anti-TREM 2 antibodies and related methods
阅读说明：本技术 抗trem2抗体和相关方法 (anti-TREM 2 antibodies and related methods ) 是由 M·斯特雷利 V·斯里拉姆 A·帕尔 L·G·普雷斯塔 于 2018-12-11 设计创作，主要内容包括：本文提供了抗TREM2抗体以及制备和使用抗TREM2抗体的相关方法。还提供了用于使免疫应答增强和/或用于治疗个体的免疫相关疾患例如癌症,包括使用抗TREM2抗体或其抗原结合片段杀灭非刺激性骨髓细胞,使非刺激性骨髓细胞丧失能力或消减的方法和组合物。(Provided herein are anti-TREM 2 antibodies and related methods of making and using anti-TREM 2 antibodies. Also provided are methods and compositions for enhancing an immune response and/or for treating an immune related disorder, such as cancer, in an individual, comprising killing, incapacitating, or depleting non-stimulatory myeloid cells using an anti-TREM 2 antibody or antigen binding fragment thereof.)
1. An isolated humanized antibody that binds human TREM2(SEQ ID NO:15) and competes with 37017 antibody (SEQ ID NOS: 31 and 32) for binding to mouse TREM2(SEQ ID NO: 17).
2. The isolated humanized antibody of claim 1, wherein the antibody comprises:
a. CDR-H1 comprising the sequence set forth in SEQ ID NO. 9,
b. CDR-H2 comprising the sequence set forth in SEQ ID NO. 10,
c. CDR-H3 comprising the sequence set forth in SEQ ID NO. 11,
d. CDR-L1 comprising the sequence set forth in SEQ ID NO. 12,
e. CDR-L2 comprising the sequence set forth in SEQ ID NO 13, and
f. CDR-L3 comprising the sequence set forth in SEQ ID NO. 14.
3. The isolated antibody of claim 2, wherein the antibody is an algal-free glycosylated antibody and comprises a VH sequence shown as SEQ ID NO: 1; a VL sequence shown as SEQ ID NO 2; and an active human IgG1 Fc region.
4. The isolated antibody of claim 2, wherein the antibody comprises an A to T substitution at position 97 of the sequence shown as SEQ ID NO 7; and a VH sequence with K to R substitutions at position 98 of the sequence shown as SEQ ID NO 7.
5. The isolated antibody of claim 3, wherein the antibody comprises a VH sequence shown as SEQ ID NO 1,3, or 5.
6. The isolated antibody of claim 5, wherein the antibody comprises a VH sequence shown as SEQ ID NO 1,3, or 5 and a VL sequence shown as SEQ ID NO 2,4, or 6.
7. The isolated antibody of claim 3, wherein the antibody comprises the VH sequence shown as SEQ ID NO 1.
8. The isolated antibody of claim 7, wherein the antibody comprises a VH sequence shown as SEQ ID NO 1 and a VL sequence shown as SEQ ID NO 2.
9. The isolated antibody of claim 8, wherein the antibody is an 37012 antibody.
10. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain sequence shown as SEQ ID NO 25 and a light chain sequence shown as SEQ ID NO 26.
11. An isolated antibody that binds human TREM2(SEQ ID NO:15), wherein the antibody
i) Competes with 37017 antibody (SEQ ID NOS: 31 and 32) for binding to mouse TREM2(SEQ ID NO: 17); and is
ii) comprises an active human Fc region.
12. An isolated humanized antibody, wherein the antibody comprises:
a. CDR-H1 comprising the sequence set forth in SEQ ID NO. 9,
b. CDR-H2 comprising the sequence set forth in SEQ ID NO. 10,
c. CDR-H3 comprising the sequence set forth in SEQ ID NO. 11,
d. CDR-L1 comprising the sequence set forth in SEQ ID NO. 12,
e. CDR-L2 comprising the sequence set forth in SEQ ID NO 13, and
f. CDR-L3 comprising the sequence set forth in SEQ ID NO. 14.
13. The isolated antibody of claim 12, wherein the antibody comprises an a to T substitution at position 97 of the sequence shown as SEQ ID No. 7; and a VH sequence with K to R substitutions at position 98 of the sequence shown as SEQ ID NO 7.
