virus (HPV) infection, dengue virus infection, malaria, sepsis, and e. Exemplary immune disorders include, but are not limited to, autoimmune diseases such as rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, guillain-barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, graves 'disease, hashimoto's disease thyroiditis, myasthenia gravis, and vasculitis.

In some examples, the subject to be treated in the methods disclosed herein can be a human cancer patient. For example, a human patient may have a cancer of B cell origin. Examples include B-line acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-hodgkin's lymphoma. Alternatively, the human patient may have breast cancer, gastric cancer, neuroblastoma or osteosarcoma.

As used herein, the term "effective amount" refers to the amount of each active agent, alone or in combination with one or more active agents, needed to confer a therapeutic effect on a subject. As will be appreciated by those skilled in the art, effective amounts will vary depending upon the particular condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex, and weight), duration of treatment, route of administration, use of excipients, co-use with other active agents (if any), and like factors within the knowledge and expertise of the health practitioner. The amount administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to generate a cell-mediated immune response. The precise amount of active ingredient that needs to be administered depends on the judgment of the practitioner. However, suitable dosage ranges are readily determined by those skilled in the art.

As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease, a symptom of a target disease, or a predisposition to a target disease, with the purpose of treating, curing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease, the symptom of the disease, or the predisposition to the disease.

In some cases, the genetically engineered immune cells disclosed herein are used to treat cancer. Such immune cells express a Chimeric Antigen Receptor (CAR) that targets a cancer antigen, such as CD19 or BCMA. In addition to any of the IL-6 antagonists disclosed herein (e.g., including the scFv of SEQ ID NO:13 or SEQ ID NO:14), the genetically engineered immune cells can further express an IL-1 antagonist, such as IL-1 RA. Genetically engineered immune cells may have knocked-out endogenous GM-CSF and/or TCR genes. Alternatively, the genetically engineered immune cells may carry wild-type endogenous GM-CSF and/or TCR genes.

An effective amount of the genetically engineered immune cells can be administered to a human patient in need of treatment by a suitable route, such as intravenous infusion. In thatIn some cases, about 1x10 may be used⁶To about 1x10⁸The CAR + T cells are administered to a human patient (e.g., a leukemia patient, a lymphoma patient, or a multiple myeloma patient). In some examples, the human patient may receive multiple doses of the genetically engineered immune cells. For example, a patient may receive two doses of immune cells on two consecutive days. In some cases, the first dose is the same as the second dose. In other cases, the first dose is lower than the second dose, and vice versa.

In any of the methods of treatment disclosed herein, including methods using genetically engineered immune cells, IL-2 can be administered to a subject concurrently with cell therapy. More specifically, an effective amount of IL-2 can be administered to a subject by a suitable route before, during or after cell therapy. In some embodiments, IL-2 is administered to the subject after administration of the immune cells.

Alternatively or additionally, subjects treated with the cell therapies disclosed herein may be immune cell infused prior to treatment with an IL-6 antagonist (in addition to IL-6 antagonists produced by immune cells used in the cell therapy).

The immune cell population comprising modified immune cells as described herein can be used in combination with other types of cancer therapies such as chemotherapy, surgery, radiation, gene therapy, and the like. Such therapies may be administered simultaneously or sequentially (in any order) with the immunotherapy described herein. When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent can be reduced due to additive or synergistic effects.

In some examples, the subject receives a suitable anti-cancer therapy (e.g., those disclosed herein) prior to the CAR-T therapy disclosed herein to reduce tumor burden. For example, a subject (e.g., a human cancer patient) can receive chemotherapy (e.g., comprising a single chemotherapeutic agent or a combination of two or more chemotherapeutic agents) at a dose that significantly reduces tumor burden. In some cases, chemotherapy may reduce the total leukocyte count of the subject to below 10⁸L, e.g. less than 10⁷And L. Monitoring after initial anti-cancer therapy can be carried out by conventional methodsAnd/or tumor burden in the patient following CAR-T cell therapy disclosed herein. If the patient shows a high growth rate of cancer cells after the initial anti-cancer therapy and/or CAR-T therapy, the patient may receive a new round of chemotherapy to reduce tumor burden, followed by any CAR-T therapy as disclosed herein.

Non-limiting examples of other anti-cancer therapeutic agents that can be used in combination with the modified immune cells described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1, and CTLA4 inhibitors), anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, metalloproteinase tissue inhibitors, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR, and FLT-1 receptor, and placental proliferation protein-related proteins); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); a chemotherapeutic compound. Exemplary chemotherapeutic compounds include pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine); purine analogs (e.g., fludarabine); folic acid antagonists (e.g., mercaptopurine and thioguanine); antiproliferative or antimitotic agents, such as vinca alkaloids; microtubule disrupting agents such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and vinorelbine, and epipodophyllotoxins; DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, pravastatin, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylenediamine oxaliplatin, ifosfamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, paclitaxel, taxotere, teniposide, triethylenethiophosphoramide, and etoposide).

In some embodiments, radiation or radiation and chemotherapy are used in combination with a cell population comprising modified immune cells as described herein. Other useful agents and therapies can be found in Physician's Desk Reference, supplementary version 59, (2005), Thomson P D R, Montvale n.j.; supplementary version of pharmaceutical Science and Practice (The Science and Practice of Pharmacy) 20 (2000), by eds. remington, (2000), Lippincott Williams and Wilkins, Baltimore Md.; branunwald et al, eds. (Harrison's Principles of Internal Medicine), supplementary 15, (2001), McGraw Hill, NY; berkow et al, eds. (The Merck Manual of Diagnosis and Therapy), (1992), The Merck research laboratory, Lawei, N.J..

Kits for therapeutic use or for the manufacture of modified immune cells

The present disclosure also provides kits for use of any of the target diseases described herein involving the immune cell populations described herein and kits for manufacturing modified immune cells as described herein.

A kit for therapeutic use as described herein may comprise one or more containers comprising a population of immune cells, which may be formulated to form a pharmaceutical composition. The immune cell population includes any of the modified immune cells described herein or a combination thereof. Populations of immune cells such as T lymphocytes, NK cells, and other cells as described herein can further express a CAR construct and/or exogenous TCR as described herein.

In some embodiments, the kit can further include instructions for using the immune cell population in any of the methods described herein. The included instructions can include a description of administering the immune cell populations or pharmaceutical compositions including them to a subject to achieve a desired activity in the subject. The kit can further include a description for selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions include a description of administering the population of immune cells or a pharmaceutical composition comprising them to a subject in need of treatment.

Instructions related to using the immune cell population or a pharmaceutical composition comprising the immune cell population as described herein typically include information about the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is for treating, delaying the onset of, and/or alleviating a disease or disorder in a subject.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Packaging for use in combination with a particular device, such as an inhaler, nasal administration device, or infusion device, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. The at least one active agent in the pharmaceutical composition is a population of immune cells (e.g., T lymphocytes or NK cells) including any modified immune cells or a combination thereof.

The kit optionally may provide additional components such as buffers and explanatory information. Typically, a kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the kit described above.

Also provided herein are kits for preparing modified immune cells as described herein. Such kits may comprise one or more containers, each container containing reagents for introducing knock-in and/or knock-out modifications into an immune cell. For example, a kit may contain one or more components of a gene editing system for performing one or more knockout modifications such as those described herein. Alternatively or additionally, the kit may include one or more exogenous nucleic acids for expressing cytokine antagonists also as described herein and reagents for delivering the exogenous nucleic acids into host immune cells. Such kits may further comprise instructions for making the desired modifications to the host immune cells.

