Humanized and affinity matured anti-CEACAM 1 antibodies
阅读说明：本技术 人源化和亲和力成熟的抗ceacam1抗体 (Humanized and affinity matured anti-CEACAM 1 antibodies ) 是由 R·S·布卢姆伯格 Y-h·黄 A·甘地 M·伯塔诺利 C·尹 R·G·E·霍尔加特 A 于 2019-12-09 设计创作，主要内容包括：本文提供了可用于结合和抑制癌胚抗原相关细胞粘附分子1(CEACAM1)的重组抗体及其抗原结合片段。还提供了使用所公开的CEACAM1抗体及其抗原结合片段降低T细胞耐受性和治疗癌症的方法。(Provided herein are recombinant antibodies and antigen-binding fragments thereof that are useful for binding to and inhibiting carcinoembryonic antigen-associated cell adhesion molecule 1(CEACAM 1). Also provided are methods of reducing T cell tolerance and treating cancer using the disclosed CEACAM1 antibodies and antigen binding fragments thereof.)
1. An antibody or antigen-binding fragment thereof that binds CEACAM1, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;
wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR 3; and is
Wherein:
the sequence of CDR1H comprises sequence X1HX2X3S(SEQ ID NO:1)；
Wherein X1 is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3 is an amino acid with a hydrophobic side chain including I or M;
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises the sequence HX4X5DYX6PX7WFAX8(SEQ ID NO:3)；
Wherein X4 is D, G or P;
wherein X5Is F or P;
wherein X6Is D or F;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F;
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L comprises the sequence QQX9X10X11X12PX13T(SEQ ID NO:6)；
Wherein X9Is W or N;
wherein X10Is S or T;
wherein X11Is a or an amino acid with a neutral hydrophilic side chain including S, N and T;
wherein X12Is L, F or N; and is
Wherein X13Is P or F.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein
The sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S(SEQ ID NO:43)；
Wherein X is any amino acid;
Wherein X1 is A, D, N or S;
wherein X2 is A or G; and is
Wherein X3 is an amino acid with a hydrophobic side chain including I or M; and is
The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8(SEQ ID NO:44)；
Wherein X4 is D, G or P;
wherein X5Is F or P;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein
The sequence of CDR1H comprises sequence X1HX2X3S(SEQ ID NO:1)；
Wherein X1 is A, D, N or S;
wherein X2 is A or G; and is
Wherein X3 is an amino acid with a hydrophobic side chain including I or M;
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8(SEQ ID NO:7)；
Wherein X of CDR3H4Is D, G or P;
wherein X of CDR3H5Is F or P; and is
Wherein X of CDR3H8Is L, H or F;
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L comprises the sequence QQX9SSX12PX13T(SEQ ID NO:8)；
Wherein X9Is W or N;
wherein X12Is L, F or N; and is
Wherein X13Is P or F.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein
The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO: 9);
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
The sequence of CDR3H comprises sequence HDFDYFPYWFAH (SEQ ID NO: 10);
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L includes sequence QQWSSNPPT (SEQ ID NO: 11).
5. The antibody or antigen-binding fragment thereof of claim 1, wherein
The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO: 9);
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises sequence HDFDYFPYWFAH (SEQ ID NO: 10);
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L includes sequence QQWTSNPPT (SEQ ID NO: 12).
6. An antibody or antigen-binding fragment thereof that binds CEACAM1, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13; and is
Wherein the sequence of the light chain variable region comprises a sequence that is at least 90% identical to a light chain variable region amino acid sequence selected from the group consisting of seq id nos:
SEQ ID NO:14、
SEQ ID NO 15 and
SEQ ID NO:16。
7. the antibody or antigen-binding fragment thereof according to claim 6,
Wherein the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13; and is
Wherein the sequence of the light chain variable region comprises a sequence that is at least 95% identical to a light chain variable region amino acid sequence selected from the group consisting of seq id nos:
SEQ ID NO:14、
SEQ ID NO 15 and
SEQ ID NO:16。
8. the antibody or antigen-binding fragment thereof according to claim 7,
wherein the sequence of the heavy chain variable region comprises SEQ ID NO 13; and is
Wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of seq id no:
SEQ ID NO:14、
SEQ ID NO 15 and
SEQ ID NO:16。
9. the antibody or antigen-binding fragment thereof according to claim 8,
wherein the sequence of the heavy chain variable region comprises SEQ ID NO 13; and is
Wherein the sequence of the light chain variable region comprises SEQ ID NO 14.
10. The antibody or antigen-binding fragment thereof according to claim 8,
wherein the sequence of the heavy chain variable region comprises SEQ ID NO 13; and is
Wherein the sequence of the light chain variable region comprises SEQ ID NO 15.
11. An antibody or antigen-binding fragment thereof that binds CEACAM1, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13;
Wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14;
wherein the sequence of the heavy variable chain comprises the sequence GXXXXXXX1HX2X3S(SEQ ID NO:43)；
Wherein X is any amino acid;
wherein X1 is A, D, N or S;
wherein X2 is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M; and is
Wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8(SEQ ID NO:44)；
Wherein X4 is D, G or P;
wherein X5Is F or P;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F.
12. An antibody or antigen-binding fragment thereof that binds CEACAM1, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13;
wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14;
wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR 3; and is
Wherein:
the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO 13,
the sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO. 13,
The sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO. 14,
the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO 14 and
the sequence of CDR3L includes residues S91 and S92 of SEQ ID NO. 14.
13. The antibody or antigen-binding fragment thereof of claim 12;
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13;
wherein the sequence of the light chain variable region comprises a sequence that is at least 90% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14.
14. The antibody or antigen-binding fragment thereof of claim 13;
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13;
wherein the sequence of the light chain variable region comprises a sequence that is at least 95% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14.
15. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment is a chimeric, CDR-grafted, or humanized antibody or antigen-binding fragment thereof.
16. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment is a multispecific or bispecific antibody or antigen-binding fragment thereof.
17. The antibody or antigen-binding fragment of claim 16, wherein the antibody or antigen-binding fragment is a bispecific antibody comprising a complementary region that binds PD-1 or PD-L1.
18. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment is an scFv, Fv, Fab ', Fab, F (ab')2Or a diabody.
19. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment has isotype IgG 4.
20. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an S241P substitution in the constant region of the heavy chain.
21. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is deglycosylated.
22. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment lacks a C-terminal lysine in the heavy chain.
23. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is conjugated to one or more of a cytotoxin, a fluorescent label, and an imaging agent.
24. An antibody or antigen-binding fragment that binds to the same epitope on CEACAM1 as the antibody or antigen-binding fragment of claim 9.
25. An antibody or antigen-binding fragment thereof that binds to the IgV-like N domain of CEACAM1, wherein the antibody or antigen-binding fragment binds to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID No. 17.
26. The antibody or antigen binding fragment of claim 25, wherein the epitope further comprises residue Q44 of SEQ ID No. 17.
27. The antibody or antigen binding fragment of claim 25, wherein the epitope further comprises one or more residues selected from the group consisting of residues S32, Q44, a49, I91, L95, and V96 of SEQ ID No. 17.
28. The antibody or antigen-binding fragment of any one of claims 25-27, wherein the antibody or antigen-binding fragment binds to the IgV-like N domain of CEACAM 1.
29. The antibody or antigen-binding fragment of any one of claims 25-27, wherein the antibody or antigen-binding fragment does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM 8.
30. The antibody or antigen-binding fragment of any one of claims 25-27, wherein the antibody or antigen-binding fragment binds at least in part to the binding site of TIM3 on CEACAM 1.
31. The antibody or antigen-binding fragment of any one of claims 25-27, wherein the antibody or antigen-binding fragment at least partially binds to the binding site of CEACAM1 on CEACAM1 during homodimerization.
32. An antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment binds to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, N42, Q89, and N97 of SEQ ID NO: 17.
33. An antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment binds to an epitope comprising one or more residues selected from the group consisting of residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and N97 of SEQ ID NO: 17.
34. The antibody or antigen binding fragment of claim 33, wherein the epitope further comprises residues F29, S32, D40, a49, T56, I91, L95, and E99 of SEQ ID NO 17.
35. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of the preceding claims.
36. A vector comprising the nucleic acid of claim 35.
37. A cell comprising the vector of claim 36.
38. A cell expressing the antibody or antigen-binding fragment of any one of claims 1 to 34.
39. A T cell having a chimeric antigen receptor comprising the CDRs of the antibody or antigen-binding fragment of any one of claims 1 to 34.
40. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 34, and a pharmaceutically acceptable excipient.
41. A method of inhibiting CEACAM1 binding to a member of the CEACAM family, the method comprising contacting CEACAM1 with the antibody or antigen-binding fragment of any one of claims 1 to 34.
42. The method of claim 41, wherein said member of the CEACAM family is selected from the group consisting of CEACAM3, CEACAM5, CEACAM6 and CEACAM 8.
43. The method of claim 41, wherein said member of the CEACAM family is CEACAM 1.
44. A method of inhibiting CEACAM1 binding to a member of the TIM family, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.
45. The method according to claim 44, wherein said member of the TIM family is TIM 3.
46. A method of inhibiting CEACAM1 binding to a bacterial adhesin, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.
47. The method of claim 46, wherein said bacterial adhesin is the helicobacter pylori adhesin HopQ, Neisseria turbidimetric protein (Opa), Neisseria meningitidis Opa, Haemophilus influenzae Outer Membrane Protein (OMP) P1, Haemophilus Egypti OMP P1, or Moraxella Opa-like protein (Olpa).
48. A method of inhibiting CEACAM1 binding to candida albicans, the method comprising contacting CEACAM1 with an antibody or antigen binding fragment of any one of claims 1 to 34.
49. A method of inhibiting CEACAM1 binding to influenza virus, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.
50. The method of claim 49, wherein the influenza virus is H5N 1.
51. A method of reducing colonization of mammalian epithelium by bacteria expressing a bacterial adhesin, the method comprising contacting CEACAM1 with the antibody or antigen-binding fragment of any one of claims 1 to 34.
52. The method of claim 51, wherein said bacterial adhesin is the helicobacter pylori adhesin HopQ, Neisseria turbidimetric protein (Opa), Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus Egypti OMP P1, or Moraxella Olpa.
53. A method of reducing colonization of mammalian epithelium by candida albicans, the method comprising contacting CEACAM1 with the antibody or antigen binding fragment of any one of claims 1 to 34.
54. A method of reducing replication of influenza virus, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.
55. A method of reducing the release of pro-inflammatory cytokines or chemokines associated with influenza virus infection, the method comprising contacting a cell population comprising epithelial cells with an antibody or antigen binding fragment according to any one of claims 1 to 34.
56. The method of claim 54 or 55, wherein the influenza virus is H5N 1.
57. A method of reducing T cell tolerance, the method comprising contacting a population of cells comprising T cells with the antibody or antigen-binding fragment of any one of claims 1 to 34.
58. A method of enhancing T cell expansion, the method comprising contacting a population of cells comprising T cells with the antibody or antigen-binding fragment of any one of claims 1 to 34.
59. A method of reducing T cell tolerance in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
60. A method of enhancing T cell expansion in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
61. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
62. The method of claim 61, wherein the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cancer, prostate cancer, breast cancer, bladder cancer, or gastric cancer.
63. A method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
64. A method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
65. A method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
66. A method of reducing the sternness of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
67. A method of reducing colonization of the epithelium of a subject by a bacterial adhesin-expressing bacterium in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
68. The method of claim 67, wherein said bacterial adhesin is the helicobacter pylori adhesin HopQ, Neisseria turbidimetric protein (Opa), Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus egypus OMP P1, or Moraxella Olpa.
69. A method of reducing colonization of the epithelium of a subject by candida albicans in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen binding fragment of any one of claims 1 to 34.
70. A method of reducing replication of influenza virus in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
71. A method of reducing the release of pro-inflammatory cytokines or chemokines associated with influenza virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 34.
72. The method of claim 70 or 71, wherein the influenza virus is H5N 1.
73. A method of reducing filarial invasion of the lymphatic system of a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
74. The method of claim 73, wherein the filaria is Wucheria bambusae.
75. A method of reducing invasion of the lymphatic system of a subject by cancer cells in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.
76. The method of any one of claims 59 to 66, further comprising administering a checkpoint inhibitor.
77. The method of claim 76, wherein the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor.
78. The method of any one of claims 59 to 66, further comprising administering one or more of an inhibitor of LAG3, TIGIT, LAP, Podoplanin, protein C receptor, ICOS, GITR, CD226 and/or CD 160.
79. The method according to any one of claims 59 to 66, further comprising administering a TIM-3 inhibitor.
80. The method of any one of claims 76-79, wherein the additional inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment.
81. The method of any one of claims 76-79, wherein the additional inhibitor is administered alone or as a mixture with the antibody or antigen-binding fragment.
82. A method of treating a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34, wherein the subject has acquired resistance to therapy with checkpoint inhibitor therapy.
83. The method of claim 82, wherein the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
Technical Field
The present invention relates generally to the fields of molecular biology and medicine. More specifically, the invention provides monoclonal antibodies and antigen-binding fragments that bind CEACAM1 and therapeutic compositions thereof, as well as methods of using such antibodies, including inhibiting homotropic and heterotropic interactions with CEACAM1, and methods for treating cancer and infectious diseases.
Statement regarding federally sponsored research or development
The invention was made with government support according to NIH DK51362 awarded by the national institutes of health. The government has certain rights in this invention.
Background
Carcinoembryonic antigen-associated cell adhesion molecule 1(CEACAM1) is a member of the carcinoembryonic antigen (CEA) family of immunoglobulin (Ig) -like transmembrane glycoproteins. Members of the CEACAM family are involved in intercellular recognition and regulate cellular processes, ranging from the formation of tissue structures and neovascularization to the modulation of insulin homeostasis and T cell proliferation.
A variety of cellular activities have been attributed to CEACAM1 protein, including roles in differentiation and arrangement of tissue three-dimensional structures, angiogenesis, apoptosis, tumor suppression, metastasis, and modulation of innate and adaptive immune responses. In addition, several cell types express CEACAM1, including tumor cells, T cells, Natural Killer (NK) cells, and certain macrophages.
For example, high CEACAM1 expression occurs in a variety of cancers, such as melanoma, colorectal, gastric, pancreatic, bladder, and thyroid cancers, and is associated with poor tumor progression, metastasis, and poor clinical prognosis. For example, non-small cell lung cancer (NSCLC) with high CEACAM1 expression exhibits high microvascular density, distant metastasis and shorter median overall survival and progression-free survival. CEACAM1 expression was also closely associated with distant metastasis of pancreatic cancer. CEACAM1 expression on tumors promoted CEACAM1 mediated inhibition of T and NK cells. Thus, inhibition of CEACAM1 activity can inhibit tumor cell metastasis and the formation of cancer stem cell niches.
CEACAM1 is also expressed in certain immune system cells and plays a role in immunosuppression and immune cell depletion. For example, high CEACAM1 expression on tumor-infiltrating lymphocytes (TILs) and other tumor-infiltrating immune cells from gastric, lung, melanoma, colorectal, and glioma is associated with poor prognosis. On T cells, CEACAM1 expression was largely excluded from resting (naive) T cells, while proteins were expressed at high levels on activated T cells. CEACAM1-L is the major isoform expressed in most T cells and acts as an inhibitory receptor that down-regulates T cell activation and inhibits T cell function. Thus, inhibition of T cells by CEACAM1 restored T cell activity and increased anti-tumor responses.
CEACAM1 was also expressed on NK cells, lymphocytes involved in innate immunity, which were involved in early control of viral infection and immune surveillance of tumors. When NK cells encounter cells expressing the major histocompatibility complex class I (MHC), inhibitory signals through receptor-ligand interactions prevent immune responses against these cells. However, when encountering cells in which MHC class I is down-regulated, such as in virally infected cells or cancer cells, NK cells are activated due to a lack of inhibitory signals, which makes "diseased" cells susceptible to NK cell-mediated killing. CEACAM1: CEACAM1 interaction results in inhibition of NK-mediated killing, independent of MHC class I expression, when CEACAM1 is present on the surface of NK and melanoma cells. Thus, disruption of this homophilic CEACAM1 interaction may be beneficial in restoring NK-mediated immune responses.
CEACAM1 expression on macrophage subpopulations was also associated with fibrosis in the tumor microenvironment. CEACAM1 also regulates other stromal cells in the tumor microenvironment, such as the vascular endothelium. Thus, inhibition of CEACAM1 interaction with its binding partner may also inhibit fibrosis and angiogenesis.
CEACAM1 also mediates intercellular adhesion by the extracellular portion of CEACAM1 containing IgV-like N domains involved in homophilic (CEACAM1: CEACAM1) and heterophilic interactions (e.g., with CEA, CEACAM5, CEACAM8, 3(TIM-3) proteins containing T-cell immunoglobulin and mucin domains, Helicobacter pylori (Helicobacter pylori) adhesin HopQ, Neisseria gonorrhoeae/meniginitis) cloudy protein (OPA), Moraxella (Moraxella sp.) Opa-like protein Olpa, Haemophilus influenzae (OMP) P1, Haemophilus aegyptius (Haemophilus aegyptius) OMP 1, Candida albicans such as Candida 1). TIM-3 was identified as a Th 1-specific cell surface protein that is expressed on a subset of activated T cells, dendritic cells and macrophages, as well as NK cells. TIM-3 is an activation-induced inhibitory molecule that is involved in tolerance and has been shown to induce T cell depletion in chronic viral infections and cancer. CEACAM1 is also expressed on activated T cells, has been shown to interact with TIM-3, and this interaction is important for TIM-3 mediated T cell inhibition.
As mentioned above, CEACAM1 also acts as a cellular receptor on the apical membrane of mucosal cells for various gram-negative bacterial pathogens associated with human mucosa and with fungal pathogens such as candida albicans. For example, neisseria gonorrhoeae, neisseria meningitidis, Moraxella catarrhalis (Moraxella catarrhalis), haemophilus influenzae, haemophilus aegypti and pathogenic Escherichia coli (Escherichia coli) strains have well characterized CEACAM1 binding adhesins. Engagement of CEACAM1 with bacterial adhesins triggers bacterial endocytosis into epithelial cells and microbial endocytosis transport through the intact epithelial layer, thus allowing the microbes to utilize CEACAM1 during mucosal colonization. In addition, CEACAM1 is associated with infection by influenza virus H5N1 and daughter nematodes such as wuchereria bancronii (Wucheria bancrofti).
Disclosure of Invention
Provided herein are antibodies and antigen-binding fragments thereof that bind CEACAM1 and block the interaction of CEACAM1 with one or more binding partners. Therapeutic compositions of such antibodies and antigen-binding fragments thereof are also provided, as are methods of using these antibodies. By blocking the interaction of CEACAM1 with one or more binding partners, the antibodies and antigen-binding fragments thereof can be used to reduce, inhibit and/or reverse T cell tolerance and/or to enhance T cell expansion. The CEACAM1 antibodies and antigen-binding fragments thereof may also be used to treat cancer, reduce tumor growth, reduce tumor metastasis, and/or reduce the sternness of cancer in a subject in need thereof. CEACAM1 antibodies and antigen binding fragments thereof may also be used to treat patients resistant to checkpoint therapy. Also provided are methods of using CEACAM1 antibodies and antigen-binding fragments thereof to reduce colonization of mammalian epithelium by bacteria expressing bacterial adhesins or candida albicans or to reduce replication of influenza virus or release of proinflammatory cytokines or chemokines associated with influenza virus infection.
In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
the CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X1HX2X3S (SEQ ID NO: 1);
wherein X1 is A, D, N or S;
wherein X2 is A or G; and is
Wherein X3 is an amino acid with a hydrophobic side chain including I or M;
the CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the CDR3 sequence (CDR3H) of the heavy chain variable region comprises the sequence HX4X5DYX6PX7WFAX8(SEQ ID NO:3)；
Wherein X4Is D, G or P;
wherein X5Is F or P;
wherein X6Is D or F;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F;
the CDR1 sequence (CDR1L) of the light chain variable region comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQX9X10X11X12PX13T(SEQ ID NO:6)；
Wherein X9Is W or N;
wherein X10Is S or T;
wherein X11Is a or an amino acid with a neutral hydrophilic side chain including S, N and T;
Wherein X12Is L, F or N; and is
Wherein X13Is P or F.
In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S(SEQ ID NO:43)；
Wherein X is any amino acid;
wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M; and is
The CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8(SEQ ID NO:44)；
Wherein X4Is D, G or P;
wherein X5Is F or P;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F;
the CDR1 sequence (CDR1L) of the light chain variable region comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQX9X10X11X12PX13T(SEQ ID NO:6)；
Wherein X9Is W or N;
wherein X10Is S or T;
wherein X11Is a or an amino acid with a neutral hydrophilic side chain including S, N and T;
Wherein X12Is L, F or N; and is
Wherein X13Is P or F.
In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
the sequence of CDR1H comprises sequence X1HX2X3S(SEQ ID NO:1)；
Wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M;
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8(SEQ ID NO:7)；
Wherein X4Is D, G or P;
wherein X5Is F or P; and is
Wherein X8Is L, H or F;
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L comprises the sequence QQX9SSX12PX13T(SEQ ID NO:8)；
Wherein X9Is W or N;
wherein X12Is L, F or N; and is
Wherein X13Is P or F.
In one embodiment, the invention relates to an antibody or antigen binding fragment thereof that binds CEACAM1, wherein the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein
The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO: 9);
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises sequence HDFDYFPYWFAH (SEQ ID NO: 10);
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L includes sequence QQWSSNPPT (SEQ ID NO: 11).
In one embodiment, the invention relates to an antibody or antigen binding fragment thereof that binds CEACAM1, wherein the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein
The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO: 9);
the sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises sequence HDFDYFPYWFAH (SEQ ID NO: 10);
the sequence of CDR1L comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The sequence of CDR3L includes sequence QQWTSNPPT (SEQ ID NO: 12).
In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable region amino acid sequence of SEQ ID No. 13, and wherein the sequence of the light chain variable region comprises a sequence at least 90% identical to the light chain variable region amino acid sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16.
In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises SEQ ID No. 13, and wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16.
In another embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises SEQ ID No. 13, and wherein the sequence of the light chain variable region comprises SEQ ID No. 14.
In another embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the sequence of the heavy chain variable region comprises SEQ ID No. 13, and wherein the sequence of the light chain variable region comprises SEQ ID No. 15.
In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region;
Wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13;
wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14;
wherein the sequence of the heavy variable chain comprises the sequence GXXXXXXX1HX2X3S(SEQ ID NO:43)；
Wherein X is any amino acid;
wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M; and is
Wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8(SEQ ID NO:44)；
Wherein X4Is D, G or P;
wherein X5Is F or P;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F.
In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region,
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85%, at least 90%, or at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13,
wherein the sequence of the light chain variable region comprises a sequence that is at least 85%, at least 90%, or at least 95% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14,
Wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR 3; and is
Wherein:
the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO 13,
the sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO. 13,
the sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO. 14,
the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO 14 and
the sequence of CDR3L includes residues S91 and S92 of SEQ ID NO. 14.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is a chimeric antibody, a CDR-grafted antibody, or a humanized antibody or antigen-binding fragment thereof.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is a multispecific or bispecific antibody or antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment is a bispecific antibody comprising a complementary region that binds to PD-1 or PD-L1.
In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof provided by the invention is an scFv, Fv, Fab ', Fab, F (ab')2, or diabody.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention has isotype IgG 4.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein contains an S241P substitution in the heavy chain constant region.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is deglycosylated.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein lacks a C-terminal lysine in the heavy chain.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention is conjugated to one or more of a cytotoxin, a fluorescent label, and/or an imaging agent.
In another aspect, the invention provides CEACAM1 antibodies and antigen binding fragments thereof, characterized by the epitope on CEACAM1 to which they bind. As noted, such antibodies include, but are not limited to, the CEACAM1 antibody and antigen binding fragments thereof described by its structural features herein, including CDR motifs, CDR sequences, and heavy and light variable chain sequences. In some embodiments, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof that bind to residues in the IgV-like N domain of CEACAM 1. In another embodiment, the antibodies and antigen binding fragments thereof provided herein also selectively bind CEACAM1 as compared to one or more CEACAM family members. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof does not exhibit significant binding to other CEACAM family members including CEACAM3, CEACAM5, CEACAM6, and/or CEACAM 8. In some embodiments, the present invention provides CEACAM1 antibodies and antigen binding fragments that bind to an epitope on the N domain of CEACAM1 that overlaps or at least partially overlaps with the CEACAM1: CEACAM1 dimer interface, thereby blocking CEACAM1 homotropic interactions. In some embodiments, the invention provides CEACAM1 antibodies and antigen-binding fragments that bind to CEACAM1 residues, the CEACAM1 residues being located in the binding site of a heterologous interaction partner on CEACAM1, including, but not limited to, other CEACAM family members, TIM family members, bacterial adhesins (such as HopQ, OPA, OMP P1, and/or OlpA), candida albicans, influenza viruses (such as H5N1), and/or daughter nematodes such as wuchereria penguinii.
In one embodiment, the contemplated CEACAM1 antibody or antigen-binding fragment thereof binds the same epitope as an antibody or antigen-binding fragment having a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises SEQ ID No. 13, and wherein the sequence of the light chain variable region comprises SEQ ID No. 14.
In one aspect, contemplated CEACAM1 antibodies or antigen binding fragments thereof bind to the IgV-like N domain of CEACAM1 and bind to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID NO: 17. In one embodiment, the epitope further comprises residue Q44 of SEQ ID NO 17. In one embodiment, the epitope further comprises one or more residues selected from the group consisting of residues S32, Q44, a49, I91, L95 and V96 of SEQ ID NO 17.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof binds to the IgV-like N domain of CEACAM 1.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM 8.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof binds at least in part to the binding site of TIM3 on CEACAM 1.
In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof at least partially binds to the binding site of CEACAM1 on CEACAM1 during homodimerization.
In one aspect, the invention provides antibodies, or antigen-binding fragments thereof, that bind CEACAM and partially or fully bind to the bacterial adhesin binding site on CEACAM1, including but not limited to, helicobacter pylori adhesin HopQ, neisseria gonorrhoeae Opa, neisseria meningitidis Opa, haemophilus influenzae OMP P1, haemophilus egypophilus OMP 1, and/or moraxella Opa-like protein OlpA. In one aspect, the CEACAM1 antibody or antigen binding fragment thereof binds to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, N42, Q89, and N97 of SEQ ID NO: 17.
In one aspect, the CEACAM1 antibody or antigen binding fragment thereof binds to an epitope comprising one or more residues selected from the group consisting of residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and N97 of SEQ ID NO: 17. In one embodiment, the epitope further comprises residues F29, S32, D40, A49, T56, I91, L95, and E99 of SEQ ID NO 17.
In one embodiment, the invention provides nucleic acid molecules encoding the CEACAM1 antibodies or antigen-binding fragments thereof described herein, as well as vectors comprising such nucleic acid molecules. Also provided are cells comprising a vector encoding the CEACAM1 antibody or antigen-binding fragment thereof described herein, and cells expressing the CEACAM1 antibody or antigen-binding fragment thereof described herein. Also provided herein are chimeric antigen receptor T cells comprising the CDRs of any of the antibodies or antigen-binding fragments disclosed herein.
In one embodiment, the invention provides a composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable excipient.
In one embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to its interaction partner and/or reducing CEACAM1 activity using a CEACAM1 antibody or antigen binding fragment thereof described herein, comprising contacting CEACAM1 with a CEACAM1 antibody or antigen binding fragment thereof described herein. For example, embodiments of the present invention may be used to inhibit the interaction between CEACAM1 and a CEACAM family member. In one embodiment, the CEACAM family member is CEACAM3, CEACAM5, CEACAM6, or CEACAM 8. In some embodiments, the CEACAM family member is CEACAM1 itself.
In one embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to a member of the TIM family using a CEACAM1 antibody or antigen binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen binding fragment thereof described herein. In some embodiments, a TIM family member is TIM-3.