14. The isolated antibody of claim 13, wherein the antibody comprises a VH sequence shown as SEQ ID NO 1,3, or 5.
15. The isolated antibody of claim 14, wherein the antibody comprises a VH sequence shown as SEQ ID No. 1,3, or 5 and a VL sequence shown as SEQ ID No. 2,4, or 6.
16. The isolated antibody of claim 15, wherein the antibody comprises the VH sequence shown as SEQ ID No. 1.
17. The isolated antibody of claim 16, wherein the antibody comprises a VH sequence shown as SEQ ID No. 1 and a VL sequence shown as SEQ ID No. 2.
18. The isolated antibody of claim 17, wherein the antibody is an 37012 antibody.
19. The isolated antibody of claim 12, wherein the antibody comprises a heavy chain sequence shown as SEQ ID NO 25 and a light chain sequence shown as SEQ ID NO 26.
20. The isolated antibody of any one of the above claims, wherein the antibody is administered at less than or equal to about 1,2, 3, 4, or 5x10-9K of MDBinding to human TREM2, as measured by Surface Plasmon Resonance (SPR) assay.
21. The isolated antibody of any one of the above claims, wherein the antibody is capable of binding to TREM2+ bone marrow cells; optionally on non-stimulatory myeloid cells; optionally specific killing, depletion of intratumoral bone marrow cells, or TREM2+ bone marrow cells; optionally non-stimulatory myeloid cells; optionally disabling bone marrow cells within the tumor.
22. The isolated antibody of any of the above claims, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
23. The isolated antibody of any one of the above claims, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity.
24. The isolated antibody of any one of the above claims, wherein the antibody has Complement Dependent Cytotoxicity (CDC) activity.
25. The isolated antibody of any one of the above claims, wherein the antibody is at least one of: monoclonal antibodies, neutralizing antibodies, antagonistic antibodies, agonistic antibodies, polyclonal antibodies, IgG1 antibodies, IgG3 antibodies, non-algal glycosylated antibodies, bispecific antibodies, human antibodies, chimeric antibodies, full length antibodies, and antigen binding fragments thereof.
26. The isolated antibody of any one of the above claims, wherein the antibody is a monoclonal antibody.
27. The isolated antibody of any one of the above claims, wherein the antibody is a multispecific antibody.
28. The isolated antibody of any one of the above claims, wherein the antibody is an algal-free glycosylated antibody.
29. The isolated antibody of any one of the above claims, wherein the antibody is an antigen binding fragment thereof, Fab ', F (ab') 2, Fv, scFv, (scFv)2, a single chain antibody molecule, a double variable domain antibody, a single variable domain antibody, a linear antibody, or a V domain antibody.
30. The isolated antibody of any one of the above claims, wherein the antibody comprises a scaffold, optionally wherein the scaffold is an Fc, optionally a human Fc.
31. The isolated antibody of any one of the above claims, wherein the antibody comprises a heavy chain constant region of a class selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.
32. The isolated antibody of any one of the above claims, wherein the antibody comprises a heavy chain constant region of the IgG class and a subclass selected from IgG1, IgG2, IgG3, and IgG 4.
33. The isolated antibody of any one of the above claims, wherein the antibody comprises a heavy chain constant region of IgG 1.
34. The isolated antibody of any one of the above claims, wherein Fc comprises one or more modifications, wherein the one or more modifications result in increased half-life, increased ADCC activity, increased ADCP activity, or increased CDC activity compared to Fc without the one or more modifications.
35. The isolated antibody of any one of the above claims, wherein the Fc binds an fey receptor selected from the group consisting of: fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIc, Fc γ RIIIa, and Fc γ RIIIb.
36. The isolated antibody of any of the above claims for use in the treatment of cancer, wherein the cancer is selected from the group consisting of a solid tumor and a hematological tumor.
37. An isolated antibody that competes for binding to human TREM2 with the antibody of any one of the above claims.
38. An isolated antibody that binds the human TREM2 epitope bound by the antibody of any one of the above claims.
39. An isolated polynucleotide or set of isolated polynucleotides encoding the antibody, VH thereof, VL thereof, light chain thereof, heavy chain thereof, or antigen binding portion thereof of any one of the above claims; optionally, wherein the polynucleotide or set of polynucleotides is cDNA.