General technique V

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989) Cold spring harbor Press; oligonucleotide synthesis (oligo synthesis) (m.j. gate, ed.1984); methods in Molecular Biology (Methods in Molecular Biology), Humana Press; cell biology: a laboratory Manual (Cell Biology: laboratory Notebook) (J.E. Cellis, ed.,1989) academic Press; animal Cell Culture (Animal Cell Culture), R.I. Freshney, ed.1987; introduction of Cell and Tissue Culture (Introduction to Cell and Tissue Culture) (J.P.Mather and P.E.Roberts,1998) Plenum Press; cell and tissue culture: laboratory programs (Cell and Tissue Culture: Laboratory Procedures) (A.Doyle, J.B.Griffiths and D.G.Newell, eds.1993-8) J.Wiley and Sons; methods in Enzymology (Methods in Enzymology) (academic Press Co.); handbook of Experimental Immunology (d.m. well and c.c. blackwell, eds.): mammalian cell Gene Transfer Vectors (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.Calos, eds., 1987); current Protocols in Molecular Biology (Current Protocols in Molecular Biology) (F.M. Ausubel et al eds. 1987); PCR: polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Mullis et al, eds. 1994); current Protocols in Immunology (j.e. coligan et al, eds., 1991); short Protocols in Molecular Biology (Short Protocols in Molecular Biology) (Wiley and Sons, 1999); immunobiology (immunology) (c.a. janeway and p.travers, 1997); antibodies (Antibodies) (p.finch, 1997); antibodies: practical methods (Antibodies: a practical approach) (D.Catty., ed., IRL Press, 1988-; monoclonal antibodies: practical methods (Monoclonal antibodies: a practical approach) (P.shepherd and C.dean, eds., Oxford university Press, 2000); using antibodies: laboratory manuals (Using Antibodies: a laboratory manual), E.Harlow and D.Lane (Cold spring harbor laboratory Press, 1999), Antibodies (The Antibodies) (M.Zantetti and J.D.Capra, eds. Hawood academic Press, 1995), "methods of DNA Cloning (DNA Cloning: A practical application), volume I and II (D.N.Glover. 1985)," Nucleic Acid Hybridization (Nucleic Acid Hybridization), B.D.Hames & S.J.Higg. eds. (1985), "Transcription and Translation (Transcription and Translation) Cell Culture (B.D.media & S.J.Higgins, 1984), Cell Culture (Cell Culture and Cell Culture), Cell Culture (Cell Culture) and Cell Culture (Cell Culture) of Cells, Cell Culture.

The disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the description herein or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Also as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Further description is omitted, and it is believed that one skilled in the art can, based on the description above, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter of the present citation.

Example 1: use of IL-6 antagonist antibodies expressed in 293T cells for inhibiting IL-6 signaling

HEK293T cells were transfected with a third generation self-inactivating (SIN) lentiviral transfer vector of Lipofectamine2000(Thermo Scientific) encoding single chain variable fragment (scFv) antibodies derived from reference antibodies 1,2, 3 and 4 disclosed herein, which target IL-6 or IL-6R. The CD8 leader sequence preceded the anti-IL 6 scFv. See the description in example 7 below. The scFv antibody was fused to the Fc fragment of human IgG 1. Supernatants of transfected cells containing scFv antibodies expressed by transfected HEK293T cells were collected, diluted and HEK-Blue IL-6 reporter cells (Invivogen) added in the presence of 2ng/ml human IL-6. HEK-Blue IL-6 reporter cells were used because they are capable of producing Secreted Embryonic Alkaline Phosphatase (SEAP) under stimulation by human IL-6. After overnight incubation, supernatants of HEK-Blue IL-6 cells were collected and incubated with Quant-Blue substrate solution. SEAP production was quantified by measuring the absorbance of the conversion substrate Quant Blue (Invivogen) at a wavelength of 650nm by a spectrophotometer.

As shown in FIG. 1, all scFv antibodies were able to inhibit IL-6 signaling by binding to IL-6 or IL-6R expressed on reporter cells. Among the 4 scFv antibodies tested, the scFv antibodies derived from antibodies 1 and 2 exhibited higher efficiency in inhibiting IL-6 signaling compared to the scFv antibodies derived from reference antibodies 3 and 4. For example, scFv antibodies derived from reference antibodies 1 and 2 showed approximately 100% inhibition of IL-6 signaling at dilution 0.5, while those derived from reference antibodies 3 and 4 showed less than 60% inhibition efficiency at the same dilution. This result indicates that antibody 1 and antibody 2 are more effective at blocking IL-6-IL6R signaling.

Example 2: combined effect of anti-IL-6 antibodies and IL-1RA expressed in 293T cells in blocking IL-6 and IL-1 signaling

The nucleic acids encoding construct 1, construct 2 and construct 3 were cloned into a generation 3 self-inactivating (SIN) lentiviral transfer vector. Construct 1 comprises, from N-to C-terminus, a T2A linker, a scFv antibody derived from reference antibody 2 (targeting IL-6), a P2A linker, and an IL-1 receptor antagonist (IL-1RA) (T2A-Sir-P2A-IL1 RA). Construct 2 contains a T2A linker, scFv antibody, (G) from N-to C-terminus₄S)₃Linker and IL1RA (T2A-Sir- (G4S)₃-IL1 RA). Construct 3 contains scFv antibody from N-terminus to C-terminus, (G)₄S)₃Linker, IL1RA and T2A linker (Sir- (G)₄S)₃IL1 RA-T2A). In constructs 1,2 and 3, the CD8 leader sequence precedes the anti-IL 6 scFv. Ginseng radix (Panax ginseng C.A. Meyer)See the description in example 7 below. In construct 1, the ahGH leader sequence is located between P2A and IL1 RA.

293T cells were transfected with the above lentivirus transfer vector by Lipofectamine2000(Thermo Scientific). Supernatants from transfected cells were collected and added to HEK-BlueIL-1R cells (Invivogen) at various dilutions as indicated in the presence of 10pg/ml IL-1B. After overnight incubation, the HEK-Blue IL-1R cell supernatant was collected, incubated with a Quant-Blue substrate solution (Invivogen), and the absorbance of the conversion substrate was measured by spectrophotometer at 650nm wavelength.

The supernatant was also added to HEK-Blue IL-6 cells (Invivogen) at the indicated different dilutions in the presence of 2ng/ml IL-6. After overnight incubation, HEK-Blue IL-6 cell supernatants were collected and incubated with Quant-Blue substrate solution (Invivogen) and the absorbance of the conversion substrate was measured by spectrophotometer at 650nm wavelength.

As shown in FIGS. 2A and 2B, the duplex structures described herein successfully block IL-1 and IL-6 signaling.

Example 3: combined effect of IL-6 and GM-CSF antagonistic antibodies expressed in 293T cells in blocking IL-6 and GM-CSF signalling

Constructs for expressing three exemplary bispecific antibodies specific for IL-6 and GM-CSF as well as IL1RA described in example 2 above were generated by conventional recombinant techniques. Each bispecific antibody contained a scFv derived from reference antibody 2 (targeting IL-6) and a scFv derived from reference antibody 7, reference antibody 8 or reference antibody 9 (both targeting GM-CSF). The two scFv fragments in the bispecific antibody are linked by a GSGGSG linker. Each bispecific antibody was linked to IL1RA via a P2A linker. These constructs were named Ab2/Ab9-P2A-IL1RA, Ab2/Ab7-P2A-IL1RA and Ab2/Ab8-P2A-IL1RA (corresponding to 1,2 and 3 in FIGS. 3A-3C, respectively). In these constructs, the CD8 leader sequence was positioned before the anti-IL 6 scFv, and the ahGH leader sequence was positioned between P2A and IL1 RA. See the description in example 7 below.