In one embodiment, the present invention provides a method of inhibiting CEACAM1 binding to a bacterial adhesin using a CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the bacterial adhesin is the helicobacter pylori adhesin HopQ, neisseria gonorrhoeae Opa, neisseria meningitidis Opa, haemophilus influenzae OMP 1, haemophilus egypus OMP 1, or moraxella adhesin OlpA. In one embodiment, the invention provides a method of inhibiting CEACAM1 binding to candida albicans using a CEACAM1 antibody or antigen binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen binding fragment thereof described herein. In one embodiment, the present invention provides a method of inhibiting CEACAM1 binding to influenza virus using a CEACAM1 antibody or antigen binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen binding fragment thereof described herein. In one embodiment, the influenza virus is H5N 1.
In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce colonization of mammalian epithelium by bacteria expressing a bacterial adhesin, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the bacterial adhesin is the helicobacter pylori adhesin HopQ, neisseria meningitidis Opa, haemophilus influenzae OMP 1, haemophilus egypus OMP 1, or moraxella adhesin OlpA.
In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce colonization of mammalian epithelium by candida albicans, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein.
In one embodiment, the present invention provides a method of reducing replication of an influenza virus, comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the invention provides a method of reducing the release of a proinflammatory cytokine or chemokine associated with an influenza virus infection, the method comprising contacting a cell population comprising epithelial cells with a CEACAM1 antibody or antigen binding fragment thereof as described herein. In some embodiments, the influenza virus is H5N 1.
In one embodiment, the invention provides methods of using the CEACAM1 antibodies or antigen binding fragments thereof described herein to reduce T cell tolerance and/or enhance T cell expansion or activation. These methods are useful for in vitro and in vivo applications.
In one embodiment, the present invention provides a method of reducing T cell tolerance and/or enhancing T cell expansion in a subject in need thereof using the CEACAM1 antibody or antigen binding fragment thereof described herein, the method comprising administering to the subject an effective amount of the antibody or antigen binding fragment thereof. In one embodiment, the present invention provides a method of treating cancer in a subject in need thereof using the CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof. In some embodiments, the cancer is glioma, glioblastoma, thymoma, mesothelioma, sarcoma, uterine carcinosarcoma, chromophobe renal cell carcinoma, adenoid cystic carcinoma, acute myeloid leukemia, melanoma, uveal melanoma, papillary renal cell carcinoma, clear cell renal cell carcinoma, cholangiocarcinoma, lung adenocarcinoma, diffuse large B-cell lymphoma, pheochromocytoma and paraganglioma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous carcinoma, breast cancer, prostate cancer, bladder cancer, stomach cancer, testicular germ cell carcinoma, ovarian cancer, head and neck cancer, uterine cancer, cervical cancer, or liver cancer. In embodiments, the present invention provides methods of using the CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce tumor growth, reduce tumor metastasis, reduce tumor-associated fibrosis, and/or reduce the sternness of cancer in a subject in need thereof by administering to the subject an effective amount of the antibody or antigen-binding fragment. In some embodiments, the invention provides methods further comprising administering a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor. In some embodiments, the invention provides methods further comprising administering one or more of an inhibitor of LAG3, TIGIT, LAP, Podoplanin, protein C receptor, ICOS, GITR, CD226, or CD 160. In some embodiments, the present invention provides methods further comprising administering a TIM-3 inhibitor. In some embodiments, the inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment. In some embodiments, the inhibitor is administered alone or as a mixture with an antibody or antigen-binding fragment.
In one embodiment, the present invention provides a method of reducing colonization of the epithelium of a subject by a bacterium that expresses a bacterial adhesin in a subject in need thereof using a CEACAM1 antibody, or antigen-binding fragment thereof, described herein, comprising administering to the subject an effective amount of a CEACAM1 antibody, or antigen-binding fragment thereof, described herein. In some embodiments, the bacterial adhesin is the helicobacter pylori adhesin HopQ, neisseria meningitidis Opa, haemophilus influenzae OMP 1, haemophilus egypus OMP 1, or moraxella OlpA.
In one embodiment, the present invention provides a method of reducing colonization of the epithelium of a subject by candida albicans in a subject in need thereof using a CEACAM1 antibody or antigen binding fragment thereof described herein, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen binding fragment thereof described herein.
In one embodiment, the present invention provides a method of reducing influenza virus replication in a subject in need thereof using the CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising administering to the subject an effective amount of the CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the present invention provides a method of reducing the release of a proinflammatory cytokine or chemokine associated with influenza virus infection in a subject in need thereof using the CEACAM1 antibody or antigen binding fragment thereof described herein, the method comprising administering to the subject an effective amount of the CEACAM1 antibody or antigen binding fragment thereof described herein. In some embodiments, the influenza virus is H5N 1.
In one embodiment, the invention provides methods of using the CEACAM1 antibodies or antigen binding fragments thereof described herein to treat subjects who are not responsive to therapy with checkpoint inhibitor therapy (primary resistance) as well as patients who initially respond to treatment but later become resistant to checkpoint inhibitor blockade (secondary or acquired resistance). Such methods of treatment comprise administering to the subject the CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor. Resistant cancers may also be referred to as refractory cancers.
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FIG. 1 shows plasmid maps of the light chain expression vector pANTV κ and the heavy chain expression vector pANTVhG4 (S241P). VHAnd vk vectors both contain genomic DNA fragments incorporating introns and polyA sequences. Expression of both strands is driven by the CMV promoter.
Figure 2 shows the selectivity of CEACAM1 antibody variants. Humanized variant intermediates were examined by flow cytometry on HeLa cells transfected with CEACAM1, 3, 5, 6 and 8. The proportion of cells staining positive based on irrelevant hIgG4 is shown. There was no evidence of any staining of the HeLa-CEACAM3 or HeLa-CEACAM8 transfectants, and thus these data were not reported.
FIGS. 3A, 3B and 3C show a heavy variable chain VH1 (fig. 3A), heavy variable chain CP08H03 (fig. 3B), and light variable chain vk 8S29A (fig. 3C). CDRs are shaded. The CDR residue numbering according to Kabat and according to the primary amino acid sequence is shown.
FIG. 4 shows chimeric CEACAM1 antibody VH0/V.kappa.0 is glycosylated in CDR 1L. The introduction of mutations N26Q and S29A abolished this glycosylation. Proteins were separated on SDS-PAGE under denaturing conditions. The molecular weights of the heavy chain, glycosylated light chain and non-glycosylated light chain are shown. Residues N26 and S29 were numbered using the Kabat numbering scheme.
FIG. 5 shows a plasmid map of phagemid expression vector pANT 43. VHAnd the vk domain are linked by a flexible glycine-serine (G4S) linker and fused in frame to the M13 gene III phage coat protein. Expression of single chain variable fragment (scFv) is driven by the Lac promoter.
FIG. 6 shows the binding of phage to CEACAM1 antigen. Will be derived from parent VHPhage prepared with 1/V.kappa.8S29A scFv or irrelevant scFv were serially diluted and incubated with plate-bound GST-CEACAM 1. Using anti-M13 horseradish peroxidase (HRP) conjugates and 3,3',5' -Tetramethylbenzidine (TMB) substrate detects binding of phage to CEACAM 1.
Figure 7 provides an overview of affinity maturation library design. CDRs (as defined by Kabat) are shown in bold, and the targeted position is denoted by X. A single position may contain all 20 amino acids or a subset thereof.
FIGS. 8A, 8B and 8C provide an overview of the library construction method to generate randomized phage libraries. A light chain CDR3 library (fig. 8A), a heavy chain CDR1 library (fig. 8B), and a heavy chain CDR3 library (fig. 8C).
Fig. 9A and 9B provide an overview of two different selection activities used during affinity maturation of CEACAM1 antibody. FIG. 9A: selecting Activity 1: the library phages were subjected to solid phase panning CEACAM5/CEACAM6 deselection and multiple rounds of selection of decreasing concentrations of biotinylated soluble CEACAM1 prior to round 2. FIG. 9B: selection activity 2: panning selection using CEACAM1 was performed in round 1, followed by 2 rounds of panning CEACAM5/CEACAM6 deselection and selection using decreasing concentrations of biotinylated soluble CEACAM 1.
Figure 10 shows an example of a scFv binding ELISA assay. Serial dilutions of purified parent scFv VH1/V.kappa.8S 29A or affinity matured scFv variants were added to GST-CEACAM 1-coated plates. Binding was detected using anti-HIS 6-HRP antibody and TMB. All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Figure 11 shows the binding selectivity of different affinity matured CEACAM1 antibodies. The affinity matured antibodies CP08H03/V κ 8S29A (labeled "CP 08_ H03/parental VL"), CP08H03/CP08F05, 8H3_9B3/CP08F05, and CP08H03/CP08E05 contain phenylalanine (F) at residue 104 of CDR 3H. Affinity matured antibodies CP09B03/CP08E05, CP09C02/CP08E05, CP09C02/CP08F05 and 9B3_9E5/CP08E05 contain an aspartic acid residue (D) at residue 104 of CDR 3H. HeLa cells were transfected with the vector alone (HeLa-Neo) or the vector expressing CEACAM1, CEACAM3, CEACAM5, or CEACAM6, respectively, and stained with the indicated antibodies. The y-axis shows the% staining of each antibody by the transfected cell set. hIgG4 ═ control antibody with the same stable hinge mutation. MOPC — mouse IgG1 control antibody. Mouse antibody as positive control for transfected CEACAM isoform: col-1 ═ CEACAM3 and CEACAM5 antibodies. 9a6 ═ CEACAM6 antibody. T84.1 ═ CEACAM cross-reactive antibody and T84.66 ═ CEACAM5 antibody. No primary antibody was present for the 2 nd FITC only, and a secondary FITC conjugated antibody only. Col-1 and 9A6 are commercially available antibodies (Dako) and T84.1 and T84.66 have been previously described (Neumaier M, J Immunol 1985; 135: 3604-9). Due to abnormally high background signals, data for affinity matured antibodies 9B3_8H3/V κ 8S29A, 8H3_9B3/CP08_ E05, and 8H3_9C2/CPO08_ F05 and CEACAM8 antibody 80H3 were omitted from the figure.
FIGS. 12A, 12B and 12C show CEACAM antibodies CP08H03/V κ 8S29A (labeled "CP 08_ H03/parent VL"), CP08H03/CP08_ F05 and VH0/V kappa 0 is selective for CEACAM 1. CP08H 03/V.kappa.8S 29A and CP08H03/CP08F05 contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain). Figure 12A shows a single cycle kinetic sensorgram and fitted curve of purified lead humanized and affinity matured IgG4 variants. Increasing concentrations of different CEACAM family members were injected and single dissociation rates were determined by single cycle kinetics (surface plasmon resonance, SPR). FIG. 12B shows chimeric antibody VHThree-point binding ELISA data for 0/V κ 0 (labeled "chimeric"), purified precursor humanized and affinity matured IgG4 variants bound to CEACAM1 and CEACAM3 family members. Three points were performed (high, medium and low, concentrations based on chimeric antibody VHBinding of 0/V κ 0 to CEACAM 1) and binding was detected using anti-human κ chain antibody and TMB substrate. FIG. 12C shows chimeric antibody VHThree-point binding ELISA data for 0/V κ 0 (labeled "chimeric") and purified lead humanized and affinity matured IgG4 variants bound to CEACAM1, 5 and 6 family members. Three points were performed (high, medium and low, concentrations based on chimeric antibody V HBinding of 0/V κ 0 to CEACAM-1) was titrated and binding was detected using anti-human κ chain antibody and TMB substrate.
FIG. 13 shows the sequence homology between the N domains of different CEACAM family members. CEACAM1(C1, UniProtKB accession number P13688), CEACAM3(C3, UniProtKB accession number P40198), CEACAM4(C4, UniProtKB accession number O75871), CEACAM5(C5, UniProtKB accession number P06731), CEACAM6(C6, UniProtKB accession number P40199), CEACAM7(C7, UniProtKB accession number Q14002), and CEACAM8(C8, UniProtKB accession number P31997). The percentage identity matrix shown is created using clustal2.1. The specific residues analyzed for each CEACAM family member are shown.
Figure 14 shows the results of CEACAM1 mutagenesis studies aimed at identifying residues in CEACAM1 involved in binding to the CEACAM1 antibody shown. CEACAM1-FLAG was expressed in Human Embryonic Kidney (HEK) cells transfected with CEACAM1 containing the indicated mutations (Y34C, V39A, G41A, N42A, R43A, Q44L, G47A and Q89H), proteins were resolved by SDS-PAGE and then immunoblotted. The following chimeric (V) was usedH0/V κ 0) and humanized CEACAM antibody detected Wild Type (WT) or mutant CEACAM1 protein. Detection of a decrease indicates that the mutated residue is involved in binding to the corresponding antibody used for detection.
FIG. 15 shows the structure of CEACAM1: CP08H 03/V.kappa.8S 29A Fab complex. In the structure of the complex, Fab is shown as the ca trace and antigen is shown as a band.
Figure 16 shows a view of the antigen (CEACAM1) towards the dimer interface. The CEACAM1 residues including D40, N42, L95, V96, N97 and E99 interacting with the CP08H03/V κ 8S29A Fab light chain (see fig. 15) and CEACAM1 residues including F29, S32, Y34, Q44, T56, Q89 and I91 interacting with the CP08H03/V κ 8S29A Fab heavy chain are labeled. The relevant side chains are drawn as rods.
FIG. 17 shows a crystal structure showing CEACAM1: CEACAM1 homodimer interface (PDB ID:4 QXW). One CEACAM1 cell is shown on the left side and the other on the right side. Residues Y34, Q44, Q89 and N97 form the YQQN pocket.
FIG. 18 shows a close-up perspective image of the CP08H03/V κ 8S29A Fab-CEACAM1 interaction. CEACAM1 was plotted as a band, and Fab chains (light and heavy) as ca traces. Regions of Fab light chain residues (including S30, Y31, Y48, L49, S51, N52, W90, S91 and N93) and heavy chain residues S52, S53, T56, Y57, Y59, D102, Y103, F104, P105, Y106 interacting with CEACAM1 residues (including F29, S32, Y34, D40, N42, Q44, a49, T56, Q89, I91, L95, V96, N97 and E99) and heavy chain residues S52, S53, T56, Y57, Y59, D102, Y103, F104, P105, Y106 were labeled. The side chain of interest is drawn as a rod and the hydrogen bond is drawn as a black dashed line. The residue numbering is based on the primary amino acid sequence of the antibody and CEACAM 1.
FIG. 19 shows a comparison of CEACAM 1F 29 and V49 or A49 residues in the crystal structure of CEACAM1 WT: CP08H03/V κ 8S29A antibody (left) or CEACAM 1A 49V/Q89H mutant (right).
The material CEACAM1 of fig. 20A and 20B antibody CP08H03/V κ 8S29A (labeled "CP 08") blocked the human CEACAM1: CEACAM1 (fig. 20A) and CEACAM: human TIM-3 interaction (fig. 20B). IgG4 ═ control antibody.
FIG. 21 shows a test for CEACAM1 antibody inducing CD45 in humanized non-obese diabetic (NOD) scid gamma mice (NSG mice)+Experimental setup of the capacity of cells to proliferate. Transplantation of human Peripheral Blood Mononuclear Cells (PBMCs) adoptively transferred to NSG host mice by intraperitoneal injection was analyzed by Fluorescence Activated Cell Sorter (FACS) for staining of human CD45 and proliferation dye 38 days after injection. On day 24 post PBMC injection, mice were treated with a single injection of human IgG4 isotype control or CP08H03/V κ 8S29A (labeled "CP 08_ H03/parental VL") or CP08H03/CP08F05 antibody at the indicated concentrations. On day 31, mice were treated with a second injection. On day 38, mice were sacrificed for data collection.
Figure 22 shows CEACAM1 antibodies CP08H 03/vk 8S29A (labeled "CP 08_ H03/parental") and CP08H03/CP08F05 do not deplete transplanted human cells in humanized NSG mice. The average percentage of human CD4 and CD 8T lymphocytes was evaluated on day 38. CP08H03/CP08F05 contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
FIG. 23 shows that administration of CEACAM1 antibodies CP08H03/V κ 8S29A (labeled "CP 08_ H03/parental VL") or CP08H03/CP08F05, respectively, resulted in antibody-induced human CD45 in humanized NSG mice+An increase in immune cell expansion. CP08H 03/Vkappa 8S29A induced amplification of human CD45 PBMC in vivo. On day 38, isotype hIgG4 control (10mg/kg), CP08H03/V were sacrificedKappa 8S29A (2 and 10mg/kg) and CP08H03/CP08F05(2 and 10mg/kg) treated mice, spleen cells were isolated and harvested for proliferation assays. Ex vivo proliferation was performed under T cell stimulation conditions, where cells were cultured for 120 hours in soluble anti-CD 3(OKT3) (indicated concentrations of 10, 5, 2.5. mu.g/ml) and rIL-2(40 units/ml). Dilution of the proliferation dye represents dividing cells/proliferation (duplex DNA is analyzed as a dilution signal when cells proliferate). CP08H03/CP08F05 contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Fig. 24A, 24B and 24C show that CEACAM1 antibody CP08H 03/vk 8S29A (labeled "CP 08_ H03/parental VL") reduced tumor growth in humanized mice. Fig. 24A provides a schematic of the experimental protocol that resulted in fig. 24B. FIG. 24B shows that 1x10 6MALME-3M (human melanoma) cells and 5X 106Mean tumor size after subcutaneous injection of human PBMC into NSG together. After 10 days, palpable tumors were recorded and mice were randomized to intraperitoneally treated with the corresponding antibody concentrations on days 10, 13, 17, 20 and 24. FIG. 24C shows statistical comparison of the hIgG4 control treated group to three different CP08H 03/Vkappa 8S29A groups (2mg/kg, 0.4mg/kg, and 0.08mg/kg) by linear regression.
Figure 25 shows that T cells from humanized mice transplanted with the human melanoma cell line MALME-3M and treated with CEACAM1 antibody CP08H 03/vk 8S29A (labeled "CP 08_ H03/parental VL") as described in figure 24 show a reduction in the number of tumor cells and a reduction in proliferation and an increase in the number of CD8 and CD4 positive T cells within the tumor. These T cells showed increased proliferation when examined ex vivo after stimulation with anti-CD 3. On the day of sacrifice, humanized NSG mice treated with isotype hIgG4 control (2mg/kg), CP08H 03/vk 8S29A (labeled "CP 08_ H03/parental VL", 0.08 and 2mg/kg) bearing melanoma tumors were sacrificed. Isolation and Collection of tumor cells and CD4+And CD8+Tumor infiltrating lymphocytes were used for proliferation assays. Identifying tumor cells as FSCs HiSSCHiCells, which were negative for human CD45, were quantitatively proliferated by evaluating diluted commercial dye for proliferation (Becton-Dickinson). Identification of human CD45 by flow cytometry+CD4+And CD45+CD8+T cells. Ex vivo measurements of T cell proliferation were performed under T cell stimulation conditions, in which cells were cultured for 6 days under soluble anti-CD 3(2g/ml) and rIL-2(40 units/ml).
Fig. 26 shows phenotypic changes of intratumoral memory CD8T cells when CEACAM1 was blocked with CEACAM1 antibody CP08H 03/vk 8S29A (labeled "CP 08_ H03/parental VL") as described in fig. 24-25. CD3 of tumor infiltration from melanoma-bearing humanized NSG mice using CD62L and CD44 cell markers+CD8+T cell populations were analyzed by flow cytometry for characterization of central memory (CD 62L)+CD44+) And effector memory (CD 62L)-CD44+CD3+CD8+) A population of T cells. The treatment conditions were isotype hIgG4 control (2mg/kg) and CP08H 03/V.kappa.8S 29A (0.08, 0.4 and 2 mg/kg).
FIG. 27 shows primary CD4 of CEACAM1 in TIL from naive (left) and PD-1 and/or CTLA-4 resistant (right) melanoma patients+(upper) and CD8+Expressed on T (lower) T cells. Similar characterization of PD1 and TIM-3 expression is also shown.
Figure 28 shows that tumor-associated cells (TAC) from patients with acquired resistance to anti-PD-1 and/or anti-CTLA-4 therapy exhibited significantly higher expression of CEACAM1 compared to TAC from patients not previously exposed to anti-PD-1 and/or anti-CTLA-4 therapy. TAC is obtained from naive melanoma patients (not previously exposed to anti-PD-1 and/or anti-CTLA-4 therapy) or those melanoma patients who acquired resistance to anti-PD-1 and/or anti-CTLA-4 therapy (acquired resistance). TAC was obtained by culturing tumor tissue in DMEM medium, and floating cells were removed from the supernatant and analyzed. Cells were stained with CD3, CD4 and CD8 and assessed for CD3 +CD4+Cells and CD3+CD8+CEACAM1 expression on cells. P ═ 0.05; a, p<0.01。
FIG. 29 shows CD8 isolated from naive patients+CD8 isolated from patients resistant to anti-PD-1 and/or anti-CTLA-4 therapy as compared to T patients+Central memory in T cells (T)cm) With respect to effect memory (T)em) Relative reduction of cells. Tumor-associated cells from naive and acquired resistant patients were subjected to central memory in TAC derived from naive and resistant patients (CCR 7)+CD62L+) And effector memory (CCR 7)-CD62L-) And (4) dyeing the marker.
Figure 30 shows CEACAM1 antibody CP08H 03/vk 8S29A (labeled "CP 08") reverses T cell depletion in PD1/CTLA-4 resistant tumors. Tumor-associated cells and PBMCs were isolated from melanoma patients with secondary resistance and stage IV disease to Pembrolizumab (Pembrolizumab), Ipilimumab (Ipilimumab) + Nivolumab (Nivolumab) and Dabrafenib (Dabrafenib) + Trametinib (Trametinib). CEACAM1, PD1 or TIM-3 staining of tumor-associated cells and PBMCs and CD8+And CD4+The proportion of T cells shows expression of these markers (left). PBMC or tumor-associated cells ("tumors") cultured with soluble anti-CD 3(2g/ml) and rIL-2(40 units/ml) in the presence of CP08H 03/Vkappa 8S29A or hIgG4 control antibody are shown on the right panel. The release of IFN γ and TNF α (a measure for reversing T cell tolerance) was determined by ELISA.
Fig. 31A and 31B show flow cytometry analysis of stable HeLa CEACAM1(HeLa C1) transfectants, stable HeLa CEACAM3 transfectants (HeLa C3), stable HeLa CEACAM5 transfectants (HeLa C5), stable HeLa CEACAM6 transfectants (HeLa C6), and stable HeLa CEACAM8 transfectants (HeLa C8). Fig. 31A: the HeLa transfectants shown in 5X10^4 were washed with staining buffer and CP08H 03/Vkappa 8S29A (labeled "CP 08", left) or CEACAM1 antibody CM-24 were incubated at room temperature for 30 minutes, washed twice with staining buffer and stained with anti-human IgG4 Fluorescein Isothiocyanate (FITC) -conjugated secondary antibody for 20 minutes at room temperature. Fluorescence intensity was determined by flow cytometry. Viable cells were determined by 4', 6-diamidino-2-phenylindole (DAPI) staining, as indicated on the y-axis. The corresponding CEACAM1 antibody staining is shown on the x-axis. For CP08H03/V κ 8S29A, note that positive signals in the phylum are shown only in the HeLa CEACAM1(C1) transfectant (left). In contrast, CM-24 (right panel) was not selective and cross-reacted with CEACAM1, CEACAM3, and CEACAM 5. FIG. 31B shows a different representation of the data shown in FIG. 31A.
Fig. 32A, 32B and 32C show that CEACAM1 antibody CP08H 03/vk 8S29A (labeled "CP 08") is more effective than CEACAM1 antibody CM-24 in reversing T cell tolerance in tumor-associated cells. CEACAM1, PD1, or TIM-3 staining of tumor-associated cells derived from primary Merckel cell carcinoma tumors and showing CD8 +And CD4+Proportion of T cells (fig. 32A and 32B). Tumor-associated cells were incubated with soluble anti-CD 3(2 g/ml) and rIL-2(40 units/ml) in the presence of CP08H 03/V.kappa.8S 29A, CM-24 or hIgG4 controls, respectively. The release of IFN- γ (a measure for reversing T cell tolerance) was determined (fig. 32C). Compare CP08 with hIgG4, P ═ 0.0138.
Figures 33A, 33B, 33C, and 33D show that CM-24 treated metastatic melanoma exhibits reduced TIL and increased tumor cells in NSG mice compared to CP08H 03/vk 8S29A (labeled "CP 08") treated metastatic melanoma. Figure 33A shows the experimental setup using a therapeutic tumor model in humanized NSG mice with human melanoma xenografts using four doses of 2mg/kg of the corresponding antibody, including a hIgG4 control containing the same stabilizing hinge mutation. FIG. 33B shows high at FSC/SCC (FSC/SCC)Hi) And a leukocyte marker human CD45 deficiency in tumor infiltration CD4+T lymphocytes (Gray), CD8+A pie chart of the percentage of T lymphocytes (black) and tumor cells (white) (left: control antibody. center: CEACAM1 antibody CP08H 03/V.kappa.8 S29A. right: CEACAM1 antibody CM-24). FIG. 33C shows tumor cell proliferation of IgG4 control, CP08H 03/Vkappa 8S29A, and CM-24, indicating that CP08H 03/Vkappa 8S29A, but not CM-24, inhibits tumor proliferation. FIG. 33D shows spleen CD4 in CP08H 03/Vkappa 8S29A treated mice +Proliferation of T cells is increased and spleen CD4 in CM-24 treated mice+The proliferation of T cells is reduced.
Fig. 34A, 34B and 34C show that CEACAM1 antibody CM-24 is an agonistic drug in a metastatic melanoma model. CD4 showing tumor infiltration from metastatic melanoma+T lymphocytes (FIG. 34A), CD8+Absolute cell counts of T lymphocytes (fig. 34B) and tumor cells characterized by a high anterior/lateral scatter (FSC/SCC Hi) (fig. 34C). Show thatThe values obtained for each experimental mouse per group (n-9 for IgG 4; n-8 for CP 08; n-6 for CM-24) were reported. P<0.05；**P<0.001. Statistical analysis refers to the data contained in fig. 33B. Note that the number of TILs increased (fig. 34A and 34B) and the number of tumor cells decreased (fig. 34C) in CP08H03/Vk 8S29A (labeled "CP 08") treated mice relative to CM-24 treated mice. This data indicates that CP08H03/Vk 8S29A is an antagonistic antibody, and CM-24 is an agonistic antibody.
FIGS. 35A and 35B show that CEACAM1 antibody CP08H03/V κ 8S29A covers CEACAM1: HopQ binding interface and is expected to block CEACAM1: HopQ or CEACAM1: Opa protein interactions. Fig. 35A shows CEACAM1: HopQ binding interface based on analysis of three crystal structures (PDB ID 6AW2, 6GBH, and 6 GBG). The CEACAM1 GFCC ' face is formed by the interaction of CEACAM1 CC ' and the FG loop ' (see Huang et al, Nature.2015, 1-15 days; 517(7534):386-90), involved in HopQ binding at CEACAM1 residues F29, Y34, N42, Q89 and N97 and producing various hydrogen bonds and hydrophobic interactions (Bonsor D, A. et al EMBO J.2018, 7-2 days; 37(13) pii: e 98664; Moonens K et al EMBO J.2018, 7-2 days; 37(13) pii: e 98665). FIG. 35B shows a superposition of the CP08H 03/V.kappa.8S 29A: CEACAM1 crystal structure and CEACAM1: HopQ crystal structure. The light and heavy chains of the CP08H 03/V.kappa.8S 29A antibody are shown in surface schematic representation. The band plots show the HopQ chains (three different crystal structures PDB ID 6AW2, 6GBH and 6GBG) and CEACAM1 from three cocrystal structures different from HopQ (PDB ID 6AW2, 6GBH, 6GBG) and CEACAM1 from a cocrystal structure different from CP08H03/vk 8S29A to highlight the superposition of CP08H03/vk 8S29A and HopQ binding epitopes.
FIG. 36 shows that CEACAM1 antibody CP08H03/V κ 8S29A increases survival in tumor-bearing mice. NSG mice were injected with MALME-3M (human melanoma) cells and human PBMCs. Treatment with CEACAM1 antibody CP08H 03/vk 8S29A or control human (H) IgG4 antibody (see arrows) was performed on days 10, 13, 17, 20 and 24, respectively. Shown as% survival. n is 4/group.