40. A vector or a set of vectors comprising a polynucleotide or a set of polynucleotides according to claim 39.
41. A host cell comprising a polynucleotide or a set of polynucleotides according to claim 39 or a vector or a set of vectors according to claim 40.
42. A method of producing an antibody, comprising expressing the antibody with the host cell of claim 41, and isolating the expressed antibody.
43. A pharmaceutical composition comprising the antibody of any one of claims 1-38 and a pharmaceutically acceptable excipient.
44. A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody of any one of claims 1 to 38 or the pharmaceutical composition of claim 43.
45. The method of claim 44, wherein the disease or disorder is cancer.
46. The method of claim 44, wherein the antibody binds to the extracellular domain of TREM2 on TREM2+ bone marrow cells, optionally wherein the bone marrow cells are intratumoral.
47. The method of claim 44, wherein the antibody binds to an extracellular domain of TREM2 on a bone marrow cell, wherein the bone marrow cells are CD45+, HLA-DR +, CD11c +, CD14+ and BDCA 3-non-stimulatory bone marrow cells, wherein the antibody kills the non-stimulatory myeloid cells by ADCC, CDC and/or ADCP, incapacitates or depletes the non-stimulatory myeloid cells, to achieve a level less than the level of non-stimulatory myeloid cells present in the cancer prior to contacting said non-stimulatory myeloid cells with said antibody, wherein the non-stimulatory myeloid cells are present in an immune cell population comprising CD45+, HLA-DR +, CD14-, CD11c +, BDCA 1-and BDCA3+ stimulatory myeloid cells and the non-stimulatory myeloid cells, and wherein killing or depleting the non-stimulatory myeloid cells treats the cancer.
48. The method of claim 44, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
49. The method of claim 44, wherein the antibody has Complement Dependent Cytotoxicity (CDC) activity.
50. The method of claim 44, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity.
51. The method of claim 44, wherein the antibody has receptor-ligand blocking activity, agonistic activity, or antagonistic activity.
52. The method of any one of claims 44-51, wherein the subject is a human.
53. The method of any one of claims 44-52, wherein the cancer is a solid cancer.
54. The method of any one of claims 44-52, wherein the cancer is a liquid cancer.
55. The method of any one of claims 44-52, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, liver and gall bladder cancer, Head and Neck Squamous Carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer, kidney cancer, bladder cancer, esophageal cancer, kidney cancer, melanoma, and mesothelioma.
56. The method of claim 55, wherein the cancer is colon cancer or breast cancer.
57. The method of any one of claims 44-56, wherein said contacting enhances an immune response in said subject.
58. The method of claim 57, wherein the enhanced immune response is an adaptive immune response.
59. The method of claim 57, wherein the enhanced immune response is an innate immune response.
60. The method of any one of claims 44-59, wherein the subject has previously received, is receiving concurrently, or will subsequently receive immunotherapy.
61. The method of claim 60, wherein the immunotherapy is at least one of: (ii) a checkpoint inhibitor; checkpoint inhibitors of T cells; an anti-PD 1 antibody; anti-PDL 1 antibody; anti-CTLA 4 antibodies; adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds both T cells and antigen presenting cells; a BiTE double antigen binding protein; a toll-like receptor ligand; a cytokine; (ii) cytotoxic therapy; chemotherapy; radiotherapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator.
62. The method of claim 61, wherein the immunotherapy is an anti-PD 1 antibody.
63. A method of killing TREM2+ bone marrow cells, incapacitating or depleting TREM2+ bone marrow cells in a subject having cancer, the method comprising contacting the bone marrow cells with the antibody of any one of claims 1 to 38 or the pharmaceutical composition of claim 43, optionally wherein the bone marrow cells are intratumoral.
64. The method of claim 63, wherein the antibody binds to the extracellular domain of TREM2, wherein the bone marrow cell is a CD45+, HLA-DR +, CD11c +, CD14+, and BDCA 3-non-stimulatory bone marrow cell, wherein the antibody kills the non-stimulatory bone marrow cell by ADCC, CDC, and/or ADCP, incapacitating or depleting the non-stimulatory bone marrow cell to a level less than the level of non-stimulatory bone marrow cells present in the cancer prior to contacting the non-stimulatory bone marrow cell with the antibody, wherein the non-stimulatory bone marrow cell is present in a population of immune cells comprising CD45+, HLA-DR +, CD14-, CD11c +, BDCA1-, and BDCA3+ stimulatory bone marrow cells and the non-stimulatory bone marrow cell, wherein the contacting does not substantially kill bone marrow cells present outside of the cancer and/or stimulatory bone marrow cells present in the cancer, (ii) does not substantially incapacitate or deplete bone marrow cells present outside of the cancer and/or stimulatory bone marrow cells present in the cancer, and wherein killing the non-stimulatory bone marrow cells, incapacitating or depleting the non-stimulatory bone marrow cells treats the cancer by enhancing the immune response to the cancer.