The nucleic acid encoding the above construct is inserted into a 3 rd generation self-inactivating (SIN) lentiviral transfer vector by recombinant techniques. The resulting lentiviral transfer vectors were transfected into 293T cells by Lipofectamine2000(Thermo Scientific). Supernatants from transfected cells were collected.

Supernatants were added to TF-1 cells at the indicated different dilutions in the presence of 2ng/ml GM-CSF for 2 days of co-culture, since TF-1 cells were completely dependent on GM-CSF for proliferation. Then passed through PromegaThe Aqueous One Solution Cell Proliferation Assay assesses the Proliferation of TF-1 cells.

The supernatant was also added to HEK-Blue IL-6 cells (Invivogen) at the indicated different dilutions in the presence of 2ng/ml IL-6. After overnight incubation, HEK-Blue IL-6 cell supernatants were collected and incubated with Quant-Blue substrate solution (Invivogen) and the absorbance of the conversion substrate was measured by spectrophotometer at 650nm wavelength.

In addition, the supernatant was added to HEK-Blue IL-1R cells (Invivogen) at the various dilutions indicated in the presence of 10pg/ml IL-1B. After overnight incubation, the HEK-Blue IL-1R cell supernatant was collected and incubated with a Quant-Blue substrate solution (Invivogen) and the absorbance of the conversion substrate was measured by spectrophotometer at 650nm wavelength.

All three constructs tested in this example showed inhibitory activity against IL-6 signaling and IL-1 signaling. Fig. 3B and 3C. On the other hand, constructs containing Ab2/Ab9 bispecific antibody showed significant blocking activity against GM-CSF signaling. Fig. 3A.

Example 4: disruption of GM-CSF by CRISPR techniques

Primary T cells (PPA research) from healthy donors were activated with anti-CD 3/28 beads (Thermo scientific). Four days later, activated T cells were electroporated with Cas9 protein (Thermo scientific) and different grnas targeting the first exon of GM-CSF as shown below, while T cells electroporated with Cas9 protein served only as negative controls.

Exemplary guide RNA template sequences for targeting human GM-CSF exon 1 (spacer sequence before PAM motif shown in bold):

gRNA1(SEQ ID NO:30):

gRNA2(SEQ ID NO:31):

gRNA3(SEQ ID NO:32):

gRNA4(SEQ ID NO:33):

gRNA5(SEQ ID NO:34):

gRNA6(SEQ ID NO:35):

gRNA7(SEQ ID NO:36):

gRNA8(SEQ ID NO:37):

four days after electroporation, theUsing CellTiterT Cell Proliferation was assessed by the AQueous One Solution Cell Proliferation Assay (MTS) (Promega). The results indicate that gene editing for GM-CSF does not significantly alter T cell proliferation after electroporation in the presence of exogenous IL 2. Fig. 4A.

T cells were also activated with PMA/Ionomycin to analyze cytokine (GM-CSF, IL-2, IFN γ, and TNF α) expression using an intracellular staining kit (Biolegend and BD Bioscience). The results indicate that GM-CSF gene editing significantly reduced GM-CSF expression, but showed no significant effect on IL2, IFN γ, or TNF α expression. Fig. 4B.

Example 5: disruption of IL-2 by CRISPR technology

Primary T cells (PPA research) from healthy donors were activated with anti-CD 3/28 beads (Thermo scientific). Four days later, activated T cells were electroporated with Cas9 protein (Thermo scientific) and a different gRNA targeting the first exon of IL2 as shown below, while T cells electroporated with Cas9 protein served only as negative controls.

An exemplary gRNA template sequence for targeting human IL2 exon 1 (spacer sequence before PAM motif is shown in bold):

gRNA1(SEQ ID NO:38):

gRNA2(SEQ ID NO:39):

gRNA3(SEQ ID NO:40):

gRNA4(SEQ ID NO:41):

gRNA5(SEQ ID NO:42):

four days after electroporation, CellTiter was usedT Cell Proliferation was assessed by the AQueous One Solution Cell Proliferation Assay (MTS) (Promega). The results indicate that knock-out IL2 does not significantly alter T cell proliferation after electroporation in the presence of exogenous IL 2. Fig. 5A.

T cells were activated with PMA/Ionomycin to analyze cytokine expression (GM-CSF, IL-2, IFN γ, and TNF α) by intracellular staining kit (Biolegend and BD Bioscience). The results indicate that IL2 gene editing significantly reduced IL2 expression, but had no significant effect on GM-CSF, IFN γ, or TNFA expression. Fig. 5B.

Example 6: disruption of TNF alpha by CRISPR technology

Primary T cells (PPA research) from healthy donors were activated with anti-CD 3/28 beads (Thermo scientific). Three days later, activated T cells were electroporated with Cas9 protein (Thermo scientific) and different grnas targeting the first exon of TNF α as shown below, while T cells electroporated with Cas9 served only as negative controls.

Exemplary gRNA template sequences for targeting human TNF α exon 1 (spacer sequence before PAM motif shown in bold):

sgRNA 1(SEQ ID NO:43):

sgRNA 2(SEQ ID NO:44):

sgRNA 3(SEQ ID NO:45):

sgRNA 4(SEQ ID NO:46):

sgRNA 5(SEQ ID NO:47):

sgRNA 6(SEQ ID NO:48):

sgRNA 7(SEQ ID NO:49):

sgRNA 8(SEQ ID NO:50):

five days after electroporation, CellTiter was usedT Cell Proliferation was assessed by the AQueous One Solution Cell Proliferation Assay (MTS) (Promega). The results indicate that gene editing for TNFA did not significantly alter T cell proliferation after electroporation in the presence of exogenous IL 2. Fig. 6A.

T cells were activated with PMA/Ionomycin to analyze cytokine expression (GM-CSF, IL-2, IFN γ, and TNF α) by intracellular staining kit (Biolegend and BD Bioscience). The results indicate that TNFA gene editing significantly reduced TNF α expression but showed no significant effect on GM-CSF, IFN γ, or IL2 expression. Fig. 6B.

Example 7: anti-CD 19 CART cells knocked out by GM-CSF and secreting IL6 blocker/IL-1 blocker exert potent cytotoxicity and IL6 and IL1B inhibitory effects

Primary T cells (PPA research) from healthy donors were activated with anti-CD 3/CD28 beads (Thermo scientific). One day later, a V encoding (i) an anti-CD 19CAR and (ii) an anti-IL 6 scFv polypeptide (SEQ ID NO:14) having the V of SEQ ID NO:3 was used_HSequence and V of SEQ ID NO 4_L(ii) sequence, (iii) a lentiviral vector for IL1RA transduced T cells.