FIGS. 37A and 38B show that CEACAM1 antibody CP08H 03/Vkappa 8S29A increased expression of various factors involved in melanoma patients with secondary resistance to immunotherapyCD8 (1)+Immune response of T cells to cancer. FIG. 37A shows a series of visNE (visually distributed random neighbor embedding) plots presented in Cytobank using Barnes-Hut implementation of the t-SNE algorithm, which depicts the specific factors shown at CD8 as defined by mass cytometry of the left plot+Intracellular expression in T cells. Quantitation of the heat map levels for each factor shown is shown on the right side of the x-axis relative to the residue associated with each factor shown on the y-axis. Figure 37B shows the fold change in the intracellular response of the indicated factors described in figure 37A in response to CP08H 03/vk 8S29A relative to the hIgG4 control antibody set at 1.0.
Fig. 38A and 38B show that CEACAM1 antibody CP08H 03/vk 8S29A re-enhances the ability of tumor-dissociated cells from two melanoma patients that were not previously treated (fig. 38B, subject 189) or have secondary resistance to immunotherapy (fig. 38A, subject 185) to secrete interferon-gamma (IFN-). In both cases, tumor samples were destroyed by mechanical dissociation (Miltenyi) and tumor dissociated cells were treated in vitro only with 2 μ g/ml CP08H03/V κ 8S29A or human IgG4 isotype control antibody. After 96 hours, significant levels of interferon- γ were detected in the supernatant of CP08H 03/vk 8S29A, but not in the human IgG4 isotype control antibody treated samples. P <0.05
Detailed Description
Antibodies
The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and antigen-binding portions thereof (e.g., paratopes, CDRs), so long as they exhibit the desired biological activity and specificity.
As used herein, "antibody variable domain" refers to the portions of the light and heavy chains of an antibody molecule that include the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the Framework Region (FR). VHRefers to the variable domain of the heavy chain. VLRefers to the variable domain of the light chain. The amino acid positions assigned to the CDRs and FRs may be according to KabatOr according to the Chothia definition. The term "framework region" (FR) refers to those variable domain residues other than CDR residues.
As used herein, the term "complementarity determining region" (CDR) refers to the portion of an antibody variable domain that is (typically) involved in antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR 3. Each CDR may comprise amino acid residues from a CDR as defined, for example, by Kabat (i.e.about residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable domain and about residues 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable domain (Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service, National Institutes of Health, Bethesda, Md. (1987,1991)). Each CDR may also comprise amino acid residues from the "high-variable loop" (i.e.about residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable domain and about residues 26-32(H1), 53-55(H2) and 96-101(H3) and 91-96(L3) in the heavy chain variable domain) (in some cases under Chbat et al, Biol 901.196), the CDRs may comprise amino acids from the CDR regions and hypervariable loops defined according to Kabat. The Kabat residue designations do not always directly correspond to the linear numbering of the amino acid residues (primary amino acid sequence). The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering, corresponding to the shortening or insertion of the structural components (whether framework or CDRs) of the basic variable domain structure. For a given antibody or antigen-binding fragment thereof, the Kabat numbering of the correct residues may be determined by aligning homologous residues in the sequence of the antibody or antigen-binding fragment thereof with a "standard" Kabat numbered sequence. Examples of how Kabat numbering correlates with the primary amino acid sequence of an antibody can be found in fig. 3A, 3B, and 3C. Alternatively, CDRs may be defined according to the immunogenetics (imgt) system (Lefranc, m. -p. et al, dev. comp. immunol.,27,55-77 (2003)).
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs wherein:
(i) the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO 9;
(ii) the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO. 2;
(iii) the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO. 10;
(iv) the CDR1 sequence of the light chain variable region comprises SEQ ID NO 4;
(v) the CDR2 sequence of the light chain variable region comprises SEQ ID NO 5; and is
(vi) The CDR3 sequence of the light chain variable region comprises SEQ ID NO: 11.
In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs wherein:
(i) the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO 9;
(ii) the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO. 2;
(iii) the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO. 10;
(iv) the CDR1 sequence of the light chain variable region comprises SEQ ID NO 4;
(v) the CDR2 sequence of the light chain variable region comprises SEQ ID NO 5; and is
(vi) The CDR3 sequence of the light chain variable region comprises SEQ ID NO 12.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises six CDRs, wherein:
(i) the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO 9
(ii) The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO. 2;
(iii) The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO. 10;
(iv) the CDR1 sequence of the light chain variable region comprises SEQ ID NO 18;
(v) the CDR2 sequence of the light chain variable region comprises SEQ ID NO 5; and is
(vi) The CDR3 sequence of the light chain variable region comprises SEQ ID NO: 11.
As shown in the examples below, affinity maturation of CDR1H, CDR3H and CDR3L of the humanized aglycosylated CEACAM1 antibody produced variants conferring significant improvement in CEACAM1 binding affinity. The obtained variants were examined and compared to the variability introduced into the affinity maturation library, indicating that certain CDR positions (where the amino acids remain relatively unchanged) and other CDR positions (where variations can be introduced), leading to improved binding.
In one aspect, the invention provides a CEACAM1 antibody or antigen binding fragment thereof comprising CDR1H, wherein the CDR1H comprises residues 31-35(Kabat definition, corresponding to residues 31 to 35 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:19 (see fig. 3A) or SEQ ID NO:13 (see fig. 3B) of a CEACAM1 antibody, and comprises the sequence X1HX2X3S (SEQ ID NO:1),
wherein X1 of CDR1H is A, D, N or S;
wherein X2 of CDR1H is A or G; and is
Wherein X3 of CDR1H is an amino acid having a hydrophobic side chain comprising I or M.
Alternatively, the IMGT definition may be used to define CDR1H, where CDR1H comprises residues 26-33 of the CEACAM1 antibody (corresponding to residues 26-33 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:19 (see FIG. 3A) or SEQ ID NO:13 (see FIG. 3B)), and comprises the sequence X14X15X16FX17X1HX2(SEQ ID NO:20),
wherein X14 of CDR1H is G or E;
wherein X15 of CDR1H is an amino acid with an aromatic side chain comprising F or Y;
wherein X16 of CDR1H is T, S or I;
wherein X17 of CDR1H is an amino acid with a polar uncharged side chain including S, T or N;
wherein X1 of CDR1H is A, D, N or S; and is
Wherein X2 of CDR1H is A or G.
In one embodiment, CDR1H (Kabat definition) of the CEACAM1 antibody or antigen binding fragment thereof comprises the sequence SHGMS (SEQ ID NO: 9).
In some embodiments, CDR1H (defined by IMGT) comprises the sequence GFIFSHG (SEQ ID NO: 21).
In one aspect, the invention provides a CEACAM1 antibody or antigen binding fragment thereof comprising a CDR1H region, wherein the CDR1H comprises residues 26-35 of CEACAM1 antibody (Kabat definition, corresponding to, e.g., SEQ ID NO:19 (see fig. 3A) or SEQ ID NO: 13 (see fig. 3B), and comprises the sequence X14X15X16FX17X1HX2X3S(SEQ ID NO:22)，
Wherein X14Is G or E;
wherein X15Is an amino acid having an aromatic side chain including F or Y;
wherein X16Is T, S or I;
wherein X17Is an amino acid with a polar uncharged side chain including S, T or N;
wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid having a hydrophobic side chain comprising I or M.
In one embodiment, the CDR1H region comprises sequence GFIFSSHGMS (SEQ ID NO: 23).
In one aspect, the invention provides a CEACAM1 antibody or antigen binding fragment thereof comprising CDR3H, wherein the CDR3H comprises residues 95-102(Kabat definition, corresponding to residues 99 to 110 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:19 (see fig. 3A) or SEQ ID NO:13 (see fig. 3B), and comprises the sequence HX4X5DYX6PX7WFAX8(SEQ ID NO:3),
wherein X4 of CDR3H is D, G or P;
wherein X5 of CDR3H is F or P;
wherein X6 of CDR3H is D or F;
wherein X7 of CDR3H is A or Y; and is
Wherein X8 of CDR3H is L, H or F.
In one embodiment, CDR3H comprises residues 95-102(Kabat definition, corresponding to residues 99 to 110 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:19 (see FIG. 3A) or SEQ ID NO:13 (see FIG. 3B)), and comprises the sequence HX4X5DYFPYWFAX8(SEQ ID NO:7),
Wherein X4 of CDR3H is D, G or P;
wherein X5 of CDR3H is F or P; and is
Wherein X8 of CDR3H is L, H or F.
In one embodiment, CDR3H comprises sequence HDFDYFPYWFAH (SEQ ID NO: 10).
In one aspect, the invention provides a CEACAM1 antibody or antigen binding fragment thereof comprising a CDR3H region, wherein the CDR3H region comprises residues 94-102(Kabat definition, corresponding to residues 98 to 110 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:19 (see fig. 3A) or SEQ ID NO:13 (see fig. 3B), and comprises the sequence X18HX4X5DYX6PX7WFAX8(SEQ ID NO:24),
wherein X18 is R or K;
wherein X4 is D, G or P;
wherein X5 is F or P;
wherein X6 is D or F;
wherein X7 is A or Y; and is
Wherein X8 is L, H or F.
In one aspect, the CDR3H region comprises sequence RHDFDYFPYWFAH (SEQ ID NO: 25).
In one aspect, the invention provides a CEACAM1 antibody or antigen binding fragment thereof comprising a CDR3L, wherein the CDR3L comprises residues 89-97(Kabat definition, corresponding to residues 88 to 96 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:14 (see fig. 3C)) and comprises the sequence qqqx9X10X11X12PX13T(SEQ ID NO:6)，
Wherein X9Is W or N;
wherein X10Is S or T;
wherein X11Is a or an amino acid with a neutral hydrophilic side chain including S, N and T;
Wherein X12Is L, F or N; and is
Wherein X13Is P or F.
In one embodiment, CDR3L comprises residues 89-97(Kabat definition, corresponding to residues 88-96 in the primary amino acid sequence of a heavy variable chain such as SEQ ID NO:14 (see FIG. 3C)), and comprises the sequence QQX9SSX12PX13T(SEQ ID NO:8)，
Wherein X9Is W or N;
wherein X12Is L, F or N; and is
Wherein X13Is P or F.
In one embodiment, CDR3L comprises sequence QQWSSNPPT (SEQ ID NO:11) or sequence QQWTSNPPT (SEQ ID NO: 12).
In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
the CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X14X15X16FX17X1HX2X3S(SEQ ID NO:22)；
Wherein X14Is G or E;
wherein X15Is an amino acid having an aromatic side chain including F or Y;
wherein X16Is T, S or I;
wherein X17Is an amino acid with a polar uncharged side chain including S, T or N;
wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M;
the CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
The CDR3 sequence (CDR3H) of the heavy chain variable region comprises the sequence HX4X5DYX6X19X7WFAX20(SEQ ID NO: 45);
wherein X4 is D, G or P;
wherein X5 is F or P;
wherein X6 is D or F;
wherein X19 is P or A;
wherein X7 is A or Y; and is
Wherein X20 is L, H, Y or F;
the CDR1 sequence (CDR1L) of the light chain variable region comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQX9X10X11X12PX13T(SEQ ID NO:6)；
Wherein X9Is W or N;
wherein X10Is S or T;
wherein X11Is a or an amino acid with a neutral hydrophilic side chain including S, N and T;
wherein X12Is L, F or N; and is
Wherein X13Is P or F; and is
Wherein
When X is present19Is A and/or X20When is Y, X10Is T, X4Is G or P, X1Is N, and/or X16Is T or S.
In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
the CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X 14FX21FX22X23HX2X3S(SEQ ID NO:46)；
Wherein X14Is G or E;
wherein X21Is T or I;
wherein X22Is N or S;
wherein X23Is A, D or S
Wherein X2 is A or G; and is
Wherein X3 is an amino acid with a hydrophobic side chain including I or M;
the CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the CDR3 sequence (CDR3H) of the heavy chain variable region comprises the sequence HX24FDYX6X19X7WFAX25(SEQ ID NO: 47);
wherein X24 is D or G;
wherein X6 is D or F;
wherein X19 is P or A;
wherein X7 is A or Y; and is
Wherein X25 is H or Y;
the CDR1 sequence (CDR1L) of the light chain variable region comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQWX10X10NPPT(SEQ ID NO:48)；
Wherein X10Is S or T;
wherein
When X21 is I, X6Is F, X19 is P and/or X7 is Y.
In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
The CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X14FTFX22X26HAX3S(SEQ ID NO:49)；
Wherein X14 is G or E;
wherein X17Is S or N;
wherein X22Is N or S;
wherein X26 is A or D and
wherein X3 is an amino acid with a hydrophobic side chain including I or M;
the CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the CDR3 sequence (CDR3H) of the heavy chain variable region comprises the sequence HX24FDYX6X19X7WFAX25(SEQ ID NO: 47);
wherein X24 is D or G;
wherein X6Is D or F;
wherein X19 is P or A;
wherein X7 is A or Y; and is
Wherein X25 is H or Y;
the CDR1 sequence (CDR1L) of the light chain variable region comprises sequence RANSAVSYMY (SEQ ID NO: 4);
the CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQWX10X10NPPT(SEQ ID NO:48)；
Wherein X10Is S or T.
In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof, the CEACAM1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRs 1H, CDR2H and CDR3H (Kabat definition), wherein the light chain variable region comprises CDRs 1L, CDR2L and CDR3L (Kabat definition), and wherein:
The sequence of CDR1H comprises sequence X1HX2X3S(SEQ ID NO:1)，
The sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2),
the sequence of CDR3H comprises the sequence HX4X5DYX6PX7WFAX8(SEQ ID NO:3)，
The sequence of CDR1L includes sequence RANSAVSYMY (SEQ ID NO:4),
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5), and
the sequence of CDR3L comprises the sequence QQX9X10X11X12PX13T(SEQ ID NO:6)。
X1-X18As previously defined.
In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises a CDR1, a CDR2, and a CDR3, and wherein:
the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S(SEQ ID NO:43)；
Wherein X is any amino acid;
wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M; and is
The CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);
the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8(SEQ ID NO:44)；
Wherein X4Is D, G or P;
wherein X5Is F or P;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F;
the CDR1 sequence (CDR1L) of the light chain variable region comprises sequence RANSAVSYMY (SEQ ID NO: 4);
The CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO: 5); and is
The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQX9X10X11X12PX13T(SEQ ID NO:6)；
Wherein X9Is W or N;
wherein X10Is S or T;
wherein X11Is a or an amino acid with a neutral hydrophilic side chain including S, N and T;
wherein X12Is L, F or N; and is
Wherein X13Is P or F.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1H, CDR2H and CDR3H (Kabat definition), wherein the light chain variable region comprises CDR1L, CDR2L and CDR3L (Kabat definition), and wherein:
the sequence of CDR1H comprises sequence X1HX2X3S(SEQ ID NO:1)，
The sequence of CDR2H comprises sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2),
the sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8(SEQ ID NO:7)，
The sequence of CDR1L includes sequence RANSAVSYMY (SEQ ID NO:4),
the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5), and
the sequence of CDR3L comprises the sequence QQX9SSX12PX13T(SEQ ID NO:8)。
X1-X18As previously defined.
According to certain embodiments, contemplated antibodies and antigen binding fragments thereof are also characterized by a humanized framework for reduced immunogenicity. In certain embodiments, the CDRs of the contemplated antibody or antigen-binding fragment thereof are located in a framework obtained from a human antibody or antigen-binding fragment thereof. In other embodiments, the surface exposed framework residues of the contemplated antibody or antigen binding fragment thereof are replaced with framework residues of a human antibody or antigen binding fragment thereof. CDRs can also be located in a murine or humanized framework linked to human constant regions (i.e., chimeric antibodies). In a preferred embodiment, the CDRs of the antibody or antigen-binding fragment thereof under consideration are located in a framework that is a complex of two or more human antibodies. In such embodiments, contemplated antibodies or antigen-binding fragments thereof comprise two or more sequence segments ("complexes") derived from V regions of unrelated human antibodies selected to maintain monoclonal antibody sequences important for antigen binding of the starting precursor anti-human CEACAM1 monoclonal antibody, and have all been used with "computer tools" (Holgate and Baker, idrugs.2009; 12(4) 233-7) filtering for the presence of potential T cell epitopes. Close matching of human sequence segments to all portions of the starting antibody V region and elimination of CD4 prior to synthesis of the antibody or antigen-binding fragment thereof+T cell epitopes allow this technology to circumvent immunogenicity while maintaining optimal affinity and specificity through prior analysis of sequences necessary for antigen specificity (Holgate and Baker, 2009).
Also provided herein are variable heavy and variable light chain sequences and pairs thereof that are similar to, but not identical to, the variable heavy and variable light chains and pairs thereof disclosed in SEQ ID NOS: 13-16.
In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID No. 13.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID No. 16. In other embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID NO. 14. In other embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID NO. 15.
In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID No. 13 and a variable light chain amino acid sequence comprising SEQ ID No. 14.
In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID No. 13 and a variable light chain amino acid sequence comprising SEQ ID No. 15.
In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID No. 13 and a variable light chain amino acid sequence comprising SEQ ID No. 16.
The term "identity", as used herein, refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the position in each sequence aligned for comparison purposes. For example, when a position in the nucleotide sequences being compared is occupied by the same base, then the molecules are identical at that position. The degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at the shared position. For example, polypeptides that are at least 85%, 90%, 95%, 98%, or 99% identical to, and preferably exhibit substantially the same function as, a particular polypeptide described herein are contemplated, as are polynucleotides encoding such polypeptides. Methods and computer programs for determining sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al, Nucleic acids ds Research 12:387,1984), BLASTP, BLASTN, FASTA (Altschul et al, J.mol.biol.215:403(1990) and the ALIGN program (version 2.0.) the well-known Smith Waterman algorithm can also be used to determine similarity.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13; and/or
(ii) A light chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO. 14.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13;
(ii) a light chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO 14
(iii) And wherein:
the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO 13,
the sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO. 13,
The sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO. 14,
the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO 14 and
the sequence of CDR3L includes residues S91 and S92 of SEQ ID NO. 14.
The numbering of residues is based on the primary amino acid sequence of the antibody, see fig. 3A, 3B and 3C, e.g., heavy and light chain sequences.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13;
(ii) a light chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO. 14; and
(iii) six CDRs, wherein:
a. the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO 9;
b. the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO. 2;
c. the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO. 10;
d. the CDR1 sequence of the light chain variable region comprises SEQ ID NO 4;
e. the CDR2 sequence of the light chain variable region comprises SEQ ID NO 5; and is
f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO: 11.
In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region;
wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 13;
wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO. 14;
wherein the sequence of the heavy variable chain comprises the sequence GXXXXXXX1HX2X3S(SEQ ID NO:43)；
Wherein X is any amino acid;
wherein X1Is A, D, N or S;
wherein X2Is A or G; and is
Wherein X3Is an amino acid with a hydrophobic side chain comprising I or M; and is
Wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8(SEQ ID NO:44)；
Wherein X4Is D, G or P;
wherein X5Is F or P;
wherein X7Is A or Y; and is
Wherein X8Is L, H or F.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13; and/or
(ii) A light chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO. 15.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13;
(ii) a light chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO. 15;
(iii) and wherein:
the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO 13,
the sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO. 13,
the sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO. 15,
the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO 15 and
the sequence of CDR3L includes residue S92 of SEQ ID NO. 15.
The numbering of residues is based on the primary amino acid sequence of the antibody, see fig. 3A, 3B and 3C, e.g., heavy and light chain sequences.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(iv) A heavy chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13;
(v) a light chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO. 15; and
(vi) six CDRs, wherein:
a. the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO 9;
b. the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO. 2;
c. the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO. 10;
d. the CDR1 sequence of the light chain variable region comprises SEQ ID NO 4;
e. the CDR2 sequence of the light chain variable region comprises SEQ ID NO 5; and is
f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO 12.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13; and/or
(ii) A light chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO 16.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(i) A heavy chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13;
(ii) a light chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO 16;
(iii) and wherein:
the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO 13,
the sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO. 13,
the sequence of CDR1L comprises residues S30 and Y31 of SEQ ID NO 16,
the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO 16 and
the sequence of CDR3L includes residues S91 and S92 of SEQ ID NO 16.
The numbering of residues is based on the primary amino acid sequence of the antibody, see fig. 3A, 3B and 3C, e.g., heavy and light chain sequences.
In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises
(vii) A heavy chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO 13;
(viii) a light chain variable domain comprising a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO 16; and
(ix) six CDRs, wherein:
a. the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO 9;
b. the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO. 2;
c. the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO. 10;
d. the CDR1 sequence of the light chain variable region comprises SEQ ID NO 18;
e. the CDR2 sequence of the light chain variable region comprises SEQ ID NO 5; and is
f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO: 11.
Obviously, any of the frameworks described herein can be used in combination with any of the CDRs and CDR motifs described herein. In some embodiments, CEACAM1 antibodies or antigen-binding fragments thereof are utilized
The framework described in table 1.
In some embodiments of the aspects described herein, amino acid sequence modifications of an antibody or antigen-binding fragment thereof that binds CEACAM1 described herein are contemplated. Amino acid sequence variants of an antibody or antigen-binding fragment thereof are prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the antibody or antigen-binding fragment thereof or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequence of the antibody or antigen-binding fragment thereof. Any combination of deletions, insertions and substitutions are made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., binding specificity, inhibition of biological activity.
One type of variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antibody or antigen-binding fragment thereof replaced with a different residue having similar side chain properties. Amino acids can be grouped according to similarity in their side chain properties (see Lehninger, BIOCHEMISTRY (2 nd edition, Worth Publishers, New York, 1975):
(1) non-polar: ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);
(2) uncharged polarity: gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);
(3) acidity: asp (D), Glu (E);
(4) alkalinity: lys (K), Arg (R), His (H).
Thus, a non-limiting example of a conservative amino acid substitution is the replacement of a non-polar amino acid with another non-polar amino acid.
Alternatively, naturally occurring residues may be grouped based on common side chain properties:
(1) hydrophobicity: ala (A), Val (V), Leu (L), Ile (I), Met (M);
(2) neutral hydrophilicity: ser (S), Thr (T), Cys (C), Asn (N), Gln (Q);
(3) acidity: asp (D), Glu (E);
(4) alkalinity: lys (K), Arg (R), His (H);
(5) residues that influence chain orientation: gly (g), pro (p);
(6) aromatic: phe (F), Trp (W), Tyr (Y).
Thus, a non-limiting example of a conservative amino acid substitution is the replacement of a hydrophobic amino acid with another hydrophobic amino acid.
Amino acid sequence insertions are also contemplated, which can include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody or antigen-binding fragment thereof having an N-terminal methionyl residue or an antibody or antigen-binding fragment thereof fused to a cytotoxic polypeptide. Other insertional variants of the antibody or antigen-binding fragment thereof include the fusion of the N-or C-terminus of the antibody or antigen-binding fragment thereof to an enzyme or polypeptide (e.g., biotin) that increases the serum half-life of the antibody or antigen-binding fragment thereof.
Any cysteine residue not involved in maintaining the correct conformation of the antibody or antigen binding fragment thereof that binds CEACAM1 may also be substituted, for example by serine or alanine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking.
Conversely, cysteine bonds may be added to the antibody or antigen-binding fragment thereof to improve its stability (particularly where the antibody or antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).
In some embodiments, the antibody or antigen-binding fragment thereof has amino acid changes that alter the original glycosylation pattern of the antibody or antigen-binding fragment thereof. By "altering the original glycosylation pattern" is meant deleting one or more carbohydrate moieties present in the antibody or antigen-binding fragment thereof, and/or adding one or more glycosylation sites not present in the antibody or antigen-binding fragment thereof. Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The addition of glycosylation sites to an antibody or antigen-binding fragment thereof that binds CEACAM1 is achieved by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration may also be made by adding or substituting one or more serine or threonine residues in the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).
In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof provided herein is deglycosylated or non-glycosylated. In some embodiments, contemplated CEACAM1 antibodies or antigen-binding fragments thereof lack a C-terminal lysine in the heavy chain and/or contain an S241P substitution in the constant region of the heavy chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks glycosylation sites in the CDRs 1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks the N-X-S/T consensus sequence in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof has a mutation in CDR residues 26 and/or 29(Kabat numbering) of CDR1 of the variable light chain. When the antibody or antigen binding fragment thereof comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies having a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody or antigen-binding fragment thereof are described. See, for example, U.S. patent publication nos. 2003/0157108; 2004/0093621. In WO 03/011878; antibodies having bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of an antibody or antigen binding fragment thereof are cited in U.S. Pat. No. 6,602,684. Antibodies having at least one galactose residue in an oligosaccharide linked to the Fc region of an antibody or antigen binding fragment thereof are reported in WO 97/30087. See also WO 98/58964; WO 99/22764 which relates to an antibody having an altered carbohydrate linked to its Fc region.
In some embodiments, it may be desirable to modify an antibody or antigen-binding fragment thereof that binds CEACAM1 described herein with respect to effector function, e.g., to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody or antigen-binding fragment thereof. This may be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody or antigen-binding fragment thereof. Alternatively or additionally, one or more cysteine residues may be introduced in the Fc region, allowing for interchain disulfide bond formation in this region. The homodimeric antibody or antigen-binding fragment thereof so produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, 176j.exp.med.1191 (1992); shop, 148j. immunol.2918 (1992). Hetero-bifunctional cross-linkers can also be used to prepare homodimeric antibodies with enhanced anti-tumor activity, as described by Wolff et al, 53Cancer Res.2560 (1993). Alternatively, an antibody or antigen-binding fragment thereof having a dual Fc region can be engineered and thus can have enhanced complement lysis and ADCC capabilities. See Stevenson et al, 3Anti-Cancer Drug Design 219 (1989).
For example, WO 00/42072 describes an antibody with improved ADCC function in the presence of human effector cells, wherein the antibody comprises an amino acid substitution in its Fc region. Preferably, the antibody or antigen-binding fragment thereof with improved ADCC comprises a substitution at position 298, 333 and/or 334 of the Fc region (Eu numbering of residues). Typically, the altered Fc region is a human IgG1 Fc region comprising or consisting of a substitution at one, two, or three of these positions. Such substitutions are optionally combined with substitutions that increase Clq binding and/or CDC. The substitution included the Asn297Ala mutation in IgG1 Fc.
Antibodies with altered Clq binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO 99/51642, U.S. Pat. nos. 6,194,551, 6,242,195, 6,528,624 and 6,538,124. The antibody comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334 of its Fc region (Eu numbering of residues).
Antibodies with improved binding to neonatal Fc receptor (FcRn) and increased half-life are described in WO 00/42072 and U.S. patent publication No. 2005/0014934. These antibodies comprise an Fc region having one or more substitutions therein which improve binding of the Fc region to CEACAM 1. For example, the Fc region may have a substitution (Eu numbering of residues) at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, or 434. Antibody variants comprising a preferred Fc region with improved CEACAM1 binding comprise amino acid substitutions at one, two or three of positions 307, 380 and 434 of its Fc region (Eu numbering of residues). In one embodiment, the antibody or antigen-binding fragment thereof has 307/434 mutations. Engineered antibodies that bind CEACAM1 having three or more (e.g., four) functional antigen binding sites are also contemplated. See, for example, U.S. patent publication No. US 2002/0004587.
Antibody fragments and types
In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab fragment comprising a variable (V) of the light chainL) And constant (C)L) Domains and variable domains of heavy chains (V)H) And a first constant domain (C)H1) Or consist essentially of it.
In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab' fragment, which refers to a fragment at CH1 domain having one or more cysteine residues at the C-terminus.
In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a CEACAM1 antibody comprising VHAnd CH1 domain or an Fd fragment consisting essentially of the same.
In some embodiments of the aspects described herein, the CEACAM1 antibody moiety is comprising VHAnd CH1 domain and at CH1 domain C-terminal one or more cysteine residues.
Single chain Fv or scFv antibody fragments comprising the V of an antibodyHAnd VLDomains consist essentially of, or consist of, such that these domains are present in a single polypeptide chain. Generally, Fv polypeptides are described at VHAnd VLAlso included between the domains is a polypeptide linker that allows the scFv to form the desired structure for antigen binding. See, for example, Pluckthun,113Pharmacology Monoclonal Antibodies 269(Rosenburg and Moore, eds., Springer-Verlag, New York, 1994). Thus, in some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a V comprising a single arm of an antibody LAnd VHA domain or an Fv fragment consisting essentially thereof.