65. The method of claim 63, wherein the antibody kills the bone marrow cells by at least one of ADCC, CDC, and ADCP.
66. The method of claim 63, wherein the antibody incapacitates the bone marrow cells by at least one of ADCC, CDC and ADCP.
67. The method of claim 63, wherein the antibody depletes the bone marrow cells by at least one of ADCC, CDC and ADCP.
68. The method of claim 63, wherein the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
69. The method of claim 63, wherein the antibody has Complement Dependent Cytotoxicity (CDC) activity.
70. The method of claim 63, wherein the antibody has antibody-mediated phagocytosis (ADCP) activity.
71. The method of claim 63, wherein the antibody has receptor-ligand blocking activity, agonistic activity, or antagonistic activity.
72. The method of any one of claims 63-71, wherein the bone marrow cells are stimulatory bone marrow cells.
73. The method of any one of claims 63-71, wherein the bone marrow cells are non-stimulatory bone marrow cells.
74. The method of one of claims 63-73, wherein the bone marrow cells comprise at least one of dendritic cells, Tumor Associated Macrophages (TAMs), neutrophils, or monocytes.
75. The method of claim 74, wherein the bone marrow cells are neutrophils.
76. The method of claim 74, wherein the bone marrow cells are tumor associated macrophages.
77. The method of any one of claims 63-76, wherein the bone marrow cells are within a tumor.
78. The method of any one of claims 63-77, wherein the bone marrow cells are in an immune cell population comprising stimulatory bone marrow cells and non-stimulatory bone marrow cells.
79. The method of any one of claims 63-78, wherein the contacting is performed in vitro or in vivo.
80. The method of any one of claims 63-79, wherein the contacting occurs in vivo in a subject in need thereof, optionally wherein the subject has cancer.
81. The method of any one of claims 31-80, wherein the subject is a human.
82. The method of any one of claims 63-81, wherein the cancer is a solid cancer.
83. The method of any one of claims 63-81, wherein the cancer is a liquid cancer.
84. The method of claim 80, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, liver and gall bladder cancer, Head and Neck Squamous Carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer, kidney cancer, bladder cancer, esophageal cancer, kidney cancer, melanoma, and mesothelioma.
85. The method of claim 84, wherein the cancer is colon cancer or breast cancer.
86. The method of claim 80, wherein said contacting enhances an immune response in said subject.
87. The method of claim 80, wherein the enhanced immune response is an adaptive immune response.
88. The method of claim 80, wherein the enhanced immune response is an innate immune response.
89. The method of any one of claims 80-88, wherein the subject has previously received, is receiving concurrently, or will subsequently receive immunotherapy.
90. The method of claim 89, wherein the immunotherapy is at least one of: (ii) a checkpoint inhibitor; checkpoint inhibitors of T cells; an anti-PD 1 antibody; anti-PDL 1 antibody; anti-CTLA 4 antibodies; adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds both T cells and antigen presenting cells; a BiTE double antigen binding protein; a toll-like receptor ligand; a cytokine; (ii) cytotoxic therapy; chemotherapy; radiotherapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator.
91. The method of claim 90, wherein the immunotherapy is an anti-PD 1 antibody.
92. A method of detecting TREM2 in a subject having or suspected of having a disease or condition, the method comprising: (a) receiving a sample from the subject; and (b) detecting the presence or level of TREM2 in the sample by contacting the sample with the antibody of any one of claims 1 to 38.
93. The method of claim 92, wherein the disease or disorder is cancer.
94. The method of claim 92 or 93, further comprising administering a checkpoint inhibitor, optionally wherein the checkpoint inhibitor is an inhibitor of the PD1: PDL1 axis, optionally wherein the inhibitor is an antibody, and optionally wherein the antibody is an anti-PD 1 antibody or an anti-PDL 1 antibody.