The anti-CD 19CAR contains, from N-terminus to C-terminus, a CD8 leader sequence, an anti-CD 19 scFv fragment, a CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta domain. Exemplary amino acid sequences for these domains are provided below:

CD8 leader sequence (SEQ ID NO: 51):

MALPVTALLLPLALLLHAARP

anti-CD 19 scFv (SEQ ID NO: 52):

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

CD8 hinge domain (SEQ ID NO: 53):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 transmembrane domain (SEQ ID NO: 54):

IYIWAPLAGTCGVLLLSLVITLYC

4-1BB costimulatory domain (SEQ ID NO: 55):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD3z(SEQ ID NO:56)：

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

the CD8 leader sequence preceded the anti-IL 6 scFv. The nucleotide sequence encoding the T2A peptide is located between the coding sequences of (i) and (ii), and the nucleotide sequence encoding the P2A peptide is located between the coding sequences of (ii) and (iii). There is a human growth hormone signal sequence between P2A and (iii). The resulting engineered T cells expressed anti-CD 19CAR and secreted anti-IL 6 scFv antibody and IL-1RA (anti-CD 19/IL6/IL 1). Two days later, T cells were electroporated with Cas9 protein (Thermo scientific) along with a gRNA targeting the first exon of GM-CSF and optionally a gRNA targeting the TCR β chain constant region. The obtained anti-CD 19 CART cell is anti-CD 19/IL6/IL1TCR^-Or anti-CD 19/IL6/IL1TCR^-/GM-CSF^-. These cells were expanded and tested for CD3 expression by FACS analysis.

The expression of anti-CD 19CAR was analyzed by primary biotinylation of goat anti-mouse IgG-F (ab')2 fragments followed by secondary staining with streptavidin coupled to R-phycoerythrin. 81.7% anti-CD 19/IL6/IL1TCR^-The cells were CD45+/CD3-, and 78.9% anti-CD 19/IL6/IL1TCR^-/GM-CSF^-The cells were CD45+/CD 3-. The CD45+/CD 3-cell population was then analyzed for CD4 and anti-CD 19CAR expression. Approximately 10.7% of CD45+/CD3-aCD19-61 TCR-cells were anti-CD 19 CART cells, and 13.1% of CD45+/CD3-aCD19-61 TCR-/GM-CSF-cells were anti-CD 19 CART cells. Finally, CART cells were analyzed for CD4/CD8 expression. anti-CD 19/IL6/IL1TCR^-Of the cells, there were approximately 12.1% CD8+ T cells and 85.3% CD4+ T cells. anti-CD 19/IL6/IL1TCR^-/GM-CSF^-Among the cells, 13.3% of CD8+ T cells and 81.2% of CD4+ T cells.

anti-CD 19/IL6/IL1 TCR-cells and anti-CD 19/IL6/IL1TCR^-/GM-CSF^-Cells were cultured for 2 days with Nalm6-GFP cells at a 1:1E: T ratio. Supernatants were collected from both co-cultures and added to HEK-Blue IL-6 reporter cells (Invivogen) in the presence of 1ng/ml human IL-6 or HEK-Blue IL1R reporter cells in the presence of 5pg/ml human IL 1B. After overnight incubation, the supernatants of HEK-Blue IL-6 cells or HEK-Blue IL1R cells were collected and mixed with Quant-Blue substrate solutionAnd incubating together. SEAP production was quantified by measuring the absorbance of the conversion substrate Quant Blue (Invivogen) at a wavelength of 650nm by a spectrophotometer. As shown in FIG. 7A, from anti-CD 19/IL6/IL1TCR^-Cell and anti-CD 19/IL6/IL1TCR^-/GM-CSF^-Supernatants from both cells were able to inhibit IL6 signaling at dilutions above 0.1. As shown in FIG. 7B, TCR-cells from anti-CD 19/IL6/IL1 and anti-CD 19/IL6/IL1^-/GM-CSF^-Supernatants from both cells were able to inhibit IL1B signaling at dilutions above 0.1.

anti-CD 19/IL6/IL1TCR was then activated by PMA/Ionomycin^-And anti-CD 19/IL6/IL1TCR^-/GM-CSF^-Cells were tested for cytokine expression (IL2, GM-CSF, IFN γ, and TNFA). FIG. 7C shows that the percentage of T cells secreting IL2, IFN γ, and TNF α is similar in both cell populations, yet is similar to the anti-CD 19/IL6/IL1TCR^-Cell comparison in anti-CD 19/IL6/IL1TCR^-/GM-CSF^-There are much fewer T cells secreting GM-CSF in the cell population.

anti-CD 19/IL6/IL1TCR was evaluated^-Cell and anti-CD 19/IL6/IL1TCR^-/GM-CSF^-The ability of cells to kill CD19+ Nalm6 cells. After co-culture, the number of tumor cells remaining was analyzed by BD turcount beads and the percent cytotoxicity was plotted in fig. 7D. anti-CD 19/IL6/IL1TCR^-Cell and anti-CD 19/IL6/IL1TCR^-/GM-CSF^-The cells all showed cytotoxicity of more than 90% to Nalm6 cells.

Example 8: anti-BCMA CAR-T cells knocked out by GM-CSF and secreting IL6 blocker/IL-1 blocker exert potent cytotoxicity and IL6 and IL1B inhibition

Primary T cells (PPA research) from healthy donors were activated with anti-CD 3/CD28 beads (Thermo scientific). One day later, the V encoding (i) anti-BCMA CAR and (ii) anti-IL 6 scFv polypeptide (SEQ ID NO:14) with a sequence of SEQ ID NO:3 was used_HSequence and V of SEQ ID NO 4_L(ii) sequence, (iii) a lentiviral vector for IL1RA transduced T cells.

The anti-BCMA CAR contains, from N-terminus to C-terminus, a CD8 leader sequence, an anti-BCMA scFv fragment, a CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta domain. The sequences of the CD8 leader sequence, CD8 hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3 ζ are provided in example 7 above. The amino acid sequence of the anti-BCMA scFv is provided below (SEQ ID NO: 57):

DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS

the CD8 leader sequence preceded the anti-IL 6 scFv. The nucleotide sequence encoding the T2A peptide is located between the coding sequences of (i) and (ii), and the nucleotide sequence encoding the P2A peptide is located between the coding sequences of (ii) and (iii). There is a human growth hormone signal sequence between P2A and (iii). Two days later, T cells were electroporated with Cas9 protein (Thermo scientific) and a gRNA targeting the first exon of GM-CSF. These cells were expanded and tested for CD3 expression by FACS analysis and the CD3+ population was gated for further analysis. anti-BCMA CAR expression was analyzed by primary biotinylation of goat anti-mouse IgG-F (ab')2 fragments followed by secondary staining with streptavidin conjugated R-phycoerythrin. There were 98.9% CD3+ cells. The CD3+ cell population was then analyzed for CD4 and anti-BCMA CAR expression. There were approximately 5.78% of anti-BCMA CAR-T cells in the CD3+ cell population. Finally, CAR-T cells were analyzed for CD4/CD8 expression. There were approximately 23.8% CD8+ T cells and 73.6% CD4+ T cells.

anti-BCMA/IL 6/IL1GM-CSF^-Cells were cultured for 2 days at a 1:1E: T ratio to RPMI-8226 cells. Supernatants were collected from the co-cultures and added to HEK-Blue IL-6 reporter cells (Invivogen) in the presence of 5pg/ml human IL1B in the presence of 1ng/ml human IL-6 or HEK-Blue IL1R reporter cells. After overnight incubation, supernatants of HEK-Blue IL-6 cells or HEK-Blue IL1R cells were collected and incubated with Quant-Blue substrate solution. SEAP production was quantified by measuring the absorbance of the conversion substrate QuantBlue (Invivogen) at 650nm wavelength by spectrophotometer. As shown in FIG. 8A, from two anti-BCMA/IL 6/IL1GM-CSF^-The cell supernatant canIL6 signaling was inhibited at dilutions above 0.1. As shown in FIG. 8B, from anti-BCMA/IL 6/IL1GM-CSF^-The cell supernatant was able to inhibit IL1B signaling at dilutions above 0.1.