In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a diabody comprising two antigen binding sites comprising a variable domain (V) linked to a light chain in the same polypeptide chainL) Heavy chain variable domain of (V)H)。
In some embodiments of the aspects described herein,the CEACAM1 antibody moiety is comprised of VHA domain or a dAb fragment consisting essentially thereof.
In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a F (ab ')2 fragment comprising a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region.
Linear antibodies are those described by Zapata et al, Protein Engin, 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of Fd fragments (V) in tandemH-CH1-VH-CH1) Which together with the complementary light chain polypeptide form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a fragment comprising a pair of tandem Fd fragments (V)H-CH1-VH-CH1) The linear antibody of (1), said tandem Fd fragment forming a pair of antigen-binding regions with a complementary light chain polypeptide.
Various techniques have been developed and can be used to generate antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies. See, e.g., Morimoto et al, 24j. biochem. biophysis. maths.107 (1992); brennan et al, 229Science 81 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from an antibody phage library as discussed herein. Alternatively, Fab '-SH fragments can be directly recovered from E.coli and chemically coupled to form F (ab')2 fragments (Carter et al, 1992). According to another approach, the F (ab')2 fragment can be isolated directly from recombinant host cell culture. Other techniques for preparing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody fragment of choice is a single chain Fv fragment (scFv). See, for example, WO 93/16185.
In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds CEACAM1 and a complementary region that binds PD-1.
In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds CEACAM1 and a complementary region that binds PD-L1.
Contemplated antibodies or antigen-binding fragments may have all types of constant regions, including IgM, IgG, IgD, and IgE, as well as any isotype, including IgG1, IgG2, IgG3, and IgG 4. In one embodiment, human isotype IgG1 is used. In another embodiment, human isotype IgG4 is used. The light chain constant region may be lambda or kappa. An antibody or antigen-binding fragment thereof may comprise sequences from more than one class or isotype.
Also disclosed herein are chimeric antigen receptor T cells (CAR T cells) that bind CEACAM 1. In one embodiment, one or more CDRs of the anti-CEACAM antibodies disclosed herein are grafted onto a Chimeric Antigen Receptor (CAR) on a T cell. Such genetically modified T cells utilize CARs (also known as chimeric T cell receptors) to target antigens expressed on tumor cells in a human leukocyte antigen-independent manner.
Antibody binding
The human CEACAM1 gene produces 11 isoforms by alternative splicing. Each isoform has a variable (V) -like Ig domain at the amino (N) terminus of the protein. In addition to the CEACAM1-1L and CEACAM1-1S isoforms, the different isoforms have 2 or 3 constant C2-like Ig domains. The eight CEACAM1 isoforms are anchored to the cell membrane through transmembrane domains, and the three CEACAM1 isoforms (CEACAM1-4C1-3 and-3C 2) lack a transmembrane domain and are secreted. The two isoforms (CEACAM1-3AL and-3 AS) have an Alu family repeat (A) between the constant C2-like Ig domain and the transmembrane domain. The transmembrane CEACAM1 isoform also has a long (L) or short (S) cytoplasmic domain, as determined by the inclusion or exclusion of exon 7 of CEACAM1 in the message. CEACAM 1L cytoplasmic domain has two ITIM motifs, which are unique to CEACAM1 among CEACAM family members. In one aspect, the present invention provides CEACAM1 antibodies or antigen-binding fragments thereof, including antibodies described herein by their structural features, that bind an extracellular variable (V) -like Ig domain, a domain common to all isoforms of CEACAM1, including CEACAM1 isoforms 1L, 1S, 3L, 3S, 4L, 4S, 3a1, 3AS, 3, 4C1, and 4C2, at the amino (N) terminus (N domain) of the protein of CEACAM 1. In some embodiments, provided antibodies and antigen binding fragments thereof bind to human CEACAM 1. In some embodiments, provided antibodies and antigen binding fragments thereof bind to mammalian CEACAM 1. The sequence of the full-length form of CEACAM1 (NCBI reference sequence NP-001703.2; UNIPROT ID P13688) is provided as SEQ ID NO:26 (signal sequence: residues 1-34 of SEQ ID NO: 26; Ig-V N domain: residues 35-142 of SEQ ID NO: 26. the mature form of CEACAM1 (NO signal sequence) is provided as SEQ ID NO: 17.
As used herein, "binding" of an antibody or antigen-binding fragment thereof to an epitope on CEACAM1, CEACAM1, or a specific residue on CEACAM1 in certain embodiments described below includes selective interaction of the antibody or antigen-binding fragment thereof with CEACAM 1. Thus, binding includes, for example, primary and secondary interactions, including hydrogen bonding, ionic interactions, salt bridges, and hydrophilic and hydrophobic interactions.
In certain embodiments, the CEACAM1 antibody or antigen-binding fragment thereof described herein is present at 10-5To 10-12mol/l、10-6To 10-12mol/l、10-7To 10-12mol/l、10-8To 10-12mol/l、10-9To 10-12mol/l、10-10To 10-12mol/l or 10-11To 10-12mol/l of KDCombined with CEACAM 1. In other embodiments, the CEACAM1 antibody or antigen-binding fragment thereof described herein is present at 10-5To 10-11mol/l、10-6To 10-11mol/l、10-7To 10-11mol/l、10-8To 10-11mol/l、10-9To 10-11mol/l or 10-10To 10-11mol/l of KDCombined with CEACAM 1. In other embodiments, the CEACAM1 antibody or antigen-binding fragment thereof described herein is present at 10-5To 10-10mol/l、10-6To 10-10mol/l、10-7To 10-10mol/l、10-8To 10-10mol/l or 10-9To 10-10mol/l of KDCombined with CEACAM 1. In other embodiments, the CEACAM1 antibody or antigen-binding fragment thereof described herein is present at 10-5To 10-8mol/l、10-6To 10-8mol/l or 10-7To 10-8mol/l of KDCombined with CEACAM 1.
The term "specific" as used herein refers to the ability of an antibody or antigen-binding fragment thereof (such as an anti-CEACAM 1 antibody or antigen-binding fragment thereof) to recognize an epitope within CEACAM1 with little or no detectable reactivity with other portions of CEACAM 1. Specificity can be determined relatively by competition assays or by epitope identification/characterization techniques described herein or equivalents thereof known in the art.
As used herein, an "epitope" may be formed of contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5, about 9, or about 8-10 amino acids in a particular spatial conformation. "epitopes" include conventional immunoglobulin VH/VLFor the structural unit of the binding. An epitope defines the minimum binding site of an antibody or antigen-binding fragment thereof and thus represents a specific target for the antibody or antigen-binding fragment thereof. In the case of single domain antibodies, the epitope represents the structural unit bound by the isolated variable domain.
In particular embodiments, the antibody or antigen-binding fragment of interest specifically binds to the same epitope as antibody CP08H03/Vk 8S 29A. In another embodiment, contemplated antibodies or antigen binding fragments bind to the same epitope as CP08H03/CP08F 05.
In one aspect, the invention provides antibodies and antigen binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen binding fragments thereof specifically bind to at least a portion of an amphotropic binding domain on CEACAM1 (i.e., a portion of CEACAM1 protein involved in CEACAM1: CEACAM1 homodimer formation), thereby blocking CEACAM1 amphotropic interactions. In certain embodiments, provided antibodies or antigen-binding fragments thereof specifically bind to one or more of CEACAM1 residues contained in the CC' and FG loops of CEACAM1 and including the YQQN pocket (i.e., Y34, Q44, Q89, N97 of SEQ ID NO: 17) at the CEACAM1: CEACAM1 dimer interface, see Huang et al, nature.2015jan15; 517(7534):386-90.
As used herein, a "blocking" antibody or antibody "antagonist" is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. For example, in some embodiments, a CEACAM1 antagonist antibody or antigen-binding fragment thereof binds CEACAM1 and inhibits the activity of CEACAM1 and/or the binding of CEACAM1 to a heterologous binding partner (such as other CEACAM proteins or TIM-3). Inhibition of activity and inhibition of binding includes partial inhibition. Methods for identifying CEACAM1 antibodies that block homotropic and heterophilic interactions of CEACAM1 are described herein and are known to those of skill in the art. For example, competing, cross-blocking and cross-blocked antibodies can be identified using any suitable method known in the art, including competition ELISAs orAn assay wherein binding of a competing antibody or a cross-blocking antibody to human CEACAM1 prevents binding of the antibodies disclosed herein and vice versa.
In one embodiment, the heavy chain of the contemplated antibody or antigen binding fragment thereof specifically binds CEACAM1 at residues F29, Y34, T56, Q89, S93 and/or D94 of SEQ ID No. 17. In another embodiment, the heavy chain of the contemplated antibody or antigen-binding fragment thereof also specifically binds CEACAM1 at residues S32, Q44, a49, and/or I91 of SEQ ID No. 17.
In one embodiment, the light chain of the contemplated antibody or antigen-binding fragment thereof specifically binds CEACAM1 at residues D40, G41, N42, N97, and/or E99 of SEQ ID No. 17. In another embodiment, the light chain of the contemplated antibody or antigen-binding fragment thereof also specifically binds CEACAM1 at residues L95 and/or V96 of SEQ ID No. 17.
In another embodiment, the CEACAM1 antibody or antigen binding fragment thereof specifically binds CEACAM1 at residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and/or E99 of SEQ ID NO: 17. In another preferred embodiment, the CEACAM1 antibody or antigen-binding fragment thereof also specifically binds CEACAM1 at residues S32, Q44, a49, I91, L95, and/or V96 of SEQ ID NO: 17.
In another embodiment, the CEACAM1 antibody or antigen binding fragment thereof specifically binds CEACAM1 at residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID No. 17.
In another embodiment, the CEACAM1 antibody or antigen binding fragment specifically binds CEACAM1 at residues F29, S32, Y34, D40, G41, N42, Q44, a49, T56, Q89, I91, S93, D94, L95, V96, N97, and E99 of SEQ ID No. 17.
In certain embodiments, not all CDRs are directly involved in binding to the antigen. In one embodiment, four of the six CDRs of the CEACAM1 antibody or antigen-binding fragment thereof are contacted with an antigen. In one embodiment, five of the six CDRs of the CEACAM1 antibody or antigen-binding fragment thereof are contacted with an antigen. In one embodiment, six of the six CDRs of the CEACAM1 antibody or antigen-binding fragment thereof are contacted with an antigen. In one embodiment, the CDR2H, CDR3H, CDR1L, CDR2L and CDR3L of the CEACAM1 antibody or antigen binding fragment thereof is directly involved in binding to an antigen.
In one embodiment, the antibodies and antigen binding fragments thereof provided herein specifically bind to an epitope of CEACAM1 located on the N domain of CEACAM 1. In one embodiment, the antibody or antigen binding fragment thereof specifically binds to a CEACAM1 epitope comprising one or more CEACAM1 residues selected from F29, S32, D40, a49, and T56 of SEQ ID NO 17. In another embodiment, the CEACAM1 antibody specifically binds to the CEACAM1 epitope comprising residues F29, S32, D40, a49, T56, and I91 of SEQ ID NO: 17.
In one embodiment, the antibodies and antigen binding fragments thereof provided herein specifically bind to an epitope of CEACAM1 located on the N domain of CEACAM 1. In one embodiment, the antibody or antigen binding fragment thereof specifically binds to a CEACAM1 epitope comprising one or more CEACAM1 residues selected from S32, D40, a49, and I91 of SEQ ID NO 17. In another embodiment, the CEACAM1 antibody specifically binds to the CEACAM1 epitope comprising residues S32, D40, a49 and I91 of SEQ ID No. 17.
In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof provided herein binds CEACAM1, wherein
CDR2H residue Y57 binds to CEACAM1 at residue F29,
CDR2H residue Y59 binds to CEACAM1 at residue S93,
CDR3H residue D102 bound CEACAM1 at residue T56,
CDR3H residue Y103 binds to CEACAM1 at residues Y34 and/or Q89,
CDR3H residue F104 bound CEACAM1 at residue F29,
CDR3H residue Y106 binds CEACAM1 at residue D94,
CDR1L residue S30 binds to CEACAM1 at residue E99,
CDR1L residue Y31 binds to CEACAM1 at residue N97,
CDR2L residue S51 binds to CEACAM1 at residue D40, and/or
CDR2L residue N52 binds CEACAM1 at residues G41 and/or N42.
The numbering of CDR residues is based on the primary amino acid sequence of the antibody, see fig. 3A, 3B and 3C, e.g., heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO: 17.
In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof provided herein binds CEACAM1, wherein
CDR2H residue Y57 binds to CEACAM1 at residue F29,
CDR2H residue Y59 binds to CEACAM1 at residue S93,
CDR3H residue D102 bound CEACAM1 at residue T56,
CDR3H residue Y103 binds CEACAM1 at residues S32, Y34, Q44 and/or Q89,
CDR3H residue F104 bound to CEACAM1 at residue F29 and/or A49,
CDR3H residue P105 bound to CEACAM1 at residue I91,
CDR3H residue Y106 binds CEACAM1 at residue D94,
CDR1L residue S30 binds to CEACAM1 at residue E99,
CDR1L residue Y31 binds to CEACAM1 at residue N97,
CDR2L residue S51 binds to CEACAM1 at residue D40,
CDR2L residue N52 binds to CEACAM1 at residue G41 and/or N42,
CDR3L residue S91 binds to CEACAM1 at residue L95, and/or
CDR3L residue S92 binds CEACAM1 at residue V96.
The numbering of residues is based on the primary amino acid sequence of the antibody, see fig. 3A, 3B and 3C, e.g., heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO: 17.
In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof provided herein binds CEACAM1, wherein
CDR2H residue Y57 binds to CEACAM1 at residue F29,
CDR2H residue Y59 binds to CEACAM1 at residue S93,
CDR3H residue D102 bound CEACAM1 at residue T56,
CDR3H residue Y103 binds CEACAM1 at residues S32, Y34, Q44 and Q89,
CDR3H residue F104 bound to CEACAM1 at residues F29 and A49,
CDR3H residue P105 bound to CEACAM1 at residue I91,
CDR3H residue Y106 binds CEACAM1 at residue D94,
CDR1L residue S30 binds to CEACAM1 at residue E99,
CDR1L residue Y31 binds to CEACAM1 at residue N97,
CDR2L residue S51 binds to CEACAM1 at residue D40,
CDR2L residue N52 binds to CEACAM1 at residues G41 and N42,
CDR3L residue S91 binds to CEACAM1 at residue L95, and
CDR3L residue S92 binds CEACAM1 at residue V96.
The numbering of residues is based on the primary amino acid sequence of the antibody, see fig. 3A, 3B and 3C, e.g., heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO: 17.
Members of the CEACAM family are widely expressed on a variety of cell types (especially leukocytes), thereby affecting the magnitude of cellular function. For example, CEACAM1 is expressed on epithelial cells, endothelial cells, lymphocytes and bone marrow cells, CEACAM3 is expressed on granulocytes and neutrophils, CEACAM5 is expressed on epithelial cells, and CEACAM6 is expressed on epithelial cells and granulocytes. However, the N domain of CEACAM1 has about 90% similarity to the N domains of CEACAM family members 3, 5 and 6, making it difficult to selectively target CEACAM 1.
Although the N domains are highly similar in CEACAM family members, in some embodiments, the antibodies provided herein, or antigen-binding fragments thereof, including antibodies described herein by their structural features, are selective for CEACAM 1. By selectively targeting CEACAM1, embodiments of the invention can avoid unwanted interference, such as the broad activation function of CEACAM 3.
The terms "selective" and "selectivity" refer herein to the preferential binding of an antibody or antigen-binding fragment thereof (i.e., CEACAM1 antibody or antigen-binding fragment thereof) to a particular region, target, or peptide; the region, target or peptide is typically a region or epitope in CEACAM1, and is not one or more other biomolecules, including other CEACAM family members.
In some embodiments, contemplated CEACAM1 antibodies or antigen-binding fragments thereof do not exhibit significant binding to CEACAM3, CEACAM5, CEACAM6, and/or CEACAM 8. In some embodiments, contemplated CEACAM1 antibodies or antigen-binding fragments thereof do not exhibit detectable binding to CEACAM3, CEACAM5, CEACAM6, and/or CEACAM 8. In some embodiments, the binding affinity of the contemplated CEACAM1 antibody or antigen-binding fragment thereof to CEACAM1 is at least 10-fold, such as at least 100-fold, and at least 1000-fold, and at most 10,000-fold or greater, the binding affinity of the contemplated CEACAM1 antibody or antigen-binding fragment thereof to another target or polypeptide.
As used herein, binding of an antigen to an antigen by the antigenEquilibrium constant (K) for dissociation of proteinsD) The expression "affinity" is a measure of the strength of binding between an antigenic determinant and an antigen-binding site on an antigen-binding protein (such as an antibody or antibody fragment thereof). K DThe smaller the value, the stronger the binding strength between the antigenic determinant and the antigen binding molecule. Alternatively, affinity can also be expressed as an affinity constant (K)A) Which is 1/KD). As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the particular antigen of interest.
In one aspect, the invention provides antibodies and antigen binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen binding fragments thereof specifically bind to at least a portion of the binding site of one or more other members of the CEACAM family on CEACAM1, thereby blocking the interaction of CEACAM1 with one or more other members of the CEACAM family. These CEACAM family members include, but are not limited to, CEACAM3, CEACAM5, CEACAM6 and CEACAM8(Ramani et al, anal. biochem. Jan.15, 2012; 420 (2); 127-38; Scheffrahn et al, J.Immunol. May.15, 2002; 168 (10); 5139-46).
In one aspect, the invention provides antibodies and antigen binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen binding fragments thereof specifically bind to at least a portion of the binding site of a TIM family member on CEACAM1, thereby blocking the interaction of CEACAM1 with the TIM family member. In some embodiments, the TIM family member is TIM-1, TIM-3 or TIM-4. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds to one or more of CEACAM1 residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and/or N97 of SEQ ID No. 17, which residues have been shown to be involved in the binding of CEACAM1 to TIM-3 (Huang et al, nature.2015jan 15; 517(7534): 386-90).
In one aspect, the invention provides antibodies and antigen binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen binding fragments thereof specifically bind to at least a portion of the binding site of a bacterial adhesion surface protein (adhesin) on CEACAM1, thereby blocking the interaction between CEACAM1 and the adhesin. In certain embodiments, the adhesin is expressed on the surface of a pathogenic bacterium that binds CEACAM1, including but not limited to escherichia coli, particularly Diffusible Adhesion Escherichia Coli (DAEC), neisseria gonorrhoeae, neisseria meningitidis, neisseria symbiosis, Moraxella catarrhalis, haemophilus influenzae, haemophilus aegypti, helicobacter pylori, and/or Salmonella (Salmonella sp.).
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and HopQ expressed on the surface of helicobacter pylori. In one embodiment, the CEACAM1 antibody or antigen binding fragment specifically binds to one or more of CEACAM1 residues F29, Y34, N42, Q89, and N97, which residues have been predicted to be involved in binding CEACAM1 to HopQ.
In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and haze associated (Opa) adhesin proteins expressed on a surface of neisseria species, including but not limited to Opas2, Opa65, Opa68, Opav0, Opa72, Opa73, Opa74, and Opa 75. In one embodiment, the CEACAM1 antibody or antigen binding fragment specifically binds to one or more of CEACAM1 residues Q44 and a49 that have been predicted to be involved in the binding of CEACAM1 to neisseria Opa proteins.
In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Opa-like protein OlpA expressed on the surface of moraxella.
In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof disrupts the interaction between CEACAM1 and haemophilus influenzae OMP 1. In one embodiment, the CEACAM1 antibody or antigen binding fragment specifically binds to one or more of CEACAM1 residues Q44 and a49, which residues have been predicted to be involved in the binding of CEACAM1 to haemophilus influenzae OMP P1.
In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and haemophilus aegypti OMP 1. In one embodiment, the CEACAM1 antibody or antigen binding fragment specifically binds CEACAM1 residue F29, which has been predicted to be involved in the binding of CEACAM1 to haemophilus aegypti OMP 1.
In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and candida albicans.
In another embodiment, the CEACAM1 antibody or antigen binding fragment thereof disrupts the interaction between CEACAM1 and influenza viruses (including but not limited to H5N 1).
In another embodiment, the invention provides a method of inhibiting CEACAM1 binding to a synechocystis using a CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the sub-nematode is wuchereria bambusicola.
Antibody conjugates
In some embodiments of the aspects described herein, the antibody or antigen-binding fragment thereof that binds CEACAM1 is conjugated to a functional moiety. Examples of functional moieties that can be used include, but are not limited to, blocking moieties, detectable moieties, diagnostic moieties, targeting moieties, and therapeutic moieties.
Exemplary blocking moieties include moieties with sufficient steric hindrance and/or charge such that glycosylation is reduced from occurring, for example, by blocking the ability of the glycosidase glycosylation antibody or antigen binding fragment thereof. Additionally or alternatively, the blocking moiety may reduce effector function, for example, by inhibiting the ability of the Fc region to bind to a receptor or complement protein. Preferred blocking moieties include cysteine adducts and PEG moieties.
In a preferred embodiment, the blocking moiety is a cysteine, preferably a cysteine associated with a free cysteine, e.g. in cell culture, e.g. during or after translation of the Fc-containing polypeptide. Other blocking cysteine adducts include cystine, mixed disulfide adducts or disulfide bonds.
In another preferred embodiment, the blocking moiety is a polyalkylene glycol moiety, such as a PEG moiety, and preferably a PEG-maleimide moiety. Preferred pegylated moieties (or related polymers) can be, for example, polyethylene glycol ("PEG"), polypropylene glycol ("PPG"), polyoxyethylated glycerol ("POG") and other polyoxyethylated polyols, polyvinyl alcohol ("PVA") and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer may be a homopolymer, random or block copolymer, terpolymer based on the monomers listed above, linear or branched, substituted or unsubstituted, so long as it has at least one active sulfone moiety. The polymer portion may be of any length or molecular weight, but these characteristics affect the biological properties. Polymers particularly useful for reducing clearance in pharmaceutical applications have an average molecular weight in the range of 2,000 to 35,000 daltons. Furthermore, if two groups are attached to the polymer, one at each end, the length of the polymer can affect the effective distance and other spatial relationships between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or impart the desired biological activity. PEG can be used for biological applications for several reasons. PEG is generally transparent, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, non-hydrolyzing, and non-toxic. Pegylation can improve the pharmacokinetic properties of a molecule by increasing the apparent molecular weight of the molecule. Increased apparent molecular weight reduces clearance from the body following subcutaneous or systemic administration. In many cases, pegylation can reduce antigenicity and immunogenicity. In addition, pegylation can increase the solubility of the bioactive molecule.
Examples of detectable moieties that may be used in the methods and antibodies and antigen binding fragments thereof contemplated by the present invention include fluorescent moieties or labels, imaging agents, radioisotope moieties, radiopaque moieties, and the like, for example, detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radioactive labels. Exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, and the like) and other luminescent molecules (e.g., luminal). The fluorophore may be environmentally sensitive such that it is located in the modified protein if it is locatedNear the residue or residues that undergo a structural change upon binding to a substrate (e.g., a dansyl probe), then its fluorescence changes. Exemplary radiolabels include those comprising a core with one or more low sensitivity(s) (ii)13C、15N、2H、125I、123I、99Tc、43K、52Fe、67Ga、68Ga、111In, etc.) of a single molecule. Other useful moieties are known in the art.
Examples of diagnostic moieties that can be used in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include detectable moieties suitable for revealing the presence of a disease or disorder. Generally, the diagnostic moiety allows for the determination of the presence, absence or level of a molecule (e.g., a target peptide, protein or proteins) associated with a disease or disorder. Such diagnosis is also useful for predicting and/or diagnosing a disease or disorder and its progression.
Examples of therapeutic moieties that can be used in the contemplated methods and antibodies and antigen-binding fragments thereof of the present invention include, for example, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-infective agents, or therapeutic agents in general. The functional part may also have one or more of the above-mentioned functions.
Exemplary therapeutic moieties include radionuclides with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA and are therefore suitable for inducing cell death (e.g., of cancer). Exemplary high energy radionuclides include:90Y、125I、131I、123I、111In、105Rh、153Sm、67Cu、67Ga、166Ho、177Lu、186re and188re. These isotopes generally produce high energy alpha or beta particles with short path lengths. Such radionuclides kill cells in close proximity thereto, e.g., neoplastic cells to which the conjugate has been attached or has entered. They have little or no effect on non-localized cells and are substantially non-immunogenic.
Exemplary therapeutic moieties also include cytotoxic agents such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or crosslinkers, or DNA-RNA transcription modulators), enzyme inhibitors, gene modulators, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, and the like.
Exemplary therapeutic moieties also include alkylating agents, such as the anthracycline family of drugs (e.g., doxorubicin, carminomycin, cyclosporin-a, chloroquine, methotrexate, mithramycin, porphyrinomycin, streptomycin, anthracenedione, and aziridine). In another embodiment, the chemotherapeutic moiety is a cytostatic agent, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and methotrexate dichloride, 3-amino-1, 2, 4-benzotriazine 1, 4-dioxide, aminopterin, cytosine β -D-arabinofuranoside, 5-fluoro-5' -deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin, melphalan, mitoxantrone, and oxaliplatin.
Exemplary therapeutic moieties also include transcriptional modulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatic agents compatible with the present invention include ansamycin benzoquinone, quinone derivatives (e.g., quinolones, genistein, bactacrine), busulfan, ifosfamide, mechlorethamine, triimine quinone, diazaquine, carboquone, indoquinone EO9, divinyliminobenzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).
Exemplary therapeutic moieties also include cytotoxic nucleosides such as, for example, adenine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, tegafur, and 6-mercaptopurine; tubulin binding agents such as taxanes (e.g., paclitaxel, docetaxel, taxanes), nocodazole, radicotoxins, dolastatins (e.g., dolastatin 10, 11, or 15), colchicines and colchicinoids (e.g., ZD6126), combretastatins (e.g., combretastatin a-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenic compounds such as angiostatin K1-3, DL- α -difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine and (±) -thalidomide.
Exemplary therapeutic moieties also include hormones and hormone antagonists such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroxyprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17- (allylamino) -17-demethoxygeldanamycin, 4-amino-1, 8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprolide), leuprolide-releasing hormone, pifithrin-alpha, rapamycin, sex hormone binding globulin, and thapsigargin.
Exemplary therapeutic moieties also include enzyme inhibitors such as S (+) -camptothecin, curcumin, (-) -deguelin, 5, 6-dichlorobenzene-imidazole 1- β -D-ribofuranoside, etoposide, fulvestrant, forskocin, milk tree alkaloid (hispidin), 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin (mevinolin), trichostatin a, tyrphostin AG 34, and tyrphostin AG 879.
Exemplary therapeutic moieties also include gene modulators such as 5-aza-2' -deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin a aldehyde), retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin a), tamoxifen, and troglitazone.
Exemplary therapeutic moieties also include cytotoxic agents such as, for example, drugs of the pteridine family, enediynes, and podophyllotoxins. Particularly useful members of these classes include, for example, methotrexate, podophyllotoxin or podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vindesine, vinblastine and the like.
Still other cytotoxins compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), calicheamicin, gramicin D, maytansinoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, imistine, teniposide, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.
Techniques For conjugating such therapeutic moieties to Antibodies are well known, see, e.g., Amon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.), pp 243-56 (Alan R.Liss, Inc.1985); hellstrom et al, "Antibodies For Drug Delivery", Controlled Drug Delivery (2 nd edition), Robinson et al (eds.), pages 623-53 (Marcel Dekker, Inc. 1987); thorpe, "Antibodies Of cytotoxin Agents In Cancer Therapy: A Review", Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al (eds.), pp.475-; "Analysis, Results, And d Future Therapeutic Of The Therapeutic Use Of radioactive Antibody In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al (eds.), pages 303-16 (Academic Press 1985), And Thorpe et al, "The prediction And cytological Properties Of Antibody-Toxin Conjugates", Immunol. Rev.,62:119-58 (1982).