95. A kit comprising the antibody of any one of claims 1 to 38 or the pharmaceutical composition of claim 43 and instructions for use.
Background
Immunity plays a role in preventing tumor outgrowth. Complex microenvironments can arise within the lesion, and despite the recruitment of T cells, there is often no effective control of the developing mass. Understanding the balance between tumor elimination and tumor escape may depend on an understanding of the differential role bone marrow cells play in the tumor microenvironment.
The myeloid population of the tumor microenvironment is comprised primarily of monocytes and neutrophils (sometimes loosely grouped as myeloid-derived suppressor cells), macrophages and dendritic cells. While intratumoral myeloid populations as a whole have long been considered non-stimulatory or inhibitory, more recently, it has been understood that not all tumor-infiltrating myeloid cells are equivalent.
In normal tissues, many of these bone marrow cells function properly for both innate and adaptive immunity, and are particularly essential for wound repair. However, in the context of cancer, macrophages are often described in significant excess and populations of these and other cell types are in a dysfunctional or migratory state. When considered as a collective population defined by a single marker such as CD68 or CD163, "macrophage" infiltration was associated with obtaining worse results in subjects across multiple tumor types ((de Visser, Cancer Immunol Immunother, 2008; 57: 1531-9); (Hanada et al, Int J Urol 2000; 7: 263-9); (Yao et al, Clin Cancer Res,520,2001; 7: 4021-6); (Ruffell et al, PNAS, 5232012; 109: 2796-. But the phenotypic and functional sub-environments of macrophages from the tumor microenvironment are complicated by the similarity of macrophages and dendritic cells and are problematic in tumor biology. Morphological criteria have often been applied to the problem; one approach to attempt to distinguish dendritic cells from macrophages is based on the fact that dendritic cells have a more spiky or dendritic morphology, while macrophages have a more cryptic or globular morphology (Bell et al, JExp Med 555,1999; 190: 1417-26). Other groups are attempting to differentiate based on genetic and cell surface markers.
There is diversity within the tumor in the antigen presenting compartment, and T cells can distinguish the characteristics of Antigen Presenting Cells (APCs). Since T cells are the primary driver of tumor immunity, it would be important to know the exact characteristics of their cognate APC. Among the cells capable of presenting tumor-derived antigens to T cells, and thereby maintaining T cells in an activated state, bone marrow cells are prominent. Antigen presentation occurs within the tumor itself and may affect the function of tumor Cytotoxic T Lymphocytes (CTLs). T cell activation by Antigen Presenting Cells (APCs) is an important component in antigen-specific immune responses and tumor cell killing. Since these myeloid populations represent the major T cell interaction partners and antigen presenting cells to obtain the next tumor-reactive cytotoxic T lymphocytes, understanding their differences can guide the therapeutic pathway.
Related patent applications include: PCT/US2015/052682 filed on 9, 28, 2015; and PCT/US2016/054104 filed on 28/9/2016; each of these patent applications is incorporated by reference herein in its entirety for all purposes.
All patents, patent applications, publications, documents and articles cited herein are hereby incorporated by reference in their entirety.
Disclosure of Invention
Described herein is an isolated antibody that binds human TREM2(SEQ ID NO:15) and competes with the 37017 antibody (SEQ ID NO:31 and 32) for binding to mouse TREM2(SEQ ID NO: 17).
In some embodiments, the antibody contains CDR-H1 comprising the sequence set forth in SEQ ID No. 9, CDR-H2 comprising the sequence set forth in SEQ ID No. 10, CDR-H3 comprising the sequence set forth in SEQ ID No. 11, CDR-L1 comprising the sequence set forth in SEQ ID No. 12, CDR-L2 comprising the sequence set forth in SEQ ID No. 13, and CDR-L3 comprising the sequence set forth in SEQ ID No. 14.
In some embodiments, the antibody is an algal-free glycosylated antibody and comprises a VH sequence shown as SEQ ID No. 1; a VL sequence shown as SEQ ID NO 2; and an active human IgG1 Fc region.
In some embodiments, the antibody comprises all 3 heavy chain CDRs of the sequence shown as SEQ ID No. 7 and all 3 light chain CDRs of the sequence shown as SEQ ID No. 8.