Cytokine expression (IL2, GM-CSF and IFN γ) was then tested against BCMA/IL6/IL1 cells with or without disrupted GM-CSF, and FIG. 8C shows that the percentage of T cells that can secrete IL2 and IFN γ was similar in both cell populations, while there were much fewer GM-CSF secreting T cells in GM-CSF knock-out cells compared to GM-CSF WT cells.

The ability of anti-BCMA/IL 6/IL1/GM-CSF KO cells to kill BCMA + RMPI-8226 cells was evaluated. After co-culture, the number of remaining tumor cells was analyzed by BD turcount beads, and the percentage of cytotoxicity was plotted in fig. 8D. anti-BCMA/IL 6/IL1/GM-CSF KO cells showed cytotoxicity of greater than 40% to RPMI-8226 cells.

Example 9: therapeutic Effect of anti-CD 19/IL6/IL1/GM-CSF KO or anti-CD 19/IL6/IL1CAR-T cells in human cancer patients

One human patient diagnosed with lymphoblastic leukemia (BCR/ABL1 fusion gene and ABL1 gene with T315I and E255K mutations) received two rounds of anti-CD 19/IL6/IL1/GM-CSF CAR-T cell therapy as described below. As shown in figure 9A, GM-CSF expression was significantly reduced in CAR-T cells compared to the wild-type counterpart.

First time of treatment

Human patients were first treated with chemotherapy to reduce tumor burden and then pre-treated with fludarabine/cyclophosphamide to deplete endogenous lymphocytes, thereby subjecting the patients to CAR-T cell transplantation. Following chemotherapy and pretreatment, total lymphocyte count (reflecting tumor burden) dropped to 0.03X10⁹And L. Thereafter, the patient received 1X10⁸anti-CD 19/IL6/IL1/GM-CSF KO CAR-T cells as disclosed herein (e.g., examples 7 and 8 above, except that the CAR-T cells used in this example were TCR positive). 46.8% of CD19+ cells were detected in the patient's peripheral blood 5 days after CAR-T cell infusion compared to 95% of CD19+ cells prior to chemotherapy. In CAR-T cell transfusionThe total number of CD19+ cells rapidly increased to about 12.4X10 day after injection⁹L, indicating only partial response to CAR-T treatment.

Due to high fever, tositumumab was injected into patients about 8 hours after CAR-T cell infusion — patients showed grade 2 to 3 CRS (fever, hypotension and hypoxia). Evidence of CRS occurred very early, i.e., about 4 hours after CAR-T cell infusion, suggesting a heavier tumor burden at the beginning of CAR-T treatment (1 st). Analysis of cytokine levels in patients by ELISA showed significantly reduced GM-CSF levels (fig. 9B), as well as moderate levels of IL1/IL1R blockers (fig. 9D). These results confirm the successful knock-out of the GM-CSF gene in CAR-T cells and the successful expression of IL1/IL1R blockers by CAR-T cells. The patient's maximum daily body temperature (Tmax, deg.C) is shown in FIG. 9H.

Quantification of CAR vector copies by qPCR indicated that CAR-T cells had limited expansion in vivo. Fig. 9E. This may be the reason for the rapid recurrence of CD19+ tumor cells in the patient. CAR-T cell infusion was followed by elevated levels of C-reactive protein (CRP) and peaked two days after infusion. Fig. 9F. High levels of IFN γ were also observed on day 2 post-infusion. The level of IL6 was also reduced (fig. 9C), probably due to a reduction in CAR-T and/or IFN γ activity.

Second treatment

The patient was then treated with very strong chemotherapy to reduce tumor burden, followed by a fludarabine/cyclophosphamide pretreatment to deplete lymphocytes. Following chemotherapy and pretreatment, the total lymphocyte count (reflecting tumor burden) drops to a level that is barely detectable by flow cytometry-based analysis. Thereafter, the patient received two consecutive doses of 0.3X10⁸(at D0) and 1X10⁸(at D1) anti-CD 19/IL6/IL1CAR-T cells (with wild-type GM-CSF gene). IL-2 is also administered to the patient one or more times during the course of therapy. B-cell hypoplasia was detected in peripheral blood at days 7 and 33 after CART infusion, indicating a complete response to CAR-T treatment.

Cytokine level analysis showed significant reduction of IL-6 in patients after T cell infusion (fig. 10A), high levels of IL1/IL1R blocker, GM-CSF and IFN γ during D5-D10 (fig. 10C, fig. 10D and fig. 10H, respectively).

The patient was diagnosed as infected after the lymph depletion pretreatment and before the T cell infusion. Potential infection may be the cause of high levels of IL6 after infusion. However, the level of IL6 dropped significantly over time and reached a minimum at the same time that secretion of the IL1/IL1R blocker reached the highest peak (fig. 10A and 10C). The IL1/IL1R blocker was co-expressed with the IL6/IL6R blocker in a ratio, the coding sequences of the two blockers being linked by a nucleotide sequence encoding a P2A peptide linker. The maximum daily body temperature (Tmax, deg.C) is shown in FIG. 10B.

Quantification of CAR vector copies by qPCR and analysis of CAR + T cells by flow cytometry showed that CAR-T cells were maximally expanded in vivo, resulting in complete eradication of CD19+ tumor cells after CART treatment. Fig. 10F and 10H. Fig. 10E shows CRP levels after T cell infusion.

From D0 to D21, patients did not receive tositumumab and were treated with ibuprofen, nasal cannula only. Patients only showed grade 1 to 2 CRS (fever and hypoxia). No symptoms of neurotoxicity were observed in the patients.

Taken together, these results indicate maximal CAR-T cell expansion, maximal cytokine secretion (IFN γ and GM-CSF) levels, but very low levels of IL6 during CRS peak, and overall little or negligible cytokine-related toxicity symptoms. CRS are ranked as shown in table 1 below.

Example 10: therapeutic Effect of anti-BCMA/IL 6/IL1/GM-CSF/TCR KO CAR-T cells in human refractory multiple myeloma patients

One refractory Multiple Myeloma (MM) patient was treated with chemotherapy to reduce tumor burden, followed by fludarabine/cyclophosphamide pretreatment to deplete lymphocytes. Thereafter, the patient received two consecutive doses of 2X10⁶(D0) And 3X10⁶(D1) An anti-BCMA CART cell disclosed herein that secretes IL6 and IL1 blockers and knocks out GM-CSF and TCR genes. The patient was also injected with human recombinant IL-2. FIG. 11A shows the efficiency of GM-CSF gene knock-out by CRISPR/Cas9 technology. Crispr/Cas9 gene editing of the TCR resulted in the production of about 80% CD3-T cells, of which CD8 is pairedT cells were gated to analyze GM-CSF secreting cells by intracellular cytokine staining. The population of GM-CSF knock-out cells contains less than 20% GM-CSF positive cells.

Prior to CAR-T cell infusion, patients had approximately 13.9% BCMA in peripheral blood⁺Plasma cells. BCMA in peripheral blood on day 15 post CAR-T infusion⁺Plasma cell levels dropped to 0.074%, indicating that the patient responded completely to CAR-T treatment. Furthermore, the abnormal level of IgA decreased from 38.6g/L before treatment to 1.25g/L on day 41 after CAR-T treatment, indicating that malignant plasma cells were eradicated. FIG. 11B.