To increase the half-life of an antibody or polypeptide containing an amino acid sequence described herein, a salvage receptor binding epitope can be linked to the antibody or antigen binding fragment thereof (particularly an antibody fragment), as described, for example, in U.S. patent No. 5,739,277. The term "salvage receptor binding epitope" may refer to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) responsible for increasing the serum half-life of the IgG molecule in vivo (e.g., Ghetie et al, 18Ann.Rev.Immunol.739 (2000). antibodies having substitutions in their Fc region and increased serum half-life are also described in WO 00/42072, WO 02/060919; Shield et al, 276J.biol.chem.6591 (2001); Hinton,279J.biol.chem.6213-6216 (2004). for example, a nucleic acid molecule encoding a salvage receptor binding epitope may be linked in-frame to a nucleic acid encoding a polypeptide sequence described herein such that a fusion protein expressed from the engineered nucleic acid molecule comprises the salvage receptor binding epitope and a polypeptide sequence described herein Such that serum albumin binds to an antibody or antigen binding fragment thereof, such as such polypeptide sequences disclosed in WO 01/45746. In one embodiment, the half-life of the Fab is increased by these methods. For additional serum albumin binding peptide sequences see also Dennis et al, 277j.biol.chem.35035 (2002).
Other types of functional moieties are known in the art and can be readily used in the methods and compositions of the present invention based on the teachings contained herein.
Nucleic acids
Also provided herein are nucleic acids encoding CEACAM1 antibodies and antigen-binding fragments thereof, as well as vectors, host cells, and expression systems. As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
The nucleic acid encoding the CEACAM1 antibody and antigen-binding fragments thereof can be, for example, DNA, cDNA, RNA, synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any of those polynucleotides, alone or in combination. For example, expression vectors are provided comprising a polynucleotide sequence encoding the CEACAM1 antibody or antigen-binding fragment thereof described herein operably linked to expression control sequences suitable for expression in eukaryotic and/or prokaryotic host cells.
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. "vector" includes but is not limited to viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules, which may be composed of chromosomal, nonchromosomal, semisynthetic, or synthetic nucleic acids. In some embodiments, the vector used is a vector capable of autonomous replication (episomal vector) and/or expression of the nucleic acid to which it is linked (expression vector). A large number of suitable carriers are known to those skilled in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, AAV), coronaviruses, negative strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and sendai virus), positive strand RNA viruses such as picornaviruses and alphaviruses, and double stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2, epstein-barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukemia-sarcoma, mammalian type C, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, and foamy viruses.
A variety of expression vectors have been developed for the efficient synthesis of antibodies and antigen-binding fragments thereof in prokaryotic cells (such as bacteria) and in eukaryotic systems (including but not limited to yeast and mammalian cell culture systems). The vector may comprise segments of chromosomal, nonchromosomal and synthetic DNA sequences. Also provided are cells comprising an expression vector for expressing the contemplated CEACAM1 antibody or antigen-binding fragment thereof.
Antibody preparation and expression system
The antibodies or antigen-binding fragments thereof of the present invention are typically produced by recombinant expression. Nucleic acids encoding the light and heavy chain variable regions, optionally linked to a constant region, are inserted into an expression vector. The light and heavy chains may be cloned in the same or different expression vectors. The DNA segment encoding the immunoglobulin chain is operably linked to control sequences in an expression vector to ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is integrated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the cross-reactive antibodies.
These expression vectors are typically replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors contain a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow for detection of those cells transformed with the desired DNA sequence (see, e.g., Itakura et al, U.S. Pat. No. 4,704,362).
Expression of the antibodies and antigen binding fragments contemplated by the present invention may occur in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian and mammalian cells in vivo or in situ or host cells of mammalian, insect, avian or yeast origin. The mammalian cell or tissue may be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, but any other mammalian cell may be used.
Coli is a prokaryotic host particularly useful for cloning the polynucleotides (e.g., DNA sequences) of the present invention. Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
Other microorganisms such as yeast may also be used for expression. Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors having expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like, as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.
In addition, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved by using, for example, a yeast ubiquitin hydrolase system. The fusion protein so produced may be processed in vivo or purified and processed in vitro, allowing the synthesis of CEACAM1 antibodies of the invention or antigen-binding fragments thereof having a specific amino-terminal sequence. Furthermore, the problems associated with retaining the start codon-derived methionine residue in direct yeast (or bacterial) expression can be avoided. Sabin et al, 7Bio/technol.705 (1989); miller et al, 7Bio/technol.698 (1989).
When yeast are grown in glucose-rich medium, any of a series of yeast gene expression systems incorporating a large amount of produced promoter and termination elements encoding active expression genes of glycolytic enzymes can be used to obtain the recombinant CEACAM1 antibody or peptide of the present invention. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be used.
Production of CEACAM1 antibodies or antigen-binding fragments thereof in insects can be achieved. For example, an insect host is infected with a baculovirus engineered to express a transmembrane polypeptide by methods known to those skilled in the art. See Ausubel et al, 1987,1993.
In addition to microorganisms, mammalian tissue cultures can also be used to express and produce the antibodies or antigen-binding fragments thereof (e.g., polynucleotides encoding immunoglobulins or fragments thereof) of the present invention. See Winnacker, From Genes to Clones, VCH Publishers, n.y., n.y. (1987). Eukaryotic cells are actually preferred because many suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art and include CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells, and hybridomas. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.89:49(1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J.Immunol.148:1149 (1992).
Alternatively, a nucleotide sequence encoding an antibody or antigen-binding fragment thereof can be incorporated into a transgene for introduction into the genome of the transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al, U.S. patent No. 5,741,957, Rosen, U.S. patent No. 5,304,489, and Meade et al, U.S. patent No. 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes such as casein or beta lactoglobulin.
In addition, plants have become a convenient, safe and economical alternative mainstream expression system for recombinant antibody production, which is based on large-scale culture of microorganisms or animal cells. The antibody or antigen-binding fragment thereof may be expressed in plant cell cultures or in routinely grown plants. Expression in plants can be systemic, restricted to subcellular plastids or restricted to seeds (endosperm). See, for example, U.S. patent publication nos. 2003/0167531; U.S. patent nos. 6,080,560 and 6,512,162; and WO 0129242. Several plant-derived antibodies have reached a mature stage of development, including clinical trials (see, e.g., Biolex, NC).
Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or virus-based transfection may be used for other cellular hosts. (see generally Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2 nd edition, 1989.) other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al, supra.) for the production of transgenic animals, transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.
The antibodies and antigen binding fragments thereof of the present invention may be expressed using a single vector or two vectors. When antibody heavy and light chains are cloned into separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, the whole antibodies of the invention, their dimers, individual light and heavy chains or other immunoglobulin forms may be purified according to standard procedures in the art including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC Purification, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, n.y., (1982)). for pharmaceutical uses, substantially pure immunoglobulins of at least about 90% to 95% homogeneity are preferred, and 98% to 99% or more homogeneity are most preferred.
Method for modulating CEACAM1 activity
In one aspect, the invention provides methods of using the antibodies and antigen binding fragments thereof described herein to reduce the interaction between CEACAM1 and another member of the CEACAM family (including but not limited to CEACAM1, CEACAM3, CEACAM5, CEACAM6, and CEACAM 8). In some embodiments, the antibody or antigen binding fragment thereof disrupts homotropic interactions between CEACAM1 monomers.
In another aspect, the invention provides methods of using the antibodies and antigen binding fragments thereof of the invention to reduce the interaction between CEACAM1 and members of the TIM family, including but not limited to TIM-1, TIM-3 and TIM-4. In some embodiments, the antibody or antigen-binding fragment thereof disrupts the heterophilic interaction between CEACAM1 and TIM-3. Disruption of the interaction between CEACAM1 and TIM-3 by using the antibodies and antigen binding fragments thereof contemplated by the present invention may reverse CEACAM1 inhibitory function while maintaining TIM-3 activation function.
Embodiments of the invention may be used to reduce immunosuppression, such as T cell tolerance. By "decrease" is meant the ability to cause an overall decrease of about 20% or greater, 30% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, or 75%, 80%, 85%, 90%, 95% or greater, as compared to an untreated control. Immunosuppression may be mediated by immunosuppressive receptors expressed on the surface of immune cells and their interaction with their ligands. For example, cytotoxic CD 8T cells may enter a "functionally depleted" or "non-responsive" state whereby they express inhibitory receptors that prevent antigen-specific responses (such as proliferation and cytokine production). Thus, by inhibiting the activity and/or expression of such inhibitory receptors, the immune response to a cancer or tumor that is suppressed, inhibited or unresponsive may be enhanced or not inhibited. Such enhancement or reversal of immune response suppression may result in greater T cell activity, responsiveness, and/or ability or acceptance for activation.
Methods for measuring T cell activity are known in the art. As non-limiting examples, T cell tolerance may be induced by contacting T cells with recall antigens, anti-CD 3 (in the absence of co-stimulation), and/or ionomycin. Levels of, for example, IL-27, LDH-A, RAB10 and/or ZAP70 (intracellular or secreted) may be monitored, for example, to determine the degree of T cell tolerogenicity (where levels of IL-2, interferon-gamma and TNF correlate with increased T cell tolerance). The response of cells pretreated with, for example, ionomycin, to an antigen can also be measured in order to determine the degree of T Cell tolerance in a Cell or population of cells, for example by monitoring the levels of secreted and/or intracellular IL-2 and/or TNF- α (see, for example, Macian et al Cell 2002109: 719-731). Other characteristics of T cells with adaptive tolerance include increased levels of Fyn and ZAP-70/Syk, Cbl-B, GRAIL, Ikaros, CREM (cAMP response element modulators), B lymphocyte-induced mature protein-1 (Blimp-1), PD1, CD5, and SHP 2; increased phosphorylation of ZAP-70/Syk, LAT, PLCyl/2, ERK, PKC- Θ/I K BETA A; an increase in activation of intracellular calcium levels; a reduction in histone acetylation or hypoacetylation and/or an increase in CpG methylation at the IL-2 locus. Thus, in some embodiments, one or more of any of these parameters may be determined to determine whether an antibody or antigen-binding fragment thereof disclosed herein that inhibits CEACAM1 reduces immune tolerance. The reduction in T cell tolerance can also be assessed by examining T lymphocytes within tumor infiltrating lymphocytes or lymph nodes draining from established tumors. Such T cells exhibit a "depletion" profile through expression of cell surface molecules (such as PD1, TIM-3 or LAG-3) and decreased secretion of cytokines (such as interferon-gamma). Thus, evidence that T cell tolerance has decreased in the presence of CEACAM1 antibody or antigen-binding fragment thereof includes, for example, an increase in the number of T cells that have (a) antigen specificity for a tumor-associated antigen (e.g., as determined by major histocompatibility complex class I or class II tetramers containing tumor-associated peptides) and (B) the ability to secrete high levels of interferon- γ and cytolytic effector molecules, such as granzyme-B, relative to that observed in the absence of an inhibitor.
CEACAM1 antibodies and antigen-binding fragments thereof may also be used to enhance T cell expansion, activation, and proliferation.
In another aspect, the invention provides methods of using the antibodies and antigen binding fragments thereof of the invention to reduce the interaction between CEACAM1 and bacterial adhesins. In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are effective to reduce and/or prevent colonization of mammalian epithelium. In some embodiments, the adhesin is expressed from Escherichia coli, particularly Diffusible Adhesion Escherichia Coli (DAEC), Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria symbiosis, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus egypus, helicobacter pylori and/or Salmonella. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and HopQ expressed on the surface of helicobacter pylori. In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and an opacity-associated (Opa) adhesin protein expressed on a surface of neisseria species. In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and OMP adhesin proteins expressed on the surface of haemophilus.
In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and candida albicans. In one embodiment, the CEACAM1 antibody or antigen binding fragment thereof disrupts the interaction between CEACAM1 and influenza viruses (including but not limited to H5N 1). In one embodiment, the invention provides a method of inhibiting CEACAM1 binding to a nematode using a CEACAM1 antibody or antigen binding fragment thereof described herein. In one embodiment, the sub-nematode is wuchereria bambusicola.
Method of treatment
In one aspect, the invention provides CEACAM1 antibodies and antigen binding fragments thereof, which may also be used to treat a subject in need thereof.
In the methods described herein, a therapeutically effective amount of an antibody, or antigen-binding portion thereof, described herein is administered to a mammal in need thereof. Although the antibodies, or antigen-binding portions thereof, described herein are particularly useful for administration to humans, they may also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. By "therapeutically effective amount" is meant an amount of an antibody, or antigen-binding portion thereof, described herein that is effective to produce a desired therapeutic effect when administered to a mammal.
In some aspects, the antibody or antigen-binding fragment thereof binds CEACAM1 expressed by depleted T cells or Natural Killer (NK) cells, thereby restoring T cell and NK cell activity and resulting in an increased anti-tumor response. In other aspects, the antibody or antigen binding fragment thereof binds CEACAM1 expressed by tumor cells, thereby inhibiting tumor cell metastasis and formation of a cancer stem cell niche. In yet another aspect, the antibody or antigen binding fragment thereof binds CEACAM1 expressed by macrophages associated with fibrosis in the tumor environment, thereby inhibiting fibrosis. In another aspect, the antibody or antigen-binding fragment thereof binds CEACAM1 expressed by other stromal cells (such as vascular endothelial cells) in the tumor microenvironment, thereby inhibiting angiogenesis.
Accordingly, also provided herein are methods of treating a subject having a cancer or tumor and/or reducing tumor growth, comprising administering an effective amount of the CEACAM1 antibody or antigen-binding fragment thereof provided herein. "reducing" includes inhibiting and/or reversing, and may refer to, for example, symptoms of the condition being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or size of dormant tumors.
The term "cancer" refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Thus, as used herein, the term "cancer" refers to the uncontrolled growth of cells that interfere with the normal function of body organs and systems, including cancer stem cells and the tumor vascular niche. A subject with cancer is a subject in which objectively measurable cancer cells are present in the subject. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that metastasize from their original location and colonize vital organs can ultimately lead to death of the subject through functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, are able to outperform the normal hematopoietic compartment of a subject, leading to hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), ultimately leading to death.
By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline, etc. Individuals and patients are also subjects herein.
As used herein, the term "treatment" refers to a therapeutic treatment wherein the objective is to slow down (alleviate) an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reducing the extent of a condition, disorder or disease; a stable (i.e., not worsening) condition, disorder or disease state; delaying the onset or slowing the progression of a condition, disorder or disease; ameliorating a condition, disorder or disease state; and alleviating (partially or completely), detectable or undetectable or enhancing or ameliorating a condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to the expected survival without treatment. The terms "prevent", "prevention" and the like refer to acting before the onset of an apparent disease or condition to prevent or minimize the development of the disease or condition, or to slow the progression of the disease or condition.
Embodiments of the invention may be used to treat metastasis, which involves the spread of cancer from its primary site to other sites in the body. Cancer cells can detach from the primary tumor, infiltrate the lymph and blood vessels, circulate through the bloodstream, and grow in distant foci (metastases) in normal tissues elsewhere in the body. Metastasis may be local or distant. Metastasis is a continuous process, depending on the shedding of tumor cells from the primary tumor, passing through the bloodstream and stopping at distant sites. At the new site, the cells establish a blood supply and can grow to form life-threatening masses. Both stimulatory and inhibitory molecular pathways within tumor cells regulate this behavior, and the interaction between tumor cells and host cells at distant sites is also important. In addition to monitoring specific symptoms, metastasis is most often detected by using Magnetic Resonance Imaging (MRI) scans, Computed Tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays, and bone scans, alone or in combination.
Also contemplated are methods of reducing the sternness of a cancer comprising administering the CEACAM1 antibody or antigen-binding fragment thereof disclosed herein. Cancer sternness may refer to the ability of a cell to self-renew and produce additional phenotypically distinct cell types. Cancer Stem Cells (CSCs) are cancer cells that exhibit stem cell-like properties. CSCs typically exhibit at least one cancer marker and are capable of producing at least one additional phenotypically distinct cell type. In addition, cancer stem cells are capable of asymmetric and symmetric replication. It is understood that cancer stem cells can be produced from differentiated cancer cells that acquire a sternness trait and/or stem cells that acquire a phenotype associated with cancer cells. Alternatively, cancer stem cells can reconstitute non-stromal cell types within a tumor.
CEACAM1 is expressed by many tumor types, and CEACAM1 can modulate tumor growth and metastatic behavior. In another embodiment, CEACAM1 inhibition will reduce tumor growth and metastasis.
CEACAM1 expression on macrophage subpopulations was associated with fibrosis during carcinogenesis. In another embodiment, CEACAM1 inhibition will reduce tumor-associated fibrosis.
Cancers that may be treated by the compositions and methods contemplated by the present invention include tumors that are not vascularized or not yet substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer to be treated include, but are not limited to, benign and malignant tumors, as well as malignant tumors such as sarcomas, carcinomas, and melanomas. Also included are adult tumors/cancers and pediatric tumors/cancers. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancers; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasms; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma); lymphomas, including hodgkin lymphoma and non-hodgkin lymphoma; melanoma; a myeloma cell; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other carcinomas and sarcomas; and B cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-lytic NHL, large volume disease NHL, mantle cell lymphoma, AIDS related lymphoma, and Waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphocytic Leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with pharyngeal disease, edema (such as that associated with brain tumors), and Meigs syndrome. A patient may have more than one type of cancer.
The efficacy of a cancer treatment method comprising a therapeutic formulation comprising a composition comprising an antibody and antigen-binding fragments thereof described herein can be measured by various endpoints commonly used in evaluating cancer treatment, including, but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, and quality of life. In the case of cancer, a therapeutically effective amount of the recombinant CEACAM1 antibody or antigen-binding fragment thereof can reduce the number of cancer cells; reducing tumor size; inhibit (i.e., slow to some extent and preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with the condition. In the case where the patient has more than one type of cancer, the therapeutically effective amount of the recombinant CEACAM1 antibody or antigen-binding fragment thereof is an amount effective to treat at least one of the cancers. For the recombinant CEACAM1 antibody or antigen-binding fragment thereof to be used to prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival duration, progression free survival duration (PFS), Response Rate (RR), response duration, and/or quality of life.
Checkpoint proteins interact with specific ligands, send signals to T cells and shut off or inhibit T cell function. By expressing high levels of checkpoint proteins on its surface, cancer cells can control the function of T cells entering the tumor microenvironment, thereby suppressing the anti-cancer immune response. The immune checkpoint protein programmed death-1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA. Two cell surface glycoprotein ligands of PD-1, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), have been identified that are expressed on antigen presenting cells as well as on many human cancers and have been shown to down-regulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al, 2000; Latchman et al, 2001). Inhibition of the PD-1/PD-L1 interaction promotes potent anti-tumor activity. Examples of PD-1 inhibitors include, but are not limited to, pembrolizumab (MK-3475), nivolumab (MDX-1106), cimiralizumab-rwlc (REGN2810), PILITHIZHUzumab (CT-011), Stbazumab (PDR001), tiselizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym021, TSR-042. Examples of PD-L1 inhibitors include, but are not limited to: attributab mab (MPDL3280A), Devolumab (MEDI4736), Abamectin (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, and SHR-1316.
However, there is a large population of cancer patients receiving checkpoint inhibitor therapy that (1) does not respond to this type of therapy (innate or primary resistance) or (2) initially responds but eventually progresses to disease progression (secondary or acquired resistance). Resistant cancers may also be referred to as refractory cancers. As shown in the examples below, tumor-associated cells isolated from patients with acquired resistance to PD-1/PD-L1 inhibitors upregulated CEACAM1 expression relative to tumor-associated cells isolated from naive patients not exposed to PD-1 inhibitors. When CEACAM1 was expressed in the case of acquired resistance, the cells carrying CEACAM1 were more likely to be effector memory cells than central memory cells, consistent with a reduction in anti-cancer response in resistant patients.
Accordingly, also provided herein are methods of treating patients who are resistant to checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, and/or CTLA-4) using CEACAM1 antibodies and antigen binding fragments thereof, including but not limited to the specific CEACAM1 antibodies and antigen binding fragments thereof provided herein. In some embodiments, the CEACAM1 antibody used to treat a patient resistant to an inhibitor of PD-1, PD-L1, and/or CTLA-4 is CP08H03/Vk 8S 29A or CP08H03/CP08F 05. In some embodiments, the resistance is innate or primary resistance. In some embodiments, the resistance is secondary or acquired resistance. In some embodiments, the CEACAM1 antibody administered (including but not limited to the CEACAM1 antibodies and antigen-binding fragments thereof provided herein) reverses T cell depletion in patients resistant to checkpoint inhibitor therapy. Any cancer that exhibits PD-1, PDL-1 and/or CTLA-4 resistance is suitable for treatment using the methods of the present invention. In some embodiments, the CEACAM1 antibody or antigen-binding fragment is administered to a patient who has not previously received checkpoint inhibitor therapy.
In another aspect, the invention provides the use of CEACAM1 antibodies and antigen binding fragments provided herein in the treatment of patients resistant to therapy with other checkpoint inhibitors, including but not limited to PD-L2, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and across all NK, γ δ and memory CD 8)+Expressed on (. alpha.beta.) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7).
In another aspect, the invention provides methods of treating a mammal in need of reduction and/or prevention of colonization of mammalian epithelium by candida albicans and/or bacteria expressing bacterial adhesins, including but not limited to escherichia coli, particularly Diffusively Adherent Escherichia Coli (DAEC), neisseria meningitidis, neisseria symbiosis, moraxella catarrhalis, haemophilus influenzae, haemophilus ehaegypti, helicobacter pylori, and/or salmonella using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein. In another aspect, the present invention provides methods of reducing replication of influenza virus and/or reducing release of pro-inflammatory cytokines or chemokines associated with influenza virus infection using the CEACAM1 antibodies and antigen binding fragments thereof disclosed herein. In some embodiments, the influenza virus is H5N 1. In another aspect, the invention provides methods of treating a subject in need of reducing and/or preventing infection by daughter nematodes (such as wuchereria bambusae) using the CEACAM1 antibody agents disclosed herein, antigen binding fragments thereof. In another aspect, the invention provides methods of treating a subject in need of reducing and/or preventing development of lymphedema and/or hydrocele tunica vaginalis associated with infection by a daughter nematode (such as wuchereria bambusae) using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein. In one embodiment, the invention provides a method of reducing filarial invasion of the lymphatic system of a subject in need thereof using a CEACAM1 antibody or antigen binding fragment thereof as described herein. In one embodiment, the sub-nematode is wuchereria bambusicola. The subject may be infected with more than one bacterium that expresses a bacterial adhesin, candida albicans, influenza virus, and/or a daughter nematode.
In another embodiment, the invention provides a method of reducing cancer cell invasion of the lymphatic system of a subject in need thereof using the CEACAM1 antibody or antigen binding fragment thereof described herein.
Screening method
Also provided herein are methods of identifying a patient population that is likely to respond to treatment with the CEACAM1 antibodies and antibody fragments provided herein, including but not limited to CP08H03/Vk 8S 29A and CP08H03/CP08F 05.
In some embodiments, cancer patients are screened for CEACAM1 expression on certain cell types, including T cells, NK cells, tumor cells, or other cells in the tumor microenvironment, such as macrophages. In some embodiments, cancer patients showing increased CEACAM1 expression on certain cell types compared to controls are selected for treatment with CEACAM1 antibodies and antibody fragments provided herein. A "control" level of CEACAM1 expression may refer to the level of CEACAM1 expression in one or more individuals not having cancer. The levels may be measured on an individual-by-individual basis, or on an aggregate basis such as an average. In some embodiments, control levels of CEACAM1 expression from the same individual whose condition is being monitored, but obtained at different times. In certain embodiments, a "control" level may refer to a level obtained from the same patient at an earlier time (e.g., weeks, months, or years ago). In some embodiments, the control level is obtained from the patient prior to the patient receiving any cancer therapy. In some embodiments, the control level is obtained from the patient prior to the patient receiving checkpoint inhibitor treatment.
In some embodiments, CEACAM1 expression is determined for patients resistant to checkpoint inhibitor therapy (including, but not limited to, therapy with PD-1/PD-L1/CTLA-4 inhibitors). In some embodiments, patients that are resistant to checkpoint inhibitor therapy and show increased CEACAM1 expression on certain cell types as compared to controls are selected for treatment with CEACAM1 antibodies and antibody fragments provided herein (including but not limited to CP08H03/Vk 8S 29A and CP08H03/CP08F 05).
In some embodiments, the patient is assayed for an allelic variant of human CEACAM 1. Based on the allelic variant of human CEACAM1 expressed by the patient, more or less anti-CEACAM 1 antibody may be administered to the patient compared to a patient expressing the wild-type variant of CEACAM 1. In some embodiments, the patient is assayed for the presence of an allelic variant of Y34C, Q44L, and/or Q89H of CEACAM 1. In some embodiments, a higher and/or more frequent dose of anti-CEACAM 1 antibody is administered to a patient expressing a Y34C, Q44L, and/or Q89H allelic variant of CEACAM1 as compared to a patient expressing a wild-type variant of CEACAM 1.
Pharmaceutical composition
In another aspect, the present invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of the CEACAM1 antibody, or antigen-binding fragment thereof, described herein, formulated with one or more pharmaceutically acceptable excipients.
The dosage of the active agent may vary depending on the reason for use, the individual subject and the mode of administration. The dosage may be adjusted based on the weight of the subject, the age and health of the subject, and the tolerability of the compound or composition. For example, depending on the disease, this may require 0.1, 1.0, 3.0, 6.0 or 10.0mg/Kg for the antibody or antigen binding fragment thereof. For IgG (two binding sites) with a molecular weight of 150,000 g/mole, these doses correspond to binding sites of approximately 18nM, 180nM, 540nM, 1.08. mu.M and 1.8. mu.M in a 5L blood volume.
The active agents and excipients can be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions of the present invention may be specifically formulated in solid or liquid form, including those suitable for parenteral administration, for example by subcutaneous, intratumoral, intramuscular or intravenous injection, for example as a sterile solution or suspension.
Therapeutic compositions comprising an antibody or antigen-binding fragment thereof that binds CEACAM1 can be formulated with one or more pharmaceutically acceptable excipients, which can be pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc, magnesium, calcium or zinc stearate, or stearic acid), solvents, or encapsulating materials, involved in carrying or transporting a therapeutic compound, filler, salt, surfactant, and/or preservative for administration to a subject. Some examples of materials that can be used as pharmaceutically acceptable excipients include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; a wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; a buffering agent; water; isotonic saline; a pH buffer solution; and other non-toxic compatible materials used in pharmaceutical formulations.
Bulking agents are compounds that increase the quality of a pharmaceutical formulation and aid in the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol and sorbitol.
The use of a surfactant may reduce aggregation of the reconstituted protein and/or reduce the formation of particles in the reconstituted formulation. The amount of surfactant added is such as to reduce aggregation of the reconstituted protein and minimize particle formation upon reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g. polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g. lauramidopropyl); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; and polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.).
Preservatives may be used in the formulations of the present invention. Suitable preservatives for use in the formulations of the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl-dimethyl ammonium chlorides, where the alkyl group is a long chain compound) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. Other suitable excipients may be found in standard Pharmaceutical texts, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19 th edition Mack Publishing Company, Easton, Pa., (1995).
A composition comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier may comprise CEACAM1 antibody or antigen-binding portion thereof described herein at various concentrations. For example, the composition can comprise from 10mg/ml to 200mg/ml, from 25mg/ml to 130mg/ml, from 50mg/ml to 125mg/ml, from 75mg/ml to 110mg/ml, or from 80mg/ml to 100mg/ml of the antibody or antigen-binding fragment thereof. The composition may further comprise about 10mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml, or 150mg/ml of the antibody or antigen-binding fragment thereof.
In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier is lyophilized and provided in a composition for reconstitution prior to administration.
Application method
Therapeutic compositions comprising contemplated antibodies or antigen-binding fragments thereof may be administered in any convenient manner, including by injection, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intracranially, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.
In certain embodiments, the antibody or antigen-binding fragment thereof is administered to the mammal by intravenous infusion, i.e., the antibody or antigen-binding fragment thereof is introduced into a vein of the mammal over a period of time. In certain embodiments, the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.
In certain embodiments, a dose of the compound or composition is administered to the subject daily, every other day, every two days, every three days, weekly, twice weekly, three times weekly, biweekly, or monthly. In other embodiments, two, three, or four doses of the compound or composition are administered to the subject daily, every two days, every three days, weekly, biweekly, or monthly. In some embodiments, a dose of the compound or composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, or 28 days. In certain embodiments, a dose of the compound or composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or longer.