In some embodiments, the antibody comprises a substitution A to T at position 97 of the sequence shown as SEQ ID NO. 7; and K to R substitutions at position 98 of the sequence shown as SEQ ID NO. 7.
In some embodiments, the antibody comprises a VH sequence shown as SEQ ID NO 1,3 or 5.
In some embodiments, the antibody comprises a VH sequence shown as SEQ ID No. 1,3 or 5 and a VL sequence shown as SEQ ID No. 2,4 or 6.
In some embodiments, the antibody comprises the VH sequence shown as SEQ ID NO 1.
In some embodiments, the antibody comprises a VH sequence shown as SEQ ID NO. 1 and a VL sequence shown as SEQ ID NO. 2.
In some embodiments, the antibody is an 37012 antibody.
In some embodiments, the antibody comprises a heavy chain sequence shown as SEQ ID NO. 25 and a light chain sequence shown as SEQ ID NO. 26.
In another aspect, described herein is an isolated antibody that binds human TREM2(SEQ ID NO:15), wherein the antibody competes with the 37017 antibody (SEQ ID NOS: 31 and 32) for binding to mouse TREM2(SEQ ID NO: 17); and comprises an active human Fc region.
In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
In some embodiments, the antibody is a humanized antibody.
In some embodiments, the antibody is administered at less than or equal to about 1,2, 3, 4, or 5x10-9K ofDBinding to human TREM2, as measured by Surface Plasmon Resonance (SPR) assay.
In some embodiments, the antibody is capable of binding to TREM2+ bone marrow cells; optionally specifically killing, depleting, or causing TREM2+ bone marrow cells of non-stimulatory bone marrow cells; optionally incapacitating non-stimulatory myeloid cells.
In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody has antibody-mediated phagocytosis (ADCP) activity. In some embodiments, the antibody has Complement Dependent Cytotoxicity (CDC) activity.
In some embodiments, the antibody kills bone marrow cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated phagocytosis (ADCP) activity, or complement-dependent cytotoxicity (CDC), disabling or depleting the bone marrow cells.
In some embodiments, the antibody is at least one of: monoclonal antibodies, neutralizing antibodies, antagonistic antibodies, agonistic antibodies, polyclonal antibodies, IgG1 antibodies, IgG3 antibodies, non-algal glycosylated antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, full length antibodies, and antigen binding fragments thereof.
In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is a multispecific antibody.
In some embodiments, the antibody is an algal glycosylation free antibody.
In some embodiments, the antibody is an antigen binding fragment thereof, Fab ', F (ab')2、Fv、scFv、(scFv)2A single chain antibody molecule, a double variable domain antibody, a single variable domain antibody, a linear antibody or a V domain antibody.
In some embodiments, the antibody comprises a scaffold, optionally wherein the scaffold is an Fc, optionally a human Fc.
In some embodiments, the antibody comprises a heavy chain constant region of a class selected from IgG, IgA, IgD, IgE, and IgM.
In some embodiments, the antibody comprises a heavy chain constant region of the IgG class and a subclass selected from IgG1, IgG2, IgG3, and IgG 4.
In some embodiments, the antibody comprises a heavy chain constant region of IgG 1.
In some embodiments, the Fc comprises one or more modifications, wherein the one or more modifications result in an increase in half-life, an increase in ADCC activity, an increase in ADCP activity, or an increase in CDC activity, as compared to the Fc without the one or more modifications.
In some embodiments, the Fc binds to an fey receptor selected from the group consisting of: fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIc, Fc γ RIIIa, and Fc γ RIIIb.
In another aspect, described herein is an isolated antibody for use in treating cancer, wherein the cancer is selected from the group consisting of a solid tumor and a hematologic tumor.
In another aspect, described herein is an isolated antibody that competes with an antibody described herein for binding to human TREM 2.
In another aspect, described herein is an isolated antibody that binds to an epitope of human TREM2 bound by an antibody described herein.
In another aspect, described herein are antibodies, V, encoding the antibodies described hereinHV thereofLAn isolated polynucleotide or a set of polynucleotides, light chain thereof, heavy chain thereof, or antigen binding portion thereofAn isolated polynucleotide; optionally a cDNA.
In another aspect, described herein is a vector or set of vectors comprising a polynucleotide or set of polynucleotides as described herein.