Serum levels of various cytokines in patients are analyzed by routine procedures such as ELISA. The results show high levels of IFN γ secretion and IL1/IL1R blocking agent, but low levels of GM-CSF secretion. FIGS. 11F, 11I and 11J. However, the level of IL6 was very low at the peak time point of IL1/IL1R blocker secretion, its synthesis was co-expressed by the P2A linker in proportion to the IL6/IL6R blocker. Fig. 11C, 11F, and 11K. Quantification of CAR vector copies by qPCR and analysis of CAR + T cells by flow cytometry showed that CAR-T cells were maximally expanded in vivo, resulting in complete eradication of BCMA following CART treatment⁺A tumor cell. FIG. 11G. The number of CAR-T cells in peripheral blood after CAR-T infusion is shown in FIG. 11H.

From day 0 to day 15, the patients did not receive tositumumab and were treated with ibuprofen, nasal cannula only. Patients showed only grade 1 to 2 CRS (fever and mild hypoxia) as shown in table 1. No neurotoxicity was observed.

TABLE 1 CRS of patients treated with anti-BCMA CAR-T cells

Taken together, these results indicate that anti-BCMA CAR-T cells in patients with refractory multiple myeloma are maximally expanded, maximal cytokine secretion (IFN γ) levels, but IL6 levels are extremely low during CRS peaks (from D9 to D11), symptoms of cytokine release syndrome are overall small or negligible, and there is no neurotoxicity. Interestingly, when the concentration of IL6/IL6R blocker was gradually decreased after peak D11, the IL6 level briefly rose to very high levels at D13, which did not cause any fever or other toxicity.

Example 11: therapeutic Effect of anti-CD 19/IL6/IL1TCR knockout CAR-T cells with or without GM-CSF knockout in xenograft mice

6 to 8 week old NSG mice were injected intravenously with 1X10⁶A Nalm6 leukemia cell modified to stably express GFP. After 6 days, mice were injected intravenously with 2X10⁶TCR KO anti-CD 19CAR-T cells, which also express IL6/IL6R blockers and IL1/IL1R blockers with GM-CSF WT (indicated as "1" in the figure, n ═ 5) or KO (indicated as "2" in the figure, n ═ 6). Mice that did not receive CAR-T cells were included as controls (indicated as "CTRL" in the figure, n-4). After T cell infusion, mice were monitored for body weight, survival and number of GFP + Nalm6 leukemia cells and CD45+ CD3-T cells in blood by Trucount beads (BD Biosciences).

Body weight of treated mice was monitored before and after CAR-T cell treatment. Mice treated with anti-CD 19CAR-T cells with or without GM-CSF KO all showed weight gain over time, while control mice showed significant weight loss. Fig. 12A. anti-CD 19CAR-T cells with or without GM-CSF KO significantly prolonged survival and reduced levels of leukemic cells in treated mice compared to control mice. Fig. 12B and 12C. Finally, the number of T cells in mice treated with CAR-T cells decreased over time. Fig. 12D.

Overall, CAR-T cells effectively eradicated leukemic cells in mice, maintaining long-term survival. Furthermore, cytokine KO T cells in treated mice gradually decreased and did not convert to tumor-like cells, indicating the safety of the CAR-T cells disclosed herein.

Example 12: therapeutic Effect of anti-CD 19/IL6/IL1/GM-CSF/TCR KO CAR-T cells in human non-Hodgkin lymphoma patients

Human patients diagnosed with non-Hodgkin's lymphoma received treatment with anti-CD 19/IL6/IL1/TCR/GM-CSF KO CAR-T cells as disclosed herein below. A human patient is treated with chemotherapy to reduce tumor burden, followed by fludarabine/cyclophosphamide pretreatment to deplete endogenous lymphocytes, thereby subjecting the patient to CAR-T cell transplantation. Thereafter, the patient received 0.2X10⁸(on day 0, D0) and 0.3X10⁸(on day 1, D1) anti-CD 19/IL6/IL1CAR-T cells disclosed herein (GM-CSF and TCR genes knocked-out). As shown in FIG. 13A, most T cells had GM-CSF knocked out. The results show the gene editing efficiency of Crispr/Cas9 of GM-CSF KO (FIG. 13A). Recombinant IL2 was injected into patients during therapy. B-cell hypoplasia was detected by flow cytometry analysis at day 7 post CART infusion, indicating a complete response to CAR-T therapy.

Cytokine level analysis showed low IL-6 levels in patients after T cell infusion (fig. 13B and 13D) and slight increases in IL1/IL1R blocker, GM-CSF and IFNG (fig. 13E, 13F and 13C, respectively). Quantification of CAR vector copies by qPCR showed significant CAR-T cell expansion in vivo (fig. 13G), which resulted in complete eradication of CD19 following CAR-T therapy⁺A tumor cell. Fig. 13I shows CRP levels after T cell infusion. From day 0 to day 9, the patients did not receive tositumumab and showed no symptoms of fever, hypoxia or hypotension. Furthermore, no neurotoxicity was detected during the treatment.

Example 13: therapeutic Effect of anti-CD 19/IL6/IL1CAR-T cells in human acute lymphoblastic leukemia patients

Human patients diagnosed with acute lymphoblastic leukemia receive treatment with anti-CD 19/IL6/IL1CAR-T cells as disclosed herein below. A human patient is treated with chemotherapy to reduce tumor burden, followed by fludarabine/cyclophosphamide pretreatment to deplete endogenous lymphocytes, thereby subjecting the patient to CAR-T cell transplantation. Thereafter, the patient received 0.35x10 as disclosed herein⁸anti-CD 19/IL6/IL1CAR-T cells (with wild-type GM-CSF and TCR genes). Recombinant IL2 was injected into patients during therapy. On day 14 post CAR-T cell infusion, patients were diagnosed as minimal residual disease negative (MRD-), indicating complete response to CAR-T therapy.

Cytokine level analysis showed low IL-6 levels in patients after T cell infusion (FIG. 14A and FIG. 14C), high IL1/IL1R blockers, GM-CSF and IFNG levels (FIG. 14D, FIG. 14E and FIG. 14B, respectively). Quantification of CAR vector copies by qPCR revealed maximal CAR-T cell expansion in vivo (figure 14G), leading to complete eradication of CD19 following CAR-T therapy⁺A tumor cell. Fig. 14H shows CRP levels after T cell infusion. From day 0 to day 15, patients did not receive toslizumab and showed only grade 1 CRS (fever) (fig. 14F), with no symptoms of hypoxia or hypotension. Furthermore, no neurotoxicity was detected during the treatment.

Taken together, these results indicate maximal expansion of CAR-T cells, maximal cytokine secretion (IFNG) levels, but very low levels of IL6 during CRS peak, and overall little to negligible cytokine-related toxicity symptoms, and no neurotoxicity.

Example 14: therapeutic efficacy of anti-BCMA/IL 6/IL1CAR-T cells in human multiple myeloma patients

Multiple Myeloma (MM) patients were treated with chemotherapy to reduce tumor burden, followed by a fludarabine/cyclophosphamide pretreatment to deplete lymphocytes. The patient then receives a single dose of 4x10⁷(D0) anti-BCMA CART cells secreting IL6/IL1 blockers and having wild-type GM-CSF and TCR genes, while patients were also injected with human recombinant IL 2. Throughout the course of treatment, the patient was not receiving tollizumab.