Combination therapy
In one aspect, the invention provides CEACAM1 antibodies or antigen-binding fragments thereof administered with an additional therapeutic agent. Such additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, and anti-infective agents. The agents used in such combination therapies may fall into one or more of the aforementioned categories. The administration of the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be simultaneous or sequential. Administration of the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be separate or as a mixture. Furthermore, the treatment methods contemplated by the present invention may relate to treatment in combination with one or more cancer therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormonal therapy, laser therapy and radiotherapy.
Exemplary additional therapeutic agents also include radionuclides with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA and are therefore suitable for inducing cell death (e.g., of cancer). Exemplary high energy radionuclides include:90Y、125I、131I、123I、111In、105Rh、153Sm、67Cu、67Ga、166Ho、177Lu、186re and188re. These isotopes generally produce high energy alpha or beta particles with short path lengths. Such radionuclides kill cells in close proximity thereto, e.g., neoplastic cells to which the conjugate has been attached or has entered. They have little or no effect on non-localized cells and are substantially non-immunogenic.
Exemplary additional therapeutic agents also include cytotoxic agents such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or crosslinkers, or DNA-RNA transcription modulators), enzyme inhibitors, gene modulators, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, and the like.
Exemplary additional therapeutic agents also include alkylating agents, such as the anthracycline family of drugs (e.g., doxorubicin, carminomycin, cyclosporin-a, chloroquine, methotrexate, mithramycin, porphyrinomycin, streptomycin, anthracenedione, and aziridine). In another embodiment, the chemotherapeutic moiety is a cytostatic agent, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and methotrexate dichloride, 3-amino-1, 2, 4-benzotriazine 1, 4-dioxide, aminopterin, cytosine β -D-arabinofuranoside, 5-fluoro-5' -deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cisplatin, melphalan, mitoxantrone, and oxaliplatin.
Exemplary additional therapeutic agents also include transcriptional modulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatic agents compatible with the present invention include ansamycin benzoquinone, quinone derivatives (e.g., quinolones, genistein, bactacrine), busulfan, ifosfamide, mechlorethamine, triimine quinone, diazaquine, carboquone, indoquinone EO9, divinyliminobenzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).
Exemplary additional therapeutic agents also include cytotoxic nucleosides such as, for example, adenine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, tegafur, and 6-mercaptopurine; tubulin binding agents such as taxanes (e.g., paclitaxel, docetaxel, taxanes), nocodazole, radicotoxins, dolastatins (e.g., dolastatin 10, 11, or 15), colchicines and colchicinoids (e.g., ZD6126), combretastatins (e.g., combretastatin a-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenic compounds such as angiostatin K1-3, DL- α -difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine and (±) -thalidomide.
Exemplary additional therapeutic agents also include hormones and hormone antagonists such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroxyprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17- (allylamino) -17-demethoxygeldanamycin, 4-amino-1, 8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene diphosphonic acid, leuprolide, luteinizing hormone releasing hormone, pifithrin-alpha, rapamycin, sex hormone binding globulin, and thapsigargin.
Exemplary additional therapeutic agents also include enzyme inhibitors such as S (+) -camptothecin, curcumin, (-) -derris-tenocin, 5, 6-dichlorobenzimidazol 1- β -D-ribofuranoside, etoposide, fulvestrant, forstrecin, milk tree alkaloid, 2-imino-1-imidazolidine acetic acid (cyclocreatine), mevinolin, trichostatin a, tyrphostin AG 34, and tyrphostin AG 879.
Exemplary additional therapeutic agents also include gene modulators such as 5-aza-2' -deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin a aldehyde), retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin a), tamoxifen, and troglitazone.
Exemplary additional therapeutic agents also include cytotoxic agents such as, for example, drugs of the pteridine family, enediynes, and podophyllotoxins. Particularly useful members of these classes include, for example, methotrexate, podophyllotoxin or podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vindesine, vinblastine and the like.
Still other additional therapeutic agents compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), calicheamicin, gramicin D, maytansinoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, imistine, teniposide, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.
In one embodiment, the CEACAM antibody or antigen-binding fragment thereof is administered in combination with an agent that is a checkpoint inhibitor. Such inhibitors may include small molecule inhibitors or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit an immune checkpoint receptor or antibodies that bind to and block or inhibit an immune checkpoint receptor ligand. Illustrative checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and across all NK, γ δ and memory CD8 +Expressed on (. alpha.beta.) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM-3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Illustrative immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX 40 and Yervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor) as well as the aforementioned PD-1 and PD-L1 inhibitors. Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.
In some embodiments, the CEACAM1 antibodies and antigen binding fragments thereof described herein are administered with TIGIT, LAP, Podoplanin, protein C receptor, ICOS, GITR, CD226, or CD160 inhibitors.
In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered with a CTLA-4, PD-1, PD-L1, or PD-L2 inhibitor. In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered with a TIM-3 inhibitor.
It is to be understood that this invention is not limited to the particular molecules, compositions, methods or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is also to be understood that the disclosure of the invention in this specification includes all possible combinations of such specific features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention or of a particular claim, that feature may also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments of the invention, and in general in the invention.
Where reference is made herein to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes that possibility), and the method may comprise one or more further steps performed before any defined step, between two defined steps or after all defined steps (unless the context excludes that possibility).
All other cited patents and applications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein controls and the definition of that term in the reference does not apply.
In order to facilitate a better understanding of the invention, the following examples of specific embodiments are given. The following examples should not be construed as limiting or restricting the full scope of the invention.
Examples
Example 1: production of fully humanized CEACAM1 antibody
1. HumanizationGeneration of antibody variants
Design of composite human antibody variable region sequence and expression of antibody
First, a structural model of the V-region of the parent murine CEACAM1 antibody was generated using Swiss PDB and analyzed to identify potential "limiting" amino acids in the V-region that may contribute to the binding properties of the antibody. For regions outside and flanking the CDRs, a wide selection of human sequence segments were identified as possible components of the novel humanized V regions.
Based on structural analysis, a large set of preliminary sequence segments useful for generating humanized CEACAM antibody variants were selected and iTope used for in silico analysis of peptides binding to human MHC class II alleles was used TMTechnique (Perry et al, 2008.Drugs RD 9(6):385-396) and use of TCEDTMKnown antibody sequence-related T cell epitopes (Bryson et al 2010, Biodrugs 21(1):1-8) were analyzed. Important non-human germline systems to be identified as human MHC class II or for TCEDTMSequence segments that achieved significant hit scores were discarded. This analysis results in a reduced set of segments and the combination of segments is re-analyzed as described above to ensure that the junctions between segments do not contain potential T cell epitopes. Selected sequence segments are assembled into complete V-region sequences that lack important T-cell epitopes. The heavy and light chains selected for gene synthesis, expression and activity testing in mammalian cells are listed in
In table 1.
Some of the heavy and light chains in table 1 contain variations at positions that are considered part of the CDRs as defined by the Kabat CDRs but not as defined by the IMGT CDRs.
Table 1. heavy and light chains selected for gene synthesis.
Next, V was synthesized for the parent murine CEACAM1 antibody and humanized CEACAM antibody variants with flanking restriction enzyme sitesHAnd V kappa sequences, pANT expression vector system for cloning into IgG4(S241P) heavy and kappa light chainsIn (fig. 1). VHThe region was cloned between MluI and HindIII restriction sites, and the V.kappa.region was cloned between BssHII and BamHI restriction sites. All constructs were confirmed by sequencing.
Thirty-nine heavy and light chain pairings were transiently transfected into HEK EBNA adherent cells using the PEI transfection method and incubated for 5-7 days after transfection. These 39 pairings included three controls: (1) chimeric antibody VH0/V.kappa.0 (from murine V fused to the constant heavy chain region of human IgG4HZone (V)H0) And the murine vk region (vk 0) fused to the constant light chain region of human IgG 4; (2) chimeric VH heavy chains (V)H0) Pairing with light chain variant vκ 1; and (3) VH1 heavy chain pairing with a chimeric vk light chain variant (vk 0). The other 36 were complex IgG 4VHAnd a vk variant: vH1 is paired with V.kappa.1 to V.kappa.12, VH2 are paired with V.kappa.1 to V.kappa.12, VH3 is paired with V.kappa.1 to V.kappa.6, and VH4 are paired with V.kappa.1 to V.kappa.6.
Antibodies were purified from cell culture supernatants on a protein a sepharose column, buffer exchanged to PBS pH 7.4, and quantified by OD 280nm using an extinction coefficient based on the predicted amino acid sequence. Mu.g of each antibody was analyzed by SDS-PAGE, and bands corresponding to typical antibody spectra were observed. The size of the light chain and the presence of a faint band at 25kDa indicate that the glycosylation motif identified in the light chain is essentially utilized.
Competitive ELISA assay for humanized variants binding to CEACAM1
Purified antibodies were assessed for binding to human CEACAM1 in a competition ELISA assay. Nunc Immuno MaxiSorp 96 well flat bottom microtiter plates were pre-coated with 1. mu.g/ml GST-CEACAM1 in 1 XPBS at 4 ℃ overnight. The next day, plates were blocked with 2% BSA/PBS for 1 hour at room temperature ("RT") and then washed 3 times with PBST pH 7.4. The chimeric antibody V of 100 to 0.07 or 0.002. mu.g/ml is addedHA3-fold dilution series of 0/V κ 0, irrelevant IgG4 antibody and humanized CEACAM1 antibody was premixed with a constant concentration of parent murine antibody (0.45 μ g/ml final concentration), added to the plate and incubated at room temperature for 1 hour. After 3x PBST wash, parental murine CEACAM1 antibody was detected with anti-mouse-HRP and TMB substratesIn combination with (1). The reaction was stopped with 3M HCl, and the absorbance was read at 450nm on a Dynex Technologies MRX TC II microplate reader and binding curves were plotted. Binding of humanized CEACAM antibody variants to CEACAM1 and chimeric antibody contained on each plate (V)H0/V κ 0) were compared. Twelve of the 36 humanized CEACAM antibody variants were shown to not bind CEACAM1 (including those of V κ 3, V κ 4 and V κ 5). With chimeric antibody VHVariants that bind CEACAM1 showed a relative IC50 value range of 0.9 to 5.2 compared to 0/V κ 0. The data are summarized in table 2.
Table 2 summary of humanized CEACAM antibody variants and control antibody titers and binding data. Antibody expression titers (μ g/ml) were from static HEK EBNA transient transfections. Normalization of IC50 values obtained in competition assays to chimeric antibody V on the same plateH0/V κ 0. Unbound antibodies are not included in the table. Antibodies in bold were used for multicycle kinetic analysis.
Variants
Expression Titers (μ g/ml)
Average relative IC50 value
Number of experiments
VH0/V κ 0 (control)
22.9
1
4
VH0/V κ 1 (control)
21.3
1.6
4
VH1/V κ 0 (control)
30.5
0.9
4
VH1/Vκ1
20.6
2.5
4
VH1/Vκ2
23.1
1.8
4
VH1/Vκ4
27.2
2.7
4
VH1/Vκ7
34.6
1.6
2
VH1/Vκ8
33.3
1.7
2
VH1/Vκ9
39.2
1.5
2
VH1/Vκ10
35.4
1.6
2
VH1/Vκ11
38.0
2.2
2
VH1/Vκ12
44.5
1.4
2
VH2/Vκ1
10.6
2.8
4
VH2/Vκ2
43.9
2.0
4
VH2/Vκ4
30.2
3.0
4
VH2/Vκ7
41.7
1.3
2
VH2/Vκ8
44.9
1.6
2
VH2/Vκ9
39.6
1.7
2
VH2/Vκ10
35.6
1.4
2
VH2/Vκ11
32.0
1.4
2
VH2/Vκ12
40.9
1.5
2
VH3/Vκ1
17.1
3.5
4
VH3/Vκ2
17.0
2.5
4
VH3/Vκ4
21.0
5.2
4
VH4/Vκ1
13.7
4.0
4
VH4/Vκ2
29.7
3.3
4
VH4/Vκ4
30.1
4.0
4
Kinetic analysis of humanized variants binding to CEACAM1
As an alternative to evaluating the binding of 36 antibody combinations and three control antibodies to CEACAM1, kinetic analyses were performed on Biacore T200 (serial No. 1909913) running Biacore T200 evaluation software V2.0.1(Uppsala, Sweden). All experiments were performed at 25 ℃ with HBS-P + running buffer (pH 7.4) (GE Healthcare, Cat. No. BR 100671). All kinetic experiments were performed using His-tagged CEACAM1 as the analyte. For all experiments, antibodies were immobilized on the S-series protein a sensor chip surface. For kinetic experiments, the amount of immobilized/captured ligand is limited to avoid mass transfer effects on the chip surface, where the surface ideally has an analyte binding level (R) of 50-150RU max). Capture settings for all sample antibodies were using MW of 45kDa of CEACAM1 analyte, antibody ligand of 150kDa (estimate of IgG), Rmax of 50RU and stoichiometry (Sm) of 2 because each antibody was able to bind 2 target molecules, a target response level of about 75 RU.
Single-cycle analysis of 36 antibody combinations and three control antibodies was performed on purified antibodies (vk 1 to vk 6 variants) or supernatants of transiently transfected HEK EBNA cells (vk 7 to vk 12 variants). In some cases, when the supernatant is not available, the purified chimeric antibody V is purifiedH0/V κ 0 was added to HEK EBNA medium to serve as a positive control. The antibody was diluted to a concentration of 1. mu.g/ml in HBS-P + (determined by IgG quantitative ELISA). At the beginning of each cycle, antibodies were loaded onto Fc2, Fc3, and Fc4 of the protein a chip and IgG was captured at a flow rate of 8 μ l/min to give an RU of about 75. The surface is then stabilized. Single cycle kinetic data was obtained at a flow rate of 50 μ Ι/min to minimize any potential mass transfer effects. For chimeric antibody VH0/V κ 0 was repeated several times to check the stability of the surface and analyte during the kinetic cycle. The signal from the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A 5-point 2-fold dilution range of 3.125 to 50nM CEACAM1 with no regeneration between concentrations was used. The associated phase of 5 increasing concentrations of CEACAM1 injections was monitored for 100 seconds and a single dissociation phase was measured 150 seconds after the last injection of CEACAM 1. Regeneration of the protein a surface was performed using 2 injections of 10mM glycine-HCl pH 1.5 followed by a 500 second stationary phase. Signals from each antibody blank run (without CEACAM1) were subtracted to correct for differences in surface stability. Single cycle kinetics (
Table 3) showed that 24 humanized variants bound to CEACAM1, while 12 variants did not. When combined with any humanized heavy chain, the light chains of vk 3, vk 5 and vk 6 abolished CEACAM1 binding. These data are consistent with competition ELISA data (table 2).
Table 3 single cycle kinetic parameters for binding of humanized CEACAM antibody variants and control antibodies to CEACAM1-HIS determined using Biacore T200. K by humanizing CEACAM1 antibody variantsDDivided by chimeric antibody V determined in the same experimentHK of 0/V kappa 0DTo calculate the antibody VHRelative K of 0/V.kappa.0 ratioD. Antibodies in bold were used for multicycle kinetic analysis. S/N, supernatant. Not included in the tableA binding variant.
Among the 24 antibodies that bind CEACAM1, it would appear to be in chimeric antibody VHAntibody variants within 2-fold of 0/V κ 0 and with binding of relative IC50 in the range of 0.9 to 1.8 were used for multi-cycle kinetic analysis using Biacore: vH1/Vκ2、VH1/Vκ7、VH1/Vκ8、VH1/Vκ9、VH1/Vκ10、VH1/Vκ12、VH2/Vκ7、VH2/Vκ8、VH2/Vκ9、VH2/Vκ10、VH2/V κ 11 and VH2/V κ 12 (see Table 2 and
table 3, highlighted in bold).
For the multi-cycle kinetic analysis, the purified antibody was immobilized in HBS-P + at a protein concentration of 1. mu.g/ml. At the beginning of each cycle, the antibody was captured on protein a to give an RU of about 75 and stabilize the surface. Kinetic data were obtained at a flow rate of 80 μ l/min to minimize any potential mass transfer effects. Multiple replicates of the blank (without CEACAM1) and replicates of a single concentration of analyte were programmed into the kinetic run in order to examine the stability of the surface and analyte in the kinetic cycle. For kinetic analysis, a 2-fold dilution range of CEACAM1 from 200 to 3.125nM or from 100 to 1.5625nM was selected. The associated phase of CEACAM1 was monitored for 50 or 150 seconds and the dissociated phase was measured for 100 seconds. At the end of each cycle, two injections of 10mM glycine-HCL pH 1.5 were used for regeneration of the protein a surface.
The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface, using the global Rmax parameter in the 1-to-1 binding model. K by humanizing CEACAM antibody variantsDDivided by chimeric antibody V on the same chipHK of 0/V kappa 0DTo calculate the sum VHRelative K of 1/V.kappa.0 ratioD. Kinetic parameters measured for the interaction of CEACAM1 with humanized CEACAM antibody variants are shown in table 4. TABLE 5 overviewThe average relative K obtained using the 12 antibody combinations of the multicycle kinetic analysis was concludedD。
Selective analysis of humanized variants binding to CEACAM1
Testing of 24 humanized antibody variants (see table 3) that bind CEACAM1 and chimeric control antibody V on HeLa cells transfected with CEACAM1, 3, 5, 6 and 8 by flow cytometryHBinding selectivity of 0/V κ 0 to CEACAM 1. As shown in fig. 2, most variants were highly selective for CEACAM1 and showed little or no binding to CEACAM3, 5, 6 or 8. With chimeric control antibody VHAll 24 variants showed reduced binding to CEACAM5 compared to 0/V κ 0. There was no evidence of any staining of the HeLa-CEACAM3 or HeLa-CEACAM8 transfectants. Therefore, this data is not reported. V was chosen because of its favorable affinity and selectivity for CEACAM1 and its favorable expression level H1/V.kappa.8 serves as a framework for affinity maturation.
Table 4. multicycle kinetic data for variants of composite human antibodies that bind CEACAM1-HIS determined using Biacore T200 (n ═ 1). K by humanizing CEACAM antibody variantsDDivided by chimeric antibody V determined on the same chipHK of 0/V kappa 0DTo calculate the antibody VHRelative K of 0/V.kappa.0 ratioD。
TABLE 5 all humanized CEACAM antibody variants and chimeric antibody V obtained from the multi-cycle kinetics using the Biacore T200 assayHRelative K of 0/V.kappa.0 ratioDTo summarize (a). By testing the K of antibody variantsDDivided by the chimeric antibody V tested on each chipHK of 0/V kappa 0DTo calculate the antibody VHK of 0/V.kappa.0 ratioDFold difference of (c). The number of independent experiments for each variant is shown.
Removal of N-linked/HEK-derived glycosylation
Sequence analysis showed a potential N-linked glycosylation motif in the original mouse hybridoma light chain CDR 1. The CDR1L of the parent murine antibody contains the N-X-S/T consensus sequence (N26 and S29 according to Kabat numbering, corresponding to residues 26 and 28 in the primary amino acid sequence of the light variable chain, see fig. 3C), which makes the N26 residue the target of N-linked glycosylation. To reduce potential glycosylation-related immunogenicity, two CDR mutations were designed to remove the N-X-S/T consensus sequence (glycosylation site): N26Q and S29A (Kabat numbering scheme). Mutation of either residue abolished glycosylation as shown in figure 4.
Competition ELISA experiments (see table 6), multiple cycle kinetic analysis (see table 7) and selective analysis (see table 8) were performed to confirm the binding of the mutant chimeras to CEACAM 1. The antibody mutant S29A (Kabat numbering scheme) showed higher expression levels and a more similar K than the unmutated antibody, compared to the antibody mutant N26QD(see table 9) while maintaining high selectivity to CEACAM 1. Thus, during further development, the S29A mutation (Kabat numbering scheme) was incorporated into the CEACAM1 leader antibody.
TABLE 6 results of competition ELISA experiments using non-glycosylated chimeric antibodies. The CDR1L residues were numbered according to the Kabat numbering scheme.
TABLE 7. analysis of the kinetics of multiple cycles using non-glycosylated chimeric antibodies. The CDR1L residues were numbered according to the Kabat numbering scheme.
Table 8 selectivity analysis of non-glycosylated humanized variants. The CDR1L residues were numbered according to the Kabat numbering scheme. The binding selectivity of the antibody variants was assessed by flow cytometry using HeLa cells transfected with vectors expressing CEACAM1, CEACAM5 and CEACAM6, respectively. Shown are the relative amounts of cells expressing the corresponding antigens bound by the indicated antibodies.
Variants
CEACAM1
CEACAM5
CEACAM6
VH0/Vκ0
78.00
8.81
0.30
VH0/Vκ0N26Q
79.90
15.50
0.18
VH0/Vκ0S29A
77.90
9.82
0.00
Control IgG4
0.37
1.02
1.39
TABLE 9 Experimental summary of non-glycosylated chimeric antibodies. The CDR1L residues were numbered according to the Kabat numbering scheme.
3. Non-glycosylated CEACAM1 antibody VH1/VK8 Affinity maturation of S29AConstruction of phage vector and parent VHBinding assay for 1/VK 8S29A scFv
For leader antibody VHAffinity maturation of one of 1/VK8, construction of the code VH1 and VK8 and converted to scFv format using overlapping PCR, with the heavy chain being via 15 amino acids (G)4S)3The linker is attached to the light chain. CDR1L residue S29 was numbered according to the Kabat numbering scheme and corresponds to residue 28 in the primary amino acid sequence of the light variable chain (fig. 3C). The scFv sequence was then cloned into the phagemid vector pANT43 using the restriction enzymes Sfi I and Not I, such that the scFv was displayed as a gene III fusion protein on the phage surface (fig. 5). The cloned scFv was transformed into E.coli (TG1) and all constructs were confirmed by sequencing. Preparation of a recombinant vector containing parent VHPhage of 1/VK 8S29A scFv or unrelated scFv and tested for binding to GST-CEACAM1 (FIG. 6). Derived from parent VHPhages of the 1/VK 8S29A sequence bound specifically to the antigen, since no binding was observed with unrelated phages.
Mutagenesis and library construction
To construct affinity maturation libraries, non-glycosylated humanized antibody V was targeted using semi-random codonsHSpecific amino acids within CDR1H, CDR3H and CDR3L of 1/V κ 8S29A were used for "hot-spot" mutagenesis. The sequence positions of the possible contact residues are analyzed and ordered sequentially within each block. This information is used together with the amino acid preference at any given position in CDR3 and the crystal structure of the parent murine antibody. In the case of possible Next, the highest ranked contact residue within each block is prioritized.
Four different libraries were generated: one library was used for mutation of CDR1H (HC), two libraries for mutation of CDR3H, and one library for mutation of CDR3L (see fig. 7).
CDR1H was identified as five amino acids (S31 to S35) in length (Kabat definition, corresponding to residues 31-35 of the primary amino acid sequence of the heavy variable chain, see fig. 3A), with IMGT CDR1H definition (G26 to G33) covering a more extended region. Considering together and in conjunction with the crystal data of the parent murine antibody, G26 to S35 are contained in a single library, wherein each position comprises a subset of amino acids.
CDR3H was identified as 12 amino acids in length (H95-Y102 according to Kabat definition, corresponding to residues 99-110 of the primary amino acid sequence of the heavy variable chain, see fig. 3A). For mutagenesis, CDR3H was split into two libraries overlapping at D100 (corresponding to residue 104 of the primary amino acid sequence of the heavy variable chain according to Kabat definition, see fig. 3A and 3B): block 1 (R94 to D100 according to the Kabat definition, corresponding to residues 98 to 104 in the primary amino acid sequence of the heavy variable chain, see fig. 3A and 3B) and block 2 (D100 to Y102 according to the Kabat definition, corresponding to residues 104 to 110 in the primary amino acid sequence of the heavy variable chain, see fig. 3A and 3B), wherein each block contains a subset of amino acids in all positions. Position R94 (corresponding to residue 98 of the primary amino acid sequence of the heavy variable chain according to the Kabat definition, see fig. 3A and 3B) was included (block 1) to allow more diversity in the germline residues of the anchored CDRs.
CDR3L was identified as 9 amino acids (Q89-T97) in length (corresponding to residues 88 to 96 of the primary amino acid sequence of the light variable chain, according to Kabat definition, see fig. 3C). The regions Q90 to P96 (corresponding to residues 89 to 97 of the primary amino acid sequence of the light variable chain, according to the Kabat definition, see fig. 3C) were comprised in a single library, wherein each position comprises a subset of amino acids. Kabat numbering is used for all protein sequence coordinates.
Fig. 8A, 8B and 8C show an overview of the library construction. Preparation of a catalyst containing VHTruncated fragment of the 1/V.kappa.8S29A parent scFv and two of the regions to be randomizedA non-expression plasmid with continuous stop codons. The purpose of this step was to reduce the probability that the parent scFv was produced and dominates the selection (as occasionally observed during affinity maturation) so that only recombinant antibody fragments produced by PCR were able to form functional scFv in the phagemid vector.
For the CDR3L library, randomization of CDR3L was performed by performing two PCRs. In the first PCR, randomized 3' primers and a V containing Sfi I restriction site were usedHFW1 specific 5' primers to amplify most scFv genes and introduce mutations into vkappa CDR 3. A second PCR adds the remainder of the scFv and appends a restriction site (Not I) for subcloning of the fragment.
For VHLibrary, V by performing two PCRs using two templates containing full-length parent scFv moietiesHPCR of the pool. Initially, V was amplified with randomized 5 'library primers and 3' primers specific for V.kappa.light chain FW4H. In a separate PCR, the heavy chain FW1 region-based 5' primer was used in addition to VH3' primer amplification of V with partial complementarity of CDR randomized primerHThe remainder of the process. Full-length V was then constructed by annealing the two amplified fragments and re-amplifying the scFv by PCR using primers that attach two restriction sites (Sfi I or Not I) for subcloning of the fragmentsHCDR randomized scFv library.
To assess the diversity of the generated libraries, purified amplified DNA of all four libraries was then digested with Sfi I and Not I and ligated into similarly cut phagemid vectors (pANT 43). The ligated DNA was pelleted, resuspended in nuclease-free water, and transformed by electroporation into freshly prepared electrocompetent TG1 cells. The next day, colonies were counted, scraped and glycerol stocks prepared. The library was electroporated multiple times to adequately cover the theoretical library diversity. In all cases, 4.0 times or more coverage was obtained. Single colonies from each of the four libraries were sequenced to confirm that the appropriate CDR blocks had mutated.
Bacteria from each library were inoculated to 150ml of 2TYCG (2%)In culture. Cultures were grown to mid-log (OD)600nm0.5-0.6) and estimate the total number of cells (based on OD)600nmIs 1 ≈ 5x108Individual cells/ml). Helper phage were added and incubated for 1 hour, then centrifuged, resuspended in 2TYCK medium and grown overnight at 30 ℃. The next day, the culture supernatant was recovered by centrifugation and then the phage was harvested using 4/10 Xvolume of cooled 20% PEG/2.5M NaCl pellet. After incubation on ice for 1 hour, the precipitated phage were recovered by centrifugation and the pellet was resuspended in 1x PBS pH 7.4. The supernatant was again centrifuged to remove any cell debris and then the supernatant was reprecipitated as described above. The precipitated phage were resuspended in 1x PBS pH 7.4 and filter sterilized. To increase the chance of obtaining scFv with increased affinity, multivalent superphage M13K07 Δ pIII helper phage was used for library resuscitation at a multiplicity of infection of 20 due to the relatively low affinity of the starting antibody. After the first round of selection, a monovalent M13K07 helper phage with a multiplicity of infection of 10 was used, due to the expected enrichment of antigen binders.
Phage selection with improved affinity
Two separate selection strategies were implemented to increase the likelihood of obtaining affinity-improved phage. CEACAM1 was either biotinylated (for soluble selection) or not biotinylated (for solid phase panning) throughout the selection. Soluble selection (activity 1) or solid phase panning (activity 2) was used in round 1 in different selection cascades to enrich for functionally bound phage and diversity. Deselection using the closely related family members CEACAM5 and CEACAM6 was performed by individual panning at 1 μ g/ml of each protein to try and reduce cross-reactivity. This is done twice during each activity by deselecting either before any one round of selection and before the 2 nd round (activity 1) or before the second and third rounds of selection (activity 2). For both campaigns, the four libraries remained separate at all stages.