In another aspect, described herein is a host cell comprising a polynucleotide or a set of polynucleotides as described herein or a vector or a set of vectors as described herein.
In another aspect, described herein is a method of producing an antibody, comprising expressing the antibody with a host cell described herein, and isolating the expressed antibody.
In another aspect, described herein is a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable excipient.
In another aspect, described herein is a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition described herein.
In some embodiments, the disease or disorder is cancer.
In some embodiments, the antibody binds to the extracellular domain of TREM2 on TREM2+ bone marrow cells, optionally wherein the bone marrow cells are intratumoral. In one embodiment, the antibody binds to the extracellular domain of TREM2 on a bone marrow cell, wherein the bone marrow cell is CD45+、HLA-DR+、CD11c+、CD14+And BDCA3-Non-stimulatory myeloid cells, wherein said antibody kills said non-stimulatory myeloid cells by ADCC, CDC and/or ADCP, incapacitates or depletes said non-stimulatory myeloid cells to a level less than that of non-stimulatory myeloid cells present in the cancer prior to contact of said non-stimulatory myeloid cells with said antibody, wherein said non-stimulatory myeloid cells are present comprising CD45+、HLA-DR+、CD14-、CD11c+、BDCA1-And BDCA3+A population of immune cells of stimulatory myeloid cells and said non-stimulatory myeloid cells, and wherein said non-stimulatory myeloid cells are killedBone marrow cells, incapacitation or depletion of said non-stimulatory bone marrow cells may treat said cancer.
In some embodiments, the antibody kills bone marrow cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated phagocytosis (ADCP) activity, or complement-dependent cytotoxicity (CDC), disabling or depleting the bone marrow cells. In some embodiments, the antibody has receptor-ligand blocking activity, agonistic activity, or antagonistic activity.
In some embodiments, the subject is a human. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, renal cancer, liver and gall bladder cancer, Head and Neck Squamous Carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer, kidney cancer, bladder cancer, esophageal cancer, kidney cancer, melanoma, and mesothelioma. In some embodiments, the cancer is colon cancer or breast cancer.
In some embodiments, the contacting enhances an immune response in the subject. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response.
In some embodiments, the subject has previously received, is receiving concurrently, or will subsequently receive immunotherapy. In some embodiments, the immunotherapy is at least one of the following: (ii) a checkpoint inhibitor; checkpoint inhibitors of T cells; an anti-PD 1 antibody; anti-PDL 1 antibody; anti-CTLA 4 antibodies; adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds both T cells and antigen presenting cells; a BiTE double antigen binding protein; a toll-like receptor ligand; a cytokine; (ii) cytotoxic therapy; chemotherapy; radiotherapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator. In some embodiments, the immunotherapy is an anti-PD 1 antibody.
In another aspect, described herein are methods of killing TREM2+ bone marrow cells, incapacitating or depleting TREM2+ bone marrow cells, of a subject having cancer, the method comprising contacting the bone marrow cells with an anti-TREM 2 antibody described herein or a pharmaceutical composition described herein, optionally wherein the bone marrow cells are intratumoral.
In some embodiments, the antibody binds to the extracellular domain of TREM2, wherein TREM2+ bone marrow cell is CD45+、HLA-DR+、CD11c+、CD14+And BDCA3-Non-stimulatory myeloid cells, wherein said antibody kills said non-stimulatory myeloid cells by ADCC, CDC and/or ADCP, incapacitates or depletes said non-stimulatory myeloid cells to a level less than that of non-stimulatory myeloid cells present in the cancer prior to contact of said non-stimulatory myeloid cells with said antibody, wherein said non-stimulatory myeloid cells are present comprising CD45+、HLA-DR+、CD14-、CD11c+、BDCA1-And BDCA3+A population of immune cells of stimulatory myeloid cells and the non-stimulatory myeloid cells, wherein the contacting does not substantially kill, does not substantially incapacitate or deplete, or does deplete bone marrow cells present outside the cancer and/or stimulatory myeloid cells present in the cancer, and wherein killing, incapacitating or depleting the non-stimulatory myeloid cells treats the cancer by enhancing an immune response to the cancer.