Temperature monitoring (fig. 15A) indicated fever from day 1 to day 6, and increased levels of CRP, ferritin, and IFNG (fig. 15A to fig. 15D). However, from day 1 to day 6, IL6 levels remained low (<100pg/ml, fig. 15E), and only grade 1 CRS (fever) (no hypoxia and hypotension), and no neurotoxicity was observed. A significant increase in IL1RA was detected during treatment (fig. 15H), consistent with CART expansion (fig. 15G). Comparing the level of IL6 with IFNG and IL1RA levels (fig. 15F, 15H and 15I) indicates that IL6 secretion is initially inhibited during CAR-T cell expansion. However, IL6 increased dramatically on day 9 and remained at a high level from day 9 to day 23. Mild neurotoxicity was observed only during days 13 to 17, during which no fever, hypoxia or hypotension was observed. Cytokine analysis showed low levels of IFNG, IL1B, IL2, IL4, IL10, IL17A, TNFA and GM-CSF from day 13 to day 17, indicating that high levels of IL6 alone may lead to neurotoxicity (fig. 15D and 15J to 15P).

In summary, fever was observed only during days 1 to 6, and mild neurotoxicity was observed in patients only during days 13 to 17. No hypoxia or hypotension was observed during CART treatment. The above results indicate that patients experienced typical CRS from day 1 to day 6 with a significant reduction in IL6 blocker production by CAR-T cells. Thereafter, IL6 increased dramatically on day 9, and the presence of high levels of IL6 from day 13 to day 17 was likely due to down-regulation of the CAR-T cell-produced IL6 blocker, which resulted in mild neurotoxicity without fever, hypoxia or hypotension. IL 6-associated neurotoxicity further supports that IL6 is an important target not only for reducing CRS severity, but also for reducing neurotoxicity.

Other embodiments

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are within the claims.

Equivalents of

Although several embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, embodiments of the invention may be practiced otherwise than as specifically described and claimed. The presently disclosed embodiments relate to each individual feature, system, article, material, kit and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents, and patent applications disclosed herein are incorporated by reference to the subject matter to which each is cited, where the entire document may be included in some cases.

The indefinite articles "a" and "an" used in the specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used in the specification and claims should be understood to mean "one or two" of the elements so combined, i.e., the elements present in combination in some cases and present in isolation in other cases. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" elements so connected. In addition to elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, a reference to "a and/or B" when used in conjunction with open language such as "comprising" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than a); in yet another embodiment, to both a and B (optionally including other elements); and so on.

As used herein, in the specification and claims, "or" is understood to have the same meaning as "and/or" as defined above. For example, when items are separated in a list, "or" and/or "should be interpreted as inclusive, i.e., containing at least one of many elements or lists of elements, but also containing more than one, and optionally containing additional unlisted items. Only terms of the contrary, such as "only one" or "exactly one," or "consisting of … …" as used in the claims, will be expressly indicated as including exactly one of a number of elements or a list of elements. In general, the term "or" as used herein can only be interpreted to mean a specific alternative (i.e., "one or the other, rather than two") when preceded by an exclusive term such as "or," one, "" only one, "or" exactly one. "consisting essentially of … …" when used in a claim shall have its ordinary meaning as used in the art of patent law.

As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. The definition also allows that elements may optionally be present, whether related or unrelated to those specifically identified elements, other than the specifically identified elements indicated by the phrase "at least one" within the list of elements. Thus, as a non-limiting example, "at least one of a and B" (or, equivalently, "at least one of a or B," or, equivalently "at least one of a and/or B) may refer in one embodiment to at least one (optionally including more than one) a, with no B present (and optionally including elements other than B); in another embodiment may refer to at least one (optionally including more than one) B, with no a present (and optionally including elements other than a); in yet another embodiment, may refer to at least one (optionally including more than one) a and at least one (optionally including more than one) B (and optionally including other elements); and so on.

It will also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Sequence listing

<110> CELLEDT LLC

<120> modified immune cells co-expressing chimeric antigen receptor and IL-6 antagonist for reducing toxicity and their use in adoptive cell therapy

<130> 103168-643327-70001WO00

<150> 62/789,311

<151> 2019-01-07

<150> 62/855,250

<151> 2019-05-31

<150> 62/928,720

<151> 2019-10-31

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

65 70 75 80

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

85 90 95

Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 10

Ala 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 Gln Ala Ser Gln Ser Ile Asn Asn Glu

20 25 30

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

35 40 45

Tyr Arg Ala Ser Thr Leu Ala 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Arg Asn

85 90 95

Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 11

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<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 Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Met Arg Asn Lys Asn Tyr Gln Tyr Gly Thr Tyr Tyr Ala Glu

50 55 60

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Ser Tyr Tyr Gly Phe Thr Ser Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<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 Gln Ala Ser Gln Asp Ile Gly Ile Ser

20 25 30

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

35 40 45

Tyr Asn Ala Asn Asn Leu Ala Asp 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 Leu Gln His Asn Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Gly Ala Ser Ser Leu Glu 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 Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

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

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Arg Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp Asn

165 170 175

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

180 185 190

Ser Arg Asp Asn Ala Glu Asn Ser Leu Phe Leu Gln Met Asn Gly Leu

195 200 205

Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Gly Arg Asp Ser

210 215 220

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

225 230 235

<210> 14

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 14

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile Ser Val Ser Tyr Met

20 25 30

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

35 40 45

Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

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

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Pro Phe Ala Met Ser Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly

165 170 175

Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

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

210 215 220

Tyr Ala Leu Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235 240

<210> 15

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 15

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

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

115 120 125

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

130 135 140

Ala Ser Gly Phe Thr Phe Ser Ser Phe Ala Met Ser Trp Phe Arg Gln

145 150 155 160

Ser Pro Glu Lys Arg Leu Glu Trp Val Ala Glu Ile Ser Ser Gly Gly

165 170 175

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

180 185 190

Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Glu Met Ser Ser Leu Arg

195 200 205

Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Leu Trp Gly Tyr

210 215 220

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

225 230 235 240

<210> 16

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 16

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

1 5 10 15

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

20 25 30

His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Ser Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Arg Ala

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr

130 135 140

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

145 150 155 160

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn Trp Tyr Gln

165 170 175

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

180 185 190

Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr

210 215 220

Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly

225 230 235 240

Thr Lys Val Glu Ile Lys

245

<210> 17

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 17

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Phe Gly Tyr Pro Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Trp Leu Asn Pro Tyr Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Thr Thr Leu Ile Ser Val Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 18

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Met Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 19

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 19

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 His

20 25 30

Trp Met His Trp Leu Arg Gln Val Pro Gly Lys Gly Pro Val Trp Val

35 40 45

Ser Arg Ile Asn Gly Ala Gly Thr Ser Ile Thr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Leu Tyr Phe Cys

85 90 95

Ala Arg Ala Asn Ser Val Trp Phe Arg Gly Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Pro Val Thr Val Ser Ser

115 120

<210> 20

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Glu Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Lys Trp Pro Asp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 21

Gln 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 Ser Tyr

20 25 30

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

35 40 45

Ser Gly Ile Glu Asn Lys Tyr Ala Gly Gly Ala Thr Tyr Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Arg Gly Phe Gly Thr Asp Phe Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 22

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

1 5 10 15

Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Ile Gly Lys Lys Tyr Ala

20 25 30

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

35 40 45

Lys Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser

50 55 60

Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Ser Ala Trp Gly Asp Lys Gly Met Val Phe Gly

85 90 95

Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105

<210> 23

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 23

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Phe Gly Tyr Pro Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Trp Leu Asn Pro Tyr Ser Gly Asp Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Thr Arg Thr Thr Leu Ile Ser Val Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

115 120 125

Gly Gly Ser Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

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

145 150 155 160

Gln Asn Ile Arg Asn Ile Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys

165 170 175

Ala Pro Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Ser Tyr Ser Met Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 24

<211> 243

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 24

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 His

20 25 30

Trp Met His Trp Leu Arg Gln Val Pro Gly Lys Gly Pro Val Trp Val

35 40 45

Ser Arg Ile Asn Gly Ala Gly Thr Ser Ile Thr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Leu Tyr Phe Cys