For soluble selection, each library was pre-blocked with PBSB, and then the phage were incubated with decreasing concentrations of biotinylated CEACAM1 antigen for up to three hours. After incubation, streptavidin paramagnetic beads (pre-blocked as above) were added to each selection and the rotation was reversed for 15 minutes. Streptavidin-antigen-phage complexes were washed with increasing numbers of PBST washes in each successive selection round, followed by PBS washes, and captured with a magnet between each step. The phage were eluted from the beads by the addition of 50mM HCl, and the solution was then neutralized by the addition of 1M Tris-HCl pH 9.0.
Solid phase panning and all deselections were performed on Nunc Immuno MaxiSorp 96 well flat-bottomed microtiter plates coated with antigen overnight at 4 ℃ and then blocked with PBSB. To deselect, the pre-blocked phage were incubated with CEACAM5, then with CEACAM6, then unbound phage were removed and used for subsequent selection. For CEACAM1 panning, pre-blocked phage were incubated with 8 μ g/ml antigen, and the plates were then washed with 3x PBST and 2x PBS. Bound phage were eluted with 50mM HCl as for soluble selection. For soluble selection and panning, eluted phage were added to mid-log E.coli TG1 and infected cells for 1 hour at 37 ℃ before plating on 2TYCG (2%) plates and growing overnight at 37 ℃. The next day, colonies were picked for screening, or plates were scraped and phages were rescued as described above. Fig. 9A and 9B show an overview of the different selection strategies used.
Expression and initial testing of scFv
Soluble scFv were initially expressed and tested as crude periplasmic extracts. Individual colonies were picked into 1ml of 2TYCG (0.1%) medium and grown by shaking at 37 ℃ for 5 hours. The culture was induced by adding IPTG to a final concentration of 1mM and then grown overnight at 30 ℃ with shaking. The next day, the culture was centrifuged and the supernatant was discarded. The bacterial pellet was resuspended in tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) buffer pH 7.4 and incubated on ice for 30 minutes. The cells were then centrifuged and the supernatant discarded. Resuspend pellet in ice cold 5mM MgSO 4In (1). The plates were then centrifuged and the supernatant containing the scFv was transferred to fresh plates for assay.
Periplasmic extracts from colonies from different rounds of selection were screened in a single point binding assay for their ability to bind GST-CEACAM 1. At each assayIncluding parent scFv (V) on plateH1/V κ 8S29A) and unrelated scFv for comparison.
The periplasmic extracts were blocked by dilution with PBSB 1:1 and then incubated for 1 hour at room temperature on Nunc Immuno MaxiSorp 96 well flat-bottomed microtiter plates pre-coated with 1.0. mu.g/ml GST-CEACAM 1. The plates were then washed and binding of the scFv was detected with anti-HIS 6-HRP antibody and TMB substrate. The reaction was stopped with 1M HCl and the absorbance read at 450nm on a Dynex Technologies MRX TC II microplate reader and binding data plotted.
Based on relative parental scFv V in binding ELISAH1/V.kappa.8S 29A (which contains mouse CDRs) and unrelated scFv activities measured on the same plate to identify improved clones. Greater than 4400 periplasmic extracts were analyzed, 34 leader sequences that bind at least 1.5 more than the parent in two independent experiments were sequenced, and unique clones were identified. Examination of the obtained sequences revealed that the parent amino acid was found at several positions, but was encoded by a different codon than the parent. This indicates that selection occurred as expected, but the parent amino acid is the preferred amino acid at this position. Based on this sequence analysis, 19 unique CDR1H, three CDR3H block 1, three CDR3H block 2, and 9 unique CDR3L clones were used for large-scale scFv expression. Table 10 shows a summary of the 34 leader sequences selected as purified scFv for further analysis, as well as the CDR mutations of these mutants.
Table 11, table 12 and table 13 highlight the conservation/variability of the affinity matured CDRs in the scFv variant leader sequence identified using GST-CEACAM1 binding ELISA.
TABLE 10 summary of 34 scFv variant leader sequences identified using GST-CEACAM1 binding ELISA. The scFv derived libraries and the rounds of deselection by scFv are summarized. The parent (V.kappa.8S 29A) CDR is shown at the top of the table. Mutations in CDR1H, CDR3H B1, CDR3H B2, and CDR3L that differ from the parent sequence are highlighted in bold. All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Variants
CDR1H
CDR3H (Block 1)
CDR3H (Block 2)
CDR3L
Parent strain
GFIFSSHGMS
RHDFDYD
DAAWFAY
QWSSNPP
CP09E05
RHGFDYD
CP09F05
QNTALPF
CP09F03
GFTFNNHGMS
CP09A04
GFSFNAHAMS
CP09E03
GFTFSAHAIS
CP09D03
GFTFSSHAIS
CP09B02
GFTFTSHAIS
CP09C02
EFTFSDHAMS
RHGFDYD
CP09B03
GFTFNAHAIS
CP09G03
GFTFNAHAMS
CP08G09
QWTAFPP
CP08D02
QWTSFPP
CP08G02
QWTNNPP
CP08C08
QNTSLPF
CP08F05
QWTSNPP
CP08E05
QWTTNPP
CP08G01
QNTNLPF
CP08E01
QWTTFPP
CP08B04
FPAWFAL
CP08H03
FPYWFAH
CP08G10
FPAWFAF
CP08H01
KHPPDYF
CP08B01
GFTFSAHAMS
CP08A08
GFIFTNHGMS
CP08A03
GFIFNNHAIS
CP08B03
GFTFTAHAIS
CP08D11
GYSFSAHGMS
CP08B11
GFTFTNHGMS
CP08C04
GFTFSSHGMS
CP08B06
GFSFNSHAIS
CP08F07
GFTFTDHAIS
CP08C01
GYSFSNHGMS
CP08A06
GYSFSSHGMS
CP08D01
GFTFNAHGMS
Table 11 CDR motifs of heavy chain CDR1 in scFv variant leader sequences were identified using GST-CEACAM1 binding ELISA.$Residue numbering is based on the Kabat numbering scheme. Residue numbering is based on the primary amino acid sequence of the heavy variable chain. The CDR1H comprises residues 31-35 as defined by the Kabat CDR and residues 26-33 as defined by the IMGT.
TABLE 12 CDR motifs of the heavy chain CDR3 in scFv variant leaders identified using GST-CEACAM1 binding ELISA.$Residue numbering is based on the Kabat numbering scheme. Residue numbering is based on the primary amino acid sequence of the heavy variable chain. #Are not part of the CDRs as defined by Kabat. Residues are included in mutagenesis to allow for more diversity among the germline residues of the anchored CDRs.
Table 13 CDR motifs of light chain CDR3 in scFv variant leaders identified using GST-CEACAM1 binding ELISA.$Residue numbering is based on the Kabat numbering scheme. Residue numbering is based on the primary amino acid sequence of the light variable chain. Residue # was not mutated during affinity maturation.
Large Scale ScFv expression and purification
Selected clones were expressed, purified and quantified for accurate testing of scFv by binding ELISA. Briefly, individual colonies were picked into 15ml of 2TYCG (2%) medium and grown overnight by shaking at 30 ℃. The starting culture was used to inoculate 500ml of 2TYCG (0.1%) and grown at 30 ℃ until OD600nm is-0.8. The culture was induced by adding IPTG to a final concentration of 1mM and then grown overnight at 30 ℃ with shaking. The next day, the culture was centrifuged and the supernatant was discarded. The bacterial pellet was resuspended in 15ml TES and incubated on ice for 15 minutes. 22.5ml TES (diluted 1:5 in cold water) was then added and incubated on ice for an additional 30 minutes. The cells were then centrifuged and the supernatant containing the scFv was transferred to a new tube, followed by the addition of MgCl 2NaCl and imidazole to final concentrations of 1mM, 300mM and 20mM, respectively, to reduce non-specific binding. Adding Ni-agarSugar beads and scFv were bound by rotary incubation at 4 ℃ for 2 hours. The beads were pelleted by centrifugation and washed twice with wash buffer (25mM Tris pH 7.4, 300mM NaCl, 20mM imidazole) before eluting the scFv from the beads using elution buffer (25mM Tris pH 7.4, 300mM NaCl, 400mM imidazole). The samples were quantified by measuring the OD 280nm and using the extinction coefficient based on the predicted amino acid sequence. Approximately 1. mu.g of each scFv was analyzed by SDS-PAGE. Bands corresponding to typical scFv spectra were observed.
Assessment of scFv binding to GST-CEACAM1 by ELISA assay
Binding of affinity matured purified scFv to human CEACAM1 was analyzed using GST-CEACAM 1. Nunc Immuno MaxiSorp 96 well flat bottom microtiter plates were pre-coated with 1.0. mu.g/ml GST-CEACAM1 overnight at 4 ℃. The next day, the mixture is added with VH1/V.kappa.8S29A parent scFv or test scFv (50. mu.g/ml to 0.8. mu.g/ml) in PBSB a two-fold dilution series was incubated for 2 hours at RT on pre-coated ELISA plates. Binding of scFv was detected with anti-HIS 6-HRP antibody and TMB substrate. The reaction was stopped with 3M HCl, and the absorbance was read at 450nm on a Dynex Technologies MRX TC II microplate reader and binding curves were plotted. Exemplary binding assay data is shown in fig. 10. Parent scFv (V) included on each ELISA plate H1/V.kappa.8S 29A scFv) as reference. Irrelevant scFv was included on at least one plate as a negative control. Observed with parent VHMany scFvs have improved binding to CEACAM1 compared to 1/V.kappa.8S 29A scFv. Derived from VHAnd scFv of the vk library. All 34 scFv variants were reformatted as intact iggs to provide greater accuracy with respect to purity and quantitation. Reformatting further allows analysis of the avidity component of antibody binding, which is influenced by the bivalent nature of IgG. As used herein, "avidity" is a measure of the strength of binding between an antigen binding molecule (such as an antibody or antibody fragment thereof described herein) and an associated antigen.
Construction and testing of affinity matured intact antibodies
Reformatting scFv into intact IgG
The 34 variants identified by scFv screening were PCR amplified using primersPrimers were introduced flanking restriction enzyme sites for cloning into IgG 4S 241P pantvg 4 vector and kappa light chain pANTVK vector. 25 affinity matured V's were identified using Mlu I and Hind III restriction sitesHThe variants were subcloned into the IgG 4S 241P patvhg 4 vector. Similarly, 9 affinity matured vk sequences were subcloned into the kappa light chain patvk vector using bsh II and BamH I restriction sites. All constructs were confirmed by sequencing.
For expression, 25 leads were humanized affinity matured IgG 4VHThe variants were combined with a parent humanized non-glycosylated light chain (V κ 8S 29A). 9 lead humanization affinity matured kappa light chains were compared to the parental humanization heavy chain (V)H1) And (4) combining. These combinations were transiently transfected into HEK EBNA adherent cells (in 6-well plates) using the PEI transfection method. Five to seven days after transfection, supernatants were harvested, quantified by ELISA and filtered for Biacore single cycle kinetic analysis.
Single cycle kinetic analysis of binding of humanized and affinity matured leader IgG to CEACAM1
To assess binding of humanized affinity matured reformatted lead IgG, single cycle kinetic analysis was performed on the crude supernatant using Biacore T200 running Biacore T200 control software V2.0.1 and Biacore T200 evaluation software V3.0. The antibody was diluted in HBS-P + to a final concentration of 0.5. mu.g/ml. At the beginning of each cycle, antibodies were loaded onto Fc2, Fc3, and Fc4 of the protein a chip. IgG was captured at a flow rate of 10. mu.l/min to give an immobilization level (RL) of about 100RU (calculated R of about 50-150RU obtained after binding of analytemaxThe level of (d). The surface is then stabilized. Single cycle kinetic data were obtained using CEACAM1 as the analyte at a flow rate of 80 μ Ι/min to minimize any potential mass transfer effects. From parent (V) H1/V κ 8S29A) antibody was repeated multiple times to check the stability of the surface and analyte in the kinetic cycle. The signal from the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. Three-point two-fold dilution range of 70nM to 280nM CEACAM1 without regeneration between concentrations was used. Subtract from each antibodySignal of blank run (without CEACAM1) to correct for differences in surface stability. The dissociation phase was monitored for 80 seconds for three increasing concentrations of CEACAM1 injections each and a single dissociation phase was measured 150 seconds after the last injection of CEACAM 1. Regeneration of protein a surface was performed using two injections of 10mM glycine-HCLpH 1.5 followed by a 250 second stationary phase.
Single cycle kinetic constants: (
Table 14) shows that all but one of the humanized affinity matured antibodies bound CEACAM 1.
TABLE 14 humanized and affinity matured variants and parents (V)H1/V κ 8S29A) single cycle kinetic constant for antibody binding to CEACAM 1. K of variants by humanization and affinity maturationDDivided by the K of the parent determined several times in the same experimentDTo calculate the relative K compared to the parent IgG D. The variants performed are shown in bold. CDRs containing the mutation are indicated by "+". All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain). Variants shown in bold have the same identity as the parent VH1/V κ 8S29A comparison>Double relative KD。
Eight humanized and affinity matured heavy and light chain variants were identified that showed comparisons to the parent>Double relative KD(in
Highlighted in bold in table 14). These include three kinds of VHVariants (CP08H03, CP09B03, and CP09C02) and five kappa light chain variants (CP08E01, CP08E05, CP08F05, CP08D02, and CP08G 09).
CP09C02 (which was derived from the CDR1H library) contained additional point mutations in the CDR3H B1(D96G), which are most likely at the library construction stageIntroduced by PCR (see "example 1,1. Generation of humanized antibody variants" section). Thus, an additional heavy chain clone CP09E05 was also obtained, as it was identified as being at VHThere was only such a single point mutation in CDR 3B 1, and thus may be helpful in identifying which region is involved in the observed increase in affinity. Four V are then obtainedHAnd five V κ variants were used to determine whether recombinant affinity matured heavy and light chains could have improved effects.
Expression of combined leader heavy and light chain antibodies
Four humanized affinity matured IgG 4V identified after expression with parental light chainHEach of the variants (CP08H03, CP09B03, CP09C02, and CP09E05) was combined with five lead humanized affinity-matured kappa light chains (CP08E01, CP08E05, CP08F05, CP08D02, and CP08G09) (i.e., 20 pairings total, see table 15). As a control, humanized affinity matured IgG 4V was usedHVariants were combined with the parent light chain (V.kappa.8S 29A) and five lead humanised affinity matured kappa light chains were combined with the parent heavy chain (V.kappa.8S 29A)H1) Combinations (i.e., 10 control antibodies in total, see table 15). The combinations were transiently transfected into HEK EBNA adherent cells in 6-well plates using the PEI transfection method and incubated for 5-7 days post transfection as described above. The supernatants were harvested, quantified by ELISA and filtered for single cycle kinetic analysis on Biacore.
TABLE 15 parent VHOr lead humanized affinity matured IgG 4VHVariants in combination with a parent vk or a leader humanized affinity matured vk light chain. Transfected antibodies with recombinant affinity matured heavy and light chains (black), affinity matured antibodies expressed with parental heavy or light chains, and parental antibodies. All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Single cycle kinetic analysis of combined leader heavy and light chain antibodies
Single cycle kinetics using transient HEK supernatants were performed as described previously. Fitting data showing single cycle dynamics
TABLE 16 fifteen heavy and light chain combinations have at least twice the relative K as compared to the parentD. Wherein, K obtained by six combinations (CP08H03/CP08E05, CP08H03/CP08F05, CP08H03/V K8S 29A, CP09B03/CP08E05, CP09C02/CP08E05 and CP09C02/CP08F05)DIs more than four times larger than the parent (in
Highlighted in bold in table 16). These six variants were used for large-scale production and protein a purification for further analysis. Single cycle kinetics also revealed that, when combined, the three variants were found to be non-functional.
TABLE 16 leader VHAnd single cycle kinetic constants for binding of vk combination and parental antibodies to CEACAM 1. By introducing a leader VHAnd K of V kappa combinatorial variantsDDivided by the K of the parent determined in the same experimentDTo calculate the relative K compared to the parentD. The variant highlighted in bold is KDIs parental>Four times preamble combining. CDRs containing the mutation are indicated by "+". All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Recombination of four affinity-matured heavy chain CDRs to yield six additional heavy chain variants
Six combinations with greater than four times improvement are contained in four different VHThree unique heavy chains with mutations in the CDRs: CP08H03(CDRH 3B 2); CP09B03(CDR1H) and CP09C02(CDR3H B2 and a single mutation in CDR3H B1, which is also uniquely present in CP08E 05), see table 17. To determine whether further improvement could be obtained, four mutations V were performedHRecombination of CDRs (table 18). Recombination of individual V's using penetration PCR Using scFv-specific primersHCDRs, which were subsequently cloned into the IgG 4S 241P heavy chain expression vector using Mlu I and Hind III restriction sites to generate six novel VHVariants (8H3_9B3, 8H3_9C2, 8H3_9E5, 9B3_9E5, 8H3_9C2(CDR1) and 9B3_9E5_8H 3).
Table 17 VH CDRs for recombination.
Table 18 recombinant heavy chain clones. Will be derived from the four preambles VHThe cloned individual CDRs are recombined to produce six recombinant affinity matured heavy chains. Mutations in CDR1H, CDR3H B1, and CDR3H B2 that differ from the parent sequence are highlighted in bold. Residue 104 was selected based on the sequence selected for CDR3H B2.
Recombination of VHExpression of CDR1 and CDR3 heavy and leader light chains:
six recombinant V HThe CDR1 and CDR3 variants (see table 18) were combined with (1) the parent light chain (vk 8S29A), (2) light chain CP08E05, or (3) light chain CP08F 05. The latter two light chains previously gave improved effects when combined with affinity matured heavy chains (see table 16). The resulting 18 combinations are summarized in table 19. These combinations were transiently transfected into HEK EBNA adherent cells in 6-well plates using the PEI transfection method and incubated 5-7 after transfectionAnd (5) day. The supernatants were harvested, quantified by ELISA and filtered for single cycle kinetic analysis on Biacore.
TABLE 19 recombinant VHCDR1 and CDR3 leading humanized affinity matured IgG4VHVariants in combination with the parent V κ or two lead humanized affinity matured κ light chains. Combinations with both affinity-matured light chains are denoted by "AM" and combinations with the parental light chain are denoted by "P". All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Recombinant VHAnd leader VLSingle cycle kinetic analysis of antibodies:
single cycle kinetics using transient HEK supernatants were performed as described previously. The fit data for the single cycle kinetics are shown in table 20.
Eight variants were found to have a relative K greater than four-fold greater than the parentD. Of these, five obtained KDMore than six times that of the parent (bold in table 20). Five antibodies (8H3_9B3/CP08E05, 8H3_9B3/CP08F05, 8H3_9B3/V kappa 8S29A, 8H3_9C2/CP08F05, and 9B3_9E5/CP08E05) were used for large-scale production and protein A purification for further analysis.
TABLE 20 recombinant leading humanized affinity matured IgG 4VHSingle cycle kinetic constants of the variants with either the parent V κ or one of the two leading humanized affinity-matured κ light chains. K of variants by humanization and affinity maturationDDivided by the K of the parent determined in the same experimentDTo calculate the relative K compared to the parentD. Highlighting in bold in comparison to the parent>A six-fold variant. CDRs containing the mutation are indicated by "+". All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Expression, purification and testing of leader antibodies
The six most improved combinatorial variants (CP08H03/CP08E05, CP08H03/CP08F05, CP08H03/V κ 8S29A, CP09B03/CP08E05, CP09C02/CP08E05 and CP09C02/CP08F05,
Highlighted in bold in table 16) with the five most improved VHCDR1 and VHRecombinant variants of CDR3 (8H3_9B3/CP08E05, 8H3_9B3/CP08F05, 8H3_9B3/V kappa 8S29A, 8H3_9C2/CP08F05, and 9B3_9E5/CP08E05, highlighted in bold in Table 20) were transiently transfected together into HEK NA adherent cells in EBflasks and incubated for 5-7 days post-transfection. Antibodies were purified from cell culture supernatants on a protein a sepharose column, buffer was changed to PBS pH 7.2, and extinction coefficient by OD based on predicted amino acid sequence was used280nmQuantification was performed. Mu.g of each antibody was analyzed by SDS-PAGE, and bands corresponding to typical antibody spectra were observed.
Single cycle kinetic analysis of purified lead humanized and affinity matured antibodies (using purified protein)
Single cycle kinetics were performed as described above using purified antibody instead of HEK supernatant. The fit data for the single cycle kinetics are shown in table 21. The expression levels of the individual mutants are provided in table 22.
All 11 lead variants bound > four-fold compared to the parent antibody (see table 21). Data obtained using purified IgG are consistent with data previously obtained using supernatant.
Table 21 single cycle kinetic constants of purified lead humanized affinity matured antibodies. K of variants by humanization and affinity maturationDDivided by the K of the parent determined in the same experimentDTo calculate the relative K compared to the parentD. Mutations of CDR1H, CDR3H B1, CDR3H B2, or CDR1L are denoted by "+" where applicable. All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to variable light chain)The S28A mutation in the primary amino acid sequence of the chain).
Table 22 expression levels of purified lead humanized affinity matured antibodies. Mutations of CDR1H, CDR3H B1, CDR3H B2, or CDR1L are denoted by "+" where applicable. All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Removal of potential CD4+ T cell epitopes
iTope using computer analysis of peptides for binding to human MHC class II allelesTMTechnology (Perry et al 2008) and TCED using T cell epitopes associated with known antibody sequencesTM(Bryson et al 2010) the sequences of 11 leader antibodies (see table 21) were analysed to ensure that no significant T cell epitopes were introduced during affinity maturation. The CDR1 mutations (G26E, according to the CDR definition of IMGT) present in the heavy chain of CP09C02 correlated with the introduction of a promiscuous homoepitope not observed in the parental sequences (see table 21).
Selective analysis of leader antibodies
Initial selectivity analysis of several precursor antibodies showed that the antibody with phenylalanine (F) at position 104 of CDR3H showed on average an increase in selectivity to CEACAM1 compared to the antibody with aspartic acid (D) at position 104 of CDR3H (fig. 11).
Multi-cycle kinetic analysis
The variants CP08H03/V κ 8S29A and CP08H03/CP08F05 were further analyzed using a Biacore T200 instrument running Biacore T200 evaluation software V3.0.1 using multi-cycle kinetic analysis. The purified antibody was diluted to a concentration of 1. mu.g/ml in HBS-P +. At the beginning of each cycle, each antibody was captured on the protein a surface to give an RL of about 100 RU. After capture, the surface was stabilized. Kinetic data were obtained using a flow rate of 80 μ l/min to minimize any potential mass transfer effects. Multiple replicates of the blank (without CEACAM1) and replicates of a single concentration of analyte were programmed into the kinetic run in order to examine the stability of the surface and analyte in the kinetic cycle. For kinetic analysis, a two-fold dilution range from 100 to 1.56nM CEACAM1 was selected. The associated phase of CEACAM1 was monitored for 150 seconds and the dissociated phase was measured for 150 seconds. At the end of each cycle, two injections of 10mM glycine-HCL pH 1.5 were used for regeneration of the protein a surface.
The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface and the global Rmax parameter was used in the 1-to-1 binding model. K of composite human antibody variants by affinity maturationDDivided by the parent K on the same chipDTo calculate the parent (V)H1/V.kappa.8S 29A) relative KD. Kinetic parameters measured for the interaction of CEACAM1 with the affinity matured CEACAM1 antibody variants CP08H03/V κ 8S29A and CP08H03/CP08F05 are shown in table 23. And VHTwo affinity matured CEACAM1 antibody variants showed a comparison of the 1/V.kappa.8S 29A parent>Four times the improvement in affinity.
TABLE 23 antibodies V determined using Biacore T200H1/V.kappa.8S 29A (parental), chimeric antibody (VH0/VK0) and data on the multi-cycle kinetics of binding of two affinity matured leader sequences to CEACAM 1. K of variants by maturation of affinityDDivided by the K of the parent determined on the same chipDTo calculate the parent (V)H1/V.kappa.8S 29A) relative KD. All variable light chains contained the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).
Example 2: selectivity of CEACAM1 antibody
To further evaluate CEACAM1 antibody VHBinding selectivity of 0/V κ 0, CP08H03/V κ 8S29A, CP08H03/CP08F05 CEACAM1 relative to other proteins binding affinities to CEACAM1, CEACAM3, CEACAM5 and CEACAM6 were compared using single cycle kinetic analysis performed as described above. Single cycle kinetics were performed using CEACAM concentrations of 280nM to 70 nM. The antibodies were loaded onto the chip at the following concentrations (taking into account the different analyte MW): CEACAM1 was 100RU, CEACAM3 was 375RU, CEACAM5 was 71.4RU, and CEACAM6 was 150 RU. No three CEACAM1 antibodies CP08H03/V κ 8S29A, CP08H03/CP08F05 and V were observed for CEACAM3, CEACAM5 and CEACAM6HSignificant binding of 0/V κ 0 (see FIG. 12A).
These results are consistent with the data obtained by measuring antibody specificity with ELISA. For ELISA experiments, 96-well plates were coated with 0.5 or 1.0. mu.g/ml CEACAM 1. Non-specific binding was blocked with 2% BSA/Dulbecco PBS. Preparation of CP08H 03/Vkappa 8S29A, CP08H03/CP08F05 or V in 2% BSA/PBSH0/V kappa 0 in a 1:3 dilution series (starting concentration 50. mu.g/mL). 100 μ L of sample was added to the pre-coated plate and incubated for 1 hour at RT. CEACAM antibodies were detected using an anti-human Ig kappa chain-peroxidase secondary antibody (AP 502P). The plate was developed with TMB and quenched with 3M HCl. The results were analyzed by subtracting the background. Essentially no three CEACAM1 antibodies CP08H03/V kappa 8S29A, CP08H03/CP08F05 and V were observed HBinding of 0/V κ 0 to CEACAM3, CEACAM5 or CEACAM6 (see FIGS. 12B and 12C).
Although the N domains of different CEACAMs have a high homology, this high selectivity can be observed: the N-domains of CEACAM1 and CEACAM3 were 88% identical, the N-domains of CEACAM1 and CEACAM5 were 89% identical, and the N-domains of CEACAM1 and CEACAM6 were 90% identical, as shown by the percentage identity matrix generated using clustal2.1 (see fig. 13).
Example 3: epitope analysis of CEACAM1 antibody
To determine which residues on CEACAM1 were involved in binding to certain CEACAM1 antibodies contemplated by the present invention, single point mutations were introduced into FLAG-labeled CEACAM 1. Each FLAG-tagged CEACAM1 mutant was transfected into 293T cells. At 48 hours post-transfection, western blotting was performed for CEACAM1 protein. CEACAM1 antibodies VH0/VK0 (chimeric antibody), VH1/VK8, VH2/VK4, VH3/VK1 and VH4/VK1 were used as detection antibodies. Mutations at CEACAM1 residues Y34, V39, G41, N42, R43, Q44, G47 and Q89, which are part of the GFCC face of CEACAM1, resulted in reduced binding of CEACAM1 to CEACAM1 antibody, suggesting that these CEACAM1 residues may be involved in binding (see fig. 14).
Example 4: CEACAM antibodies and crystal structures of CEACAM1
To more accurately delineate the binding interface between CEACAM1 and CP08H03/VK 8S 29A, the crystal structure of human CEACAM1 complexed with CP08H03/VK 8S 29A Fab fragment was determined.
CEACAM1 was expressed from escherichia coli transformed with a pET 21D-based plasmid expressing a marker-free form of CEACAM 1. The protein was refolded in arginine-containing buffer and purified. Fab fragments were prepared by antibody digestion, concentrated to about 18mg/ml, and then purified by protein a affinity and gel filtration chromatography using immobilized papain resin. Purified CEACAM1 and Fab were mixed in a 1:1 molar ratio prior to crystallization screening. Initial crystallization hits of CEACAM1: Fab complexes were determined and subsequently optimized. Diffraction quality crystals were grown at room temperature in the presence of 18-20% PEG 6000, 50mM potassium dihydrogen phosphate, 20mM Tris pH 7.0 and 1% β -octyl glucoside. SDS-PAGE analysis and silver staining of the washed crystals were used to confirm the crystallization of the complex. An Advanced Photon Source (Advanced Photon Source of Argonne National Laboratory) at the Algong National Laboratory collects X-ray data for many crystals from the beam line NE-CAT 24-ID-E. Combining the best data from two non-twinned isomorphous crystals to produce For structure determination and refinement. Resolving the complex structure by molecular replacement and refining to final R and RFreedom of movementThe values were 24.9% and 32.8%, respectively.