85 90 95

Ala Arg Ala Asn Ser Val Trp Phe Arg Gly Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Pro Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ser Gln Ser Val Ser Thr Asn Leu Ala Trp Tyr Gln Gln Lys Leu

165 170 175

Gly Gln Gly Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr

180 185 190

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

195 200 205

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys

210 215 220

Gln Gln Tyr Asp Lys Trp Pro Asp Thr Phe Gly Gln Gly Thr Lys Leu

225 230 235 240

Glu Ile Lys

<210> 25

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 25

Gln 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 Ser Tyr

20 25 30

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

35 40 45

Ser Gly Ile Glu Asn Lys Tyr Ala Gly Gly Ala Thr Tyr Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

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

85 90 95

Tyr Cys Ala Arg Gly Phe Gly Thr Asp Phe Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

115 120 125

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

130 135 140

Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Ile Gly

145 150 155 160

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

165 170 175

Leu Val Ile Tyr Lys Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser

180 185 190

Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln

195 200 205

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Gly Asp Lys Gly

210 215 220

Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

225 230 235

<210> 26

<211> 487

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 26

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile Ser Val Ser Tyr Met

20 25 30

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

35 40 45

Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

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

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Pro Phe Ala Met Ser Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly

165 170 175

Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

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

210 215 220

Tyr Ala Leu Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235 240

Gly Ser Gly Gly Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu

245 250 255

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Phe Gly

260 265 270

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

275 280 285

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

290 295 300

Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr

305 310 315 320

Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

325 330 335

Thr Ala Val Tyr Tyr Cys Thr Arg Thr Thr Leu Ile Ser Val Tyr Phe

340 345 350

Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser

355 360 365

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Gln Met

370 375 380

Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr

385 390 395 400

Ile Ala Cys Arg Ala Ser Gln Asn Ile Arg Asn Ile Leu Asn Trp Tyr

405 410 415

Gln Gln Arg Pro Gly Lys Ala Pro Gln Leu Leu Ile Tyr Ala Ala Ser

420 425 430

Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

435 440 445

Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala

450 455 460

Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Met Pro Arg Thr Phe Gly Gly

465 470 475 480

Gly Thr Lys Leu Glu Ile Lys

485

<210> 27

<211> 489

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 27

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile Ser Val Ser Tyr Met

20 25 30

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

35 40 45

Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

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

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Pro Phe Ala Met Ser Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly

165 170 175

Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

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

210 215 220

Tyr Ala Leu Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235 240

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

245 250 255

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

260 265 270

Phe Thr Phe Ser Arg His Trp Met His Trp Leu Arg Gln Val Pro Gly

275 280 285

Lys Gly Pro Val Trp Val Ser Arg Ile Asn Gly Ala Gly Thr Ser Ile

290 295 300

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

305 310 315 320

Ala Asn Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Asp Asp

325 330 335

Thr Ala Leu Tyr Phe Cys Ala Arg Ala Asn Ser Val Trp Phe Arg Gly

340 345 350

Leu Phe Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser Gly

355 360 365

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Glu Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

450 455 460

Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Lys Trp Pro Asp Thr Phe

465 470 475 480

Gly Gln Gly Thr Lys Leu Glu Ile Lys

485

<210> 28

<211> 484

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 28

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile Ser Val Ser Tyr Met

20 25 30

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

35 40 45

Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

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

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

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

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Pro Phe Ala Met Ser Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly

165 170 175

Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

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

210 215 220

Tyr Ala Leu Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235 240

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

245 250 255

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

260 265 270

Phe Thr Phe Ser Ser Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly

275 280 285

Lys Gly Leu Glu Trp Val Ser Gly Ile Glu Asn Lys Tyr Ala Gly Gly

290 295 300

Ala Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

305 310 315 320

Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

325 330 335

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Phe Gly Thr Asp Phe

340 345 350

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly

355 360 365

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Ile Glu Leu Thr Gln

370 375 380

Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys

385 390 395 400

Ser Gly Asp Ser Ile Gly Lys Lys Tyr Ala Tyr Trp Tyr Gln Gln Lys

405 410 415

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

420 425 430

Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu

435 440 445

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

450 455 460

Ala Trp Gly Asp Lys Gly Met Val Phe Gly Gly Gly Thr Lys Leu Thr

465 470 475 480

Val Leu Gly Gln

<210> 29

<211> 159

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 29

Met Ala Leu Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys

1 5 10 15

Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu

20 25 30

Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn

35 40 45

Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe

50 55 60

Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly

65 70 75 80

Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser

85 90 95

Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser

100 105 110

Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu

115 120 125

Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro

130 135 140

Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu

145 150 155

<210> 30

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 30

gctgcagagc ctgctgctct gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 31

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 31

ggagcatgtg aatgccatcc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 32

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 32

gcatgtgaat gccatccagg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 33

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 33

gagacgccgg gcctcctgga gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 34

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 34

gatggcattc acatgctccc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 35

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 35

gctcccaggg ctgcgtgctg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 36

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 36

gcgtgctggg gctgggcgag gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 37

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 37

gctggggctg ggcgagcggg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 38

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 38

gacttagtgc aatgcaagac gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 39

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 39

gatttacaga tgattttgaa gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 40

<211> 99

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 40

aagaaaacac agctacaacg ttttagagct agaaatagca agttaaaata aggctagtcc 60

gttatcaact tgaaaaagtg gcaccgagtc ggtgctttt 99

<210> 41

<211> 99

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 41

caactggagc atttactgcg ttttagagct agaaatagca agttaaaata aggctagtcc 60

gttatcaact tgaaaaagtg gcaccgagtc ggtgctttt 99

<210> 42

<211> 99

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 42

tctttgtaga acttgaagtg ttttagagct agaaatagca agttaaaata aggctagtcc 60

gttatcaact tgaaaaagtg gcaccgagtc ggtgctttt 99

<210> 43

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 43

gagcactgaa agcatgatcc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 44

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 44

ggacgtggag ctggccgagg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 45

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 45

gaggcgctcc ccaagaagac gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 46

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 46

gggggcccca gggctccagg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 47

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 47

gctgaggaac aagcaccgcc gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 48

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 48

ggcgcctgcc acgatcagga gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 49

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 49

gtgcagcagg cagaagagcg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 100

<210> 50

<211> 99

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of oligonucleotide

<400> 50

ggagtgatcg gcccccagag ttttagagct agaaatagca agttaaaata aggctagtcc 60

gttatcaact tgaaaaagtg gcaccgagtc ggtgctttt 99

<210> 51

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 51

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 52

<211> 242

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 52

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

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

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 53

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 53

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 54

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 54

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 55

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 55

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 56

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 56

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 57

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 57

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

1 5 10 15

Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu

20 25 30

Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

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

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala

195 200 205

Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe

210 215 220

Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 58

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 58

Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp

1 5 10 15

Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala

20 25 30

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

35 40 45

Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly Lys

50 55 60

Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu

65 70 75 80

Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys

85 90 95

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

100 105 110

Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala Asp

115 120 125

Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val Thr

130 135 140

Lys Phe Tyr Phe Gln Glu Asp Glu

145 150

<210> 59

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 59

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala

20 25

<210> 60

<211> 178

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 60

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Arg Pro Ser Gly Arg Lys

20 25 30

Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr

35 40 45

Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro

50 55 60

Asn Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His

65 70 75 80

Ala Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val

85 90 95

Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr

100 105 110

Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg

115 120 125

Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly

130 135 140

Trp Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr

145 150 155 160

Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu

165 170 175

Asp Glu