The CEACAM1 CP08H03/V kappa 8S29A Fab complex has the structure determined asResolution. CP08H03/V κ 8S29A Fab binds CEACAM1 at 1:1 stoichiometry (see FIG. 15). The molecular surface representation of CEACAM1 in fig. 16 shows the part of the epitope of Fab fragments on CEACAM 1. The major and minor interactions between Fab molecules and CEACAM1 are listed in tables 24 and 25.
TABLE 24 major interactions between CEACAM1 CP08H 03/Vkappa 8S29A FabResidue number # is based on the Kabat numbering scheme. Residue numbering is based on the primary amino acid sequence of the heavy variable chain.
TABLE 25 Secondary interaction between CEACAM1 CP08H 03/V.kappa.8S 29A FabResidue number # is based on the Kabat numbering scheme. Residue numbering is based on the primary amino acid sequence of the heavy variable chain.
Referring to the existing structure of CEACAM1 dimer (fig. 17), it is evident that Fab binds to the interface of CEACAM1 involved in self-association. It is speculated that this competitive interaction results in dissociation of the dimers in solution. Residues targeting CEACAM1 include four residues forming the YQQN pocket at the CEACAM1: CEACAM1 dimer interface (Y34, Q44, Q89, N97). Notably, several residues on CEACAM1 that bind antibodies are also predicted to participate in TIM-3 binding, including CEACAM1 residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and/or N97(Huang et al, nature.2015jan 15; 517(7534): 386-90).
In the Fab light chain, residues in CDR1, CDR2 and CDR3 (fig. 18) interact mainly with residues in the two loops between the β chains of the main β sheet in CEACAM1 and also with residues of the β chains in the sheet. In the Fab heavy chain, the residues of CDR2 and CDR3 interact primarily with residues distributed on the four different β chains of the central β sheet.
The interacting surfaces have a shape complementarity of 0.5 and the complex forms a total solvent accessible surface buried 1607a 1. No interaction was observed between the antigen and the Fab heavy chain CDR 1.
Alignment of human CEACAM family members indicates that CEACAM3, 5, 6, 7 and 8 all contain a valine residue at position 49, whereas human CEACAM1 contains an alanine at this position. In addition, human CEACAM5 contains histidine at position 89. Polymorphisms of these residues in hCEACAM-1 include Ala49Val (rs8110904) and Gln89His (rs 8111468). To further examine the selective properties of CEACAM1 antibody CP08H03/V κ 8S29A, the human CEACAM1a49V/Q89H mutant was expressed and purified as described above. It is noted that there are natural human allelic variants of human CEACAM1 that convert Q89 to H89, as exemplified by Huang et al, nature.2015jan 15; 517(7534) 386-90. Determination of the Structure of the CEACAM1A49V/Q89H mutant Resolution and comparison with CEACAM1 wild-type CP08H 03/V.kappa.8S 29A Fab complex. As described above, CDR3H residue F104 of CP08H 03/vk 8S29A was in contact with residue F29 in wild-type CEACAM1 (see fig. 19, left panel). Note that F29 of one CEACAM1 monomer binds F29 of a second monomer at the CEACAM1: CEACAM1 homodimer interface. Binding of CDR3H residue F104 of CP08H03/V κ 8S29A blocked the F29-F29 interaction. CEACAM1 residue A49 is located near the F104/F29 interaction site. Human CEAC due to the increased hydrophobicity of valine in non-CEACAM 1 family members compared to alanine in human CEACAM1Mutation of AM1 residue a49 to valine resulted in the movement of hydrophobic CEACAM1 residue F29 closer to CEACAM 1V 49 residue. This rotameric shift of F29 was also observed in the crystal structures of human CEACAM5(PDB encodes 2QSQ) and human CEACAM3(PDB encodes 6AW1) and predicted to conflict with CDR3H residue F104 (see fig. 19, right panel). This is illustrated by the change in orientation shown by the CEACAM 1F 29 loop that moves closer to the space previously occupied by CDR3H residue F104 (see fig. 19, right panel). These data indicate that this steric hindrance caused by the a49V mutation interferes with the binding of CEACAM1 antibody CP08H03/V κ 8S29A to other CEACAM1 family members containing a valine at position 49, and thus its major role in antibody selectivity. This rotameric shift of the F29 loop is also predicted to affect the interaction between CDR2H residues Y57 and F29. Furthermore, the CEACAM1 Ala49Val polymorphism (rs8110904) was associated with lymphedema caused by Wuzella bambusae (filarial worm invading the lymphatic system) (Debrah L, B., et al Hum genomics.2017Nov 9; 11(1): 26). The progression of the disease was associated with the Ala49Val polymorphism, which marks that alanine 49 residue was found to be involved in binding to the CP08H 03/V.kappa.8S 29A antibody. Thus, CP08H 03/vk 8S29A is also expected to interfere with other related pathogens of wuchereria bambusae and phenotypic helminth interaction with lymphatic vessels or cancer processes, such as tumor invasion.
Example 5: CEACAM1 antibody blocks CEACAM1 CEACAM1 interaction
The CEACAM1 antibody was tested for its ability to block CEACAM1 homodimerization. A CEACAM1-CEACAM1 competition ELISA study was performed in triplicate to determine the ability of CP08H 03/V.kappa.8S 29A antibody (concentration range 0-1000nM) to inhibit the binding of human CEACAM1 IgV domain unlabeled protein (1pg/ml) and human CEACAM1-GST protein (37.5 pg/ml). Furthermore, IgG4 antibody was used as a control (0-1000 nM). Goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used and assayed by addition of TMB solution (Life technologies). OD was read at 450nm on a microplate reader. Data were plotted in Graphpad and best-fit IC-50 values were determined.
CEACAM1 antibody CP08H03/V κ 8S29A was shown to block CEACAM1: CEACAM1 homeotropic interactions (see FIG. 20A).
Example 6: CEACAM1 antibody blocks CEACAM1: TIM-3 interaction
The CEACAM1 antibody was tested for its ability to reduce binding of CEACAM1 to TIM-3. CEACAM1/TIM-3 competition ELISA studies were performed in triplicate to determine the ability of CP08H 03/Vkappa 8S29A antibody (concentration range 0-300nM) to inhibit the binding of human TIM-3IgV domain unlabeled protein (3pg/ml) and human CEACAM1-GST protein (37.5 pg/ml). In addition, human IgG4 antibody was used as a control (0-1000 nM). Goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used and assayed by addition of TMB solution (Life technologies). OD was read at 450nm on a microplate reader. Data were plotted in Graphpad and best-fit IC-50 values were determined. As shown in FIG. 20B, CEACAM antibody CP08H03/V κ 8S29A blocked CEACAM1: TIM-3 heterophilic interaction.
Example 7: CEACAM1 antibody induces T cell proliferation
The ability of CEACAM antibodies CP08H03/V κ 8S29A and CP08H03/CP08F05 to induce T cell proliferation was studied in humanized NOD scid γ mice (NSG mice). The experimental setup is seen in fig. 21. Transfer of freshly isolated human PBMC (5x10^6) to NOD.Cg-PrMc by intraperitoneal (i.p.) injectionscld I/2rgtm1Wjlin/SzJ (NSG) mice. NSG animals were examined for human immune cell engraftment by tail bleeding 21 days after PBMC injection. The humanized NSG mice were administered a first and second dose of the CEACAM1 antibody or isotype control antibody at the indicated concentrations by intraperitoneal injection 24 and 31 days after PBMC injection. After termination of the study (34 days after PBMC injection), mice were sacrificed and spleens were surgically dissected for further analysis. Single cell suspensions from transplanted mice were stained with cell proliferation dyes and cultured in vitro for an additional 2 days in complete RPMI medium in the presence of a soluble form of anti-human CD3 challenge (2. mu.g/ml, OKT3 clone) and rIL-2 (40U/ml). Cells were maintained at a concentration of 10^7 cells/ml. After in vitro stimulation, cells were stained with an antibody against the human CD45 leukocyte marker and evaluated by flow cytometry.
No antibody-dependent cell-mediated cytotoxicity (ADCC) was observed in any of the test groups (see fig. 22). Administration of CEACAM antibodies CP08H03/VK 8S29A or CP08H03/CP08F05, respectively, resulted in an increase in antibody-induced T cell expansion in vivo (see FIG. 23).
Example 8: CEACAM1 antibody reduces tumor growth in melanoma models
To assess the ability of CEACAM1 antibody to reduce tumor growth, 1x10 was used6MALME-3M (human melanoma) and 5x106The individual PBMCs are injected subcutaneously into 7-8 week old male NSG (NOD. Cg-PrMc)scld I/2rgtm1WjlSzJ) mice. MALME-3M (BRAFV)600E) Cell lines were established in 1975 from metastatic sites (lungs) in 43 year old white men with metastatic melanoma. On days 7-9, all mice were confirmed to exhibit a reconstituted T cell population (see figure 24A for experimental setup). Animals were treated intraperitoneally with CEACAM antibody CP08H03/VK 8S 29A or hIgG4 control antibody on days 10, 13, 17, 20, and 24.
The human melanoma cell line MALME-3M was gifted by Nicole Beauchemin, Phd.McGill University, Montreal, Canada. MALME-3M was established in 1975 from metastatic sites (lungs) in 43 year old white men with metastatic melanoma with BRAFV 600E. Injecting 2x10^7 MALME-3M subcutaneously (s.c.) into NOD.Cg-PrMcscldI/2rgtm1Wjlin/SzJ (NSG) mice. After a 30 minute acclimation period, freshly isolated human PBMCs (1x10^ 8) were then transferred to tumor-bearing NSG mice by intraperitoneal (i.p.) injection. NSG animals were examined for human immune cell engraftment by tail bleeding 7 to 9 days after PBMC injection. At 10, 13, 17, 20 and 24 days after human cell injection, humanized NSG mice bearing tumors received a total of five doses of either CEACAM1 antibody or isotype control antibody at the indicated concentrations by intraperitoneal injection. After termination of the study (34 days after human cell injection), mice were sacrificed and surgically dissected.
CEACAM1 antibody CP08H03/VK 8S29A was effective at various concentrations to reduce tumor growth and proliferation (see fig. 24B, 24C and 25) while depleting the T cell population (see fig. 22). In addition, human CD4 was restored by administration of CEACAM1 antibody+And CD8+Proliferative capacity of tumor infiltrating lymphocytes, see fig. 25. A bias of CD 8T memory cells predominantly towards central memory T cells was also observed upon in vivo treatment with CP08H 03/Vkappa 8S29A (FIG. 26). It is noted that, relative to that observed in control-treated animals,CEACAM1 antibody CP08H03/VK 8S29A increases TcmAnd TemConsistent with an enhancement in anti-cancer response.
Example 9: CEACAM1 antibodies are useful for treating cancers resistant to checkpoint inhibitors
CEACAM1 is expressed on a majority of TILs derived from naive melanoma patients or anti-PD-1 and/or anti-CTLA-4 therapy resistant melanoma patients; and CEACAM1 expression level was greater than that of PD-1 or TIM-3 (see fig. 27). About 80% of the samples were at greater than 20% of CD4 isolated from TIL+Expression of CEACAM1 was shown on the T cell population. To compare CEACAM1 expression in patients with acquired resistance to anti-PD-1 and/or anti-CTLA-4 therapy with patients not previously exposed to anti-PD-1 and/or anti-CTLA-4 therapy, tumor-associated cells (TAC) were obtained from naive melanoma patients (not previously exposed to anti-PD-1 and/or anti-CTLA-4 therapy) or those patients with acquired resistance to anti-PD-1 and/or anti-CTLA-4 therapy (acquired resistance). TAC was obtained by culturing tumor tissue in DMEM medium and removing floating cells from the supernatant. Cells were stained with CD3, CD4 and CD8 and assessed for CD3 +CD4+And CD3+CD8+CEACAM1 expression above. These studies show that expression of CEACAM1 is upregulated by tumor-associated cells deprived of the tumor microenvironment in acquired resistance relative to that observed in naive patients (see fig. 28), suggesting that patients resistant to anti-PD-1 and/or anti-CTLA-4 therapy may benefit from anti-CEACAM 1 antibodies or antigen-binding fragments thereof, such as those contemplated in this disclosure.
As expected, CD8 was present in patients resistant to anti-PD-1 and/or anti-CTLA-4 therapy compared to patients not previously exposed to anti-PD-1 and/or anti-CTLA-4 therapy+Central memory in T cells (T)cm) Cellular versus effector memory (T)em) There was a relative decrease, which is consistent with a decrease in the anti-cancer response in resistant patients (see fig. 29). Tumour-associated cells from naive melanoma patients (untreated patients) or melanoma patients resistant to immune checkpoint inhibitors (treatment-failed patients) were stained for CD44, CCR7 and CD62L, and central memory Tcm(CD44Height of、CD62LHeight of、CCR7Height of) Sum effect memory Tem(CD44Height of、CD62LIs low in、CCR7Is low in) The relative amount of (a) is expressed as a percentage of total CD 8T cells present in a large tumor.
To assess the ability of CEACAM1 antibodies to reverse T cell depletion in patients resistant to treatment with checkpoint inhibitors such as PD-1/PD-L1 and CTLA-4 inhibitors, PBMCs and tumor-associated cells were isolated from melanoma patients with secondary resistance to pembrolizumab (PD-1 inhibitor), ipilimumab (CTLA-4 inhibitor) + nivolumab (PD-1 inhibitor) and dalafenib (B-Raf inhibitor) + trametinib (MEK inhibitor) and stage IV disease. CEACAM1, PD1 or TIM-3 staining of tumor-associated cells and PBMCs and showing CD8 +And CD4+Proportion of T cells (see figure 30, left panel). Tumor biopsies were subjected to enzymatic digestion or a commercial mechanical/enzymatic dissociation system (GentleMeCs dissociator, Miltenyi Biotec). Enzymatic digestion is based on a previously established method for producing melanoma TIL (Dudley et al, 2003, 2008). Briefly, tumor biopsies were cut into small pieces approximately 2-3mm long and placed in a container consisting of 100U ml-1DNase, 10mg ml-1Enzyme digestion mixture of collagenase VIII (Sigma-Aldrich) and continuous rotary incubation at 37 ℃ for 45 minutes. GentleMACS dissociation was performed according to the manufacturer's protocol. Briefly, tumors were cut into small fragments of approximately 2-3mm in length and placed in C tubes (Miltenyi Biotech) with RPMI1640(Lonza, Slough, UK) and solutions 1, 2 and 3 (all from Miltenyi Biotech) according to the manufacturer's recommendations; the digestion mixture containing the tumor was then subjected to three 36 second mechanical disaggregation steps (procedures h _ tumor _01.01, 02.01 and 03.01) in a GentleMeS ACS dissociator, interspersed with two 30 minute incubations at 37 ℃ after the first and second disaggregation steps, respectively. After disaggregation, TIL from enzymatic digestion and GentleMeC dissociation was passed through a 100 μm filter for further analysis. Dissociated tumor cells and autologous PBMCs were stained with the following antibodies according to standard procedures: fluorochrome-conjugated monoclonal antibodies specific for human CD3, CD4, CD8, TIM-3, PD1, CEACAM1, CD45, and reactive dyes. Data were obtained using a Cytoflex Flow cytometer (Invitrogen) and Flow Jo software (TreeStar, V7.6.5 by Windows) analysis. PBMC or tumor-associated cells were combined with soluble anti-CD 3(2pg/ml) and rIL-2(40 units/ml) in 96-well plates in the presence of CP08H03/VK 8S 29A or hIgG4 controls, supplemented with 10% Fetal Calf Serum (FCS), 1% glutamine, 100IU ml-1Penicillin, 100. mu.g ml-1Streptomycin (Life Technologies), 25mM HEPES (Sigma-Aldrich) in complete medium (RPMI 1640 (Lonza)). After 96 hours, cell culture supernatants were collected for further TNF-and IFN-ELISA (BD) analysis according to the manufacturer's protocol. CP08H03/VK 8S 29A reversed T cell depletion in PD-1/CTLA-4 resistant tumors as evidenced by increased TNF-. alpha.and IFN-. gamma.production in both tumor-associated cells and PBMCs (see FIG. 30, right panel).
Example 10: CEACAM1 antibodies contemplated by the present invention exhibit improved efficacy compared to previously known CEACAM1 antibodies
The properties of the antibody CP08H03/VK 8S 29A were compared with the anti-CEACAM 1 antibody CM-24(WO 2015/166484). Unlike CEACAM1 antibody CP08H03/VK 8S 29A disclosed herein, CM-24(i) binds CEACAM1 away from the dimer interface based on modeling, (ii) exhibits cross-reactivity with CEACAM3 and CEACAM5, (iii) exhibits limited ability to reverse T cell tolerance in TIL, and (iv) functions as an agonistic rather than an antagonistic antibody in a mouse model of metastatic melanoma.
CP08H 03/vk 8S29A is selective for CEACAM1 and does not show significant binding to CE ACAM3, CEACAM5, CEACAM6, or CEACAM 8. CM-24, on the other hand, showed significant cross-reactivity with CEACAM3 and CEACAM5 at higher antibody concentrations (fig. 31A and 31B). The cervical adenocarcinoma cell line HeLa (ATCC No CCL-2) as well as the transfected cell lines HeLaCEACAM1, HeLaCEACAM3, HeLaCEACAM5, HeLaCEACAM6 and HeLaCEAC AM8 were cultured at 37 ℃ in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum penicillin (100U/ml) and dihydrostreptomycin (100pg/ml) at 5.0% CO2 for flow cytometry experiments. Cell lines were stained with the indicated antibodies, followed by fluorescent dye-conjugated monoclonal antibodies (such as human IgG4 or mouse IgG1) and a vital Dye (DAPI) specific for the indicated antibody isotype. Data were obtained using a Cytoflex flow cytometer (Invitrogen) and analyzed using FlowJo software (treestar, V7.6.5 by Windows).
In addition, CM-24 showed limited ability to reverse T cell tolerance in tumor-associated cells. Incubation of tumor-associated cells with antibody CP08H03/V κ 8S29A resulted in a broader reversal of T-cell tolerance over a range of antibody concentrations than CM-24 in naive Merkel cell carcinoma tumor cells (FIGS. 32A, 32B and 32C). The mercker cell carcinoma biopsies were subjected to a commercial mechanical/enzymatic dissociation system (gentlemecs acs dissociator, Miltenyi Biotec) according to the manufacturer's protocol. Briefly, tumors were cut into small fragments of approximately 2-3mm in length and placed in C tubes (Miltenyi Biotech) with RPMI 1640(Lonza, Slough, UK) and solutions 1, 2 and 3 (all from Miltenyi Biotech) according to the manufacturer's recommendations; the digestion mixture containing the tumor was then subjected to three 36 second mechanical disaggregation steps (procedures h _ tumor _01.01, 02.01 and 03.01) in a GentleMeS ACS dissociator, interspersed with two 30 minute incubations at 37 ℃ after the first and second disaggregation steps, respectively. After depolymerization, TILs from enzymatic digestion and GentleMeC dissociation were passed through a 100pm filter for further analysis. In vitro assay of T cell function in tumor environment: dissociated tumor cells and autologous PBMCs were supplemented with 10% Fetal Calf Serum (FCS), 1% glutamine, 100IU ml in 96-well plates in the presence of various concentrations of antibody or related isotype controls -1Penicillin, 100. mu.g ml-1Streptomycin (Life Technologies), 25mM HEPES (Sigma-Aldrich) in complete medium (RPMI 1640(Lonza)) with 40IU ml-1Recombinant IL-2(NIH) was incubated with soluble CD3(2 g/ml). After 96 hours, cell culture supernatants were collected for further TNF-and IFN-ELISA (BD) analysis following the manufacturing procedure.
In a metastatic melanoma model (experimental setup see figure 33A), mice treated in vivo with CP08H 03/vk 8S29A showed a significant reduction in tumor cells and TIL CD4 compared to control mice treated in vivo with human IgG4(hIgG4)+And CD8+Lymphocytes were significantly increased (fig. 33B, 34A, 34B and 34C). On the other hand, mice treated in vivo with CM-24 showed an in comparison to control mice treated with hIgG4 controlA large increase in tumor cells and almost complete absence of TIL CD4+And CD8+Lymphocytes (fig. 33B, 34A, 34B and 34C). Furthermore, tumor cells showed reduced proliferation in CP08H 03/vk 8S29A treated animals relative to hIgG4 control or CM24 treated animals (fig. 33C). Furthermore, CD4 in the spleen of CP08H 03/vk 8S29A treated animals versus hIgG4 treated animals or CM24 treated mice+T cells showed increased proliferation (fig. 33D). In contrast to animals treated with CP08H 03/Vk 8S29A, animals treated with CM24 exhibited reduced splenic CD4 +Proliferation of T cells (fig. 33D). The human melanoma cell line MALME-3M was supplied by Nicole Beauchemin PhD.A. (McGill University). MALME-3M was since having BRAF in 1975V600EMetastatic melanoma of (2) established at the metastatic site (lung) in a 43 year old white male. 2x10^7 MALME-3M were injected subcutaneously (s.c.) into NSG mice. Mice were allowed to take up tumor cells after 30 minutes, and then freshly isolated human PBMC (1x10^ 8) were transferred to tumor-bearing NSG mice by intraperitoneal (i.p.) injection. NSG animals were examined for human immune cell engraftment by tail bleeding 7-9 days after PBMC injection. On day 14, palpable tumor nodules were detected. Starting on day 17 after human cell injection, humanized tumor-bearing NSG mice received a total of four doses of CP08H 03/Vkappa 8S29A antibody (2mg/kg), CM-24(2mg/kg), or isotype control antibody (2mg/kg) by intraperitoneal injection twice weekly. After termination of the study (30 days after human cell injection), mice were sacrificed and dissected surgically. Metastatic tumors were preserved with spleen and lung and liver for further analysis. The total cell count and proliferation and frequency of CD4, CD8, and tumor cells were characterized by high FSC and SSC, and negative expression of the human leukocyte marker CD 45.
34A, 34B, and 34C provide a statistical comparison of the results shown in FIG. 33B by the scheme shown in FIG. 33A. Animals treated with CM-24 showed larger tumors and no evidence of tumor-associated T cells. On the other hand, an increased amount of infiltrating T cells and a reduction in tumor cells were observed in mice treated with CP08H 03/vk 8S29A (fig. 33B). As shown, the evaluation of tumor cell proliferation showed that CP08H 03/Vkappa 8S29A inhibited tumor proliferation, but CM-24 did not inhibit tumor proliferationCloning (FIG. 33C). In addition, splenic CD4 was observed in CP08H 03/Vkappa 8S29A treated mice+Increased proliferation of T cells, whereas splenic CD4 was observed in CM-24 treated mice+Proliferation of T cells was reduced (fig. 33D).
Example 11: CEACAM1 antibody blocked the interaction between CEACAM1 and HopQ.
HopQ is expressed on the surface of H.pylori, a bacterium that specifically colonizes the human gastric epithelium and is a major causative agent in the development of ulcers and gastric cancer. The HopQ-CEACAM1 interaction has been shown to promote gastric colonization and Hp-induced pathology, for example, by enabling bacterial virulence factors to translocate into host cells and enhance the release of pro-inflammatory mediators.
Published crystal structure data (PDB ID 6AW2, 6GBH, 6GBG, see Bonsor, D, et al EMBO J.2018Jul 2; 37(13)) and Moonens K, et al EMBO J.2018Jul 2; 37(13)) indicate that the GFCC loop of CEACAM1 is involved in binding to HopQ, and that CEACAM1 residues F29, Y34, N42, Q89, and N97 undergo various hydrogen bonding and hydrophobic interactions with HopQ residues (see FIG. 35A). Modeling based on CECAM1: HopQ eutectic and CEACAM1: CP08H 03/V.kappa.8S 29A Fab eutectic showed that CEACAM1 antibody CP08H 03/V.kappa.8S 29A covered the CEACAM1 binding site of HopQ (see FIG. 35B) and was therefore able to disrupt CEACAM1: HopQ interaction.
Example 12: CEACAM1 antibodies promote long-term survival
The ability of CEACAM1 antibody CP08H 03/vk 8S29A to promote long-term survival of tumor-bearing mammals was studied using a mouse melanoma model.
Will 106MALME-3M (human melanoma) cells and 5x 106Personal PBMCs (from HLA-a2 matched donors) were injected subcutaneously into NSG mice. On day 10, tumors reached 2-2.5mm3And mice were randomly grouped (n-4/group). anti-CEACAM 1 antibody CP08H03/V κ 8S29A or control human IgG4 antibody were administered intraperitoneally on days 10, 13, 17, 20 and 24, respectively. Survival was monitored for 104 days, at which time surviving animals exhibiting intense clinical activity were sacrificed (arrows).
As shown in figure 36, treatment with anti-CEACAM 1 antibody significantly improved survival of tumor-bearing mice. Furthermore, at necropsy, the antibody-treated animals exhibited localized tumors with no visible metastasis, consistent with disease control. This data demonstrates that the anti-CEACAM 1 antibodies and fragments thereof disclosed herein are useful for treating cancer and increasing survival.
Example 13: CP08H 03/Vkappa 8S29A increases immune responses in tumor cells of naive patients or patients with secondary resistance to immunotherapy
The ability of CEACAM1 antibody CP08H 03/vk 8S29A to increase immune responses in tumors from naive melanoma patients or melanoma patients exhibiting secondary resistance to immunotherapy was examined using isolated tumor specimens.
In one example, an isolated tumor sample from a patient with secondary resistance was destroyed by mechanical dissociation and the dissociated cells were treated with CP08H 03/vk 8S29A or hIgG4 control antibody (2 μ g/ml) in the presence of 2 μ g/ml anti-CD 3 and 40 units/ml recombinant IL-2 in culture medium for 4 days. Cells were then examined by mass cytometry using the following antibodies to detect CD8 using standard techniques+Various intracellular factors in T cells associated with the immune response to tumors: IFN gamma (clone B27; 168Er), IL-17A (clone N49-653; 164Dy), IL-17F (clone SHLR 17; 166 Er); granzyme B (clone GB 11; 171 Yb); perforin (clone B-D48; 175 Lu); MIP1 β (clone D21-1351; 150 Nd); TNF α (clone Mab 11; 152Sm), CD3 (clone UCHT 1; 170 Er); CD8 (clone RPA 78; 146 Nd); an intercalator (103 Rh).
As shown in FIGS. 37A and 37B, treatment with the CP08H 03/Vkappa 8S29A antibody resulted in a significant induction of CD8 compared to the control human IgG4 antibody+Intracellular indicative factors in T cells. These results directly indicate that the CP08H03/V kappa 8S29A antibody is in CD8+The production of various factors is induced in T cells, which are potentially associated with a productive anti-tumor immune response.
In another example, tumor samples associated with two melanoma patients who had not previously received treatment (subject 189) or had secondary resistance to immunotherapy (subject 185) were destroyed by mechanical dissociation (Miltenyi). Mix 8x10 5The dissociated tumor cells per ml were placed in culture dishes. Will be freshly separatedTumor-dissociated cells were exposed only to 2 μ g/ml CP08H 03/Vkappa 8S29A or human IgG4 isotype control antibody. After 96 hours, the supernatant was removed and ELISA assays were performed in triplicate to detect the presence of interferon-gamma.
As shown in fig. 38A and 38B, treatment with the CP08H 03/vk 8S29A antibody induced a significant level of cytokine interferon- γ secretion into the supernatant of tumor dissociated cells isolated from patients with secondary resistance to immunotherapy treatment (fig. 38A, subject 185) or patients not treated with immunotherapy (fig. 38B, subject 189), relative to that observed with the control human IgG4 antibody.
Taken together, these data indicate that the anti-CEACAM 1 antibodies and fragments thereof disclosed herein can be used to treat both naive cancer patients and those patients with secondary resistance to immunotherapy.
Sequence overview
While the above written description of the invention enables one of ordinary skill in the art to make and use what is presently considered to be the best mode thereof, those of ordinary skill in the art will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples herein.
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