In some embodiments, the antibody kills bone marrow cells by at least one of ADCC, CDC, and ADCP. In some embodiments, the antibody disables bone marrow cells by at least one of ADCC, CDC, and ADCP. In some embodiments, the antibody depletes bone marrow cells via at least one of ADCC, CDC and ADCP. In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the antibody has Complement Dependent Cytotoxicity (CDC) activity. In some embodiments, the antibody has antibody-mediated phagocytosis (ADCP) activity. In some embodiments, the antibody has receptor-ligand blocking activity, agonistic activity, or antagonistic activity.
In some embodiments, the bone marrow cells are stimulatory bone marrow cells. In some embodiments, the bone marrow cells are non-stimulatory bone marrow cells. In some embodiments, the bone marrow cells comprise at least one of dendritic cells, tumor-associated macrophages (TAMs), neutrophils, or monocytes. In some embodiments, the bone marrow cells are neutrophils. In some embodiments, the bone marrow cells are tumor-associated macrophages. In some embodiments, the bone marrow cells are within a tumor. In some embodiments, the bone marrow cells are in an immune cell population comprising stimulatory bone marrow cells and non-stimulatory bone marrow cells.
In some embodiments, the subject is a human. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, renal cancer, liver and gall bladder cancer, Head and Neck Squamous Carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast cancer, ovarian cancer, stomach cancer, kidney cancer, bladder cancer, esophageal cancer, kidney cancer, melanoma, and mesothelioma. In some embodiments, the cancer is colon cancer or breast cancer.
In some embodiments, the contacting enhances an immune response in the subject. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response.
In some embodiments, the subject has previously received, is receiving concurrently, or will subsequently receive immunotherapy. In some embodiments, the immunotherapy is at least one of the following: (ii) a checkpoint inhibitor; checkpoint inhibitors of T cells; an anti-PD 1 antibody; anti-PDL 1 antibody; anti-CTLA 4 antibodies; adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds both T cells and antigen presenting cells; a BiTE double antigen binding protein; a toll-like receptor ligand; a cytokine; (ii) cytotoxic therapy; chemotherapy; radiotherapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and an epigenetic modulator. In some embodiments, the immunotherapy is an anti-PD 1 antibody.
In another aspect, described herein is a method of detecting TREM2 in a subject having or suspected of having a disease or condition, the method comprising: (a) receiving a sample from the subject; and (b) detecting the presence or level of TF in the sample by contacting the sample with an antibody described herein.
In some embodiments, the disease or disorder is cancer.
In some embodiments, the methods described herein comprise administering a checkpoint inhibitor, optionally wherein the checkpoint inhibitor is an inhibitor of the PD1: PDL1 axis, optionally wherein the inhibitor is an antibody, and optionally wherein the antibody is an anti-PD 1 antibody or an anti-PDL 1 antibody.
In another aspect, described herein is a kit comprising an antibody or pharmaceutical composition disclosed herein and instructions for use.
Drawings
1A-1C anti-TREM 2 PI-7012 mediated anti-tumor activity in a CT-26 syngeneic mouse tumor model in combination with an anti-PD-1 antibody. (A) The non-algal glycosylation of PI-7012 in combination with anti-PD-1 antibodies resulted in improved anti-tumor activity. Mean tumor volumes (10 mice/group) are shown. Shown are (B) the individual tumor volumes for PI-7012 and (C) the individual tumor volumes for non-fucosylated PI-7012 (afuc-PI-7012).
Figure 2 no significant weight loss in the case of combination treatment. Ten mice in each group were treated with the indicated antibodies and body weights were recorded at frequent intervals. The average body weight of each group was plotted against the study days.
FIG. 3 tissue macrophages were stained with anti-CD 68 antibody in addition to H & E staining. The intracellular marker CD68 has been used in the literature as a reliable cytochemical marker for immunostaining monocytes/macrophages in inflamed tissues and tumors. In the lung (panel E), as well as in other tissues analyzed, no discernible change in CD68+ macrophage numbers was observed in any of the treatment groups compared to controls, indicating that anti-TREM 2-mediated depletion occurred specifically in TME.
Fig. 4A-fig. 4B. (a) anti-CD 68 staining of FFPE lung tissue from indicated treatment groups. (B) Eight to nine fields per section were used for quantification by light microscopy.
FIG. 5 TREM2 expression is absent or very low on cells in selected tissues. Shaded histograms are from TREM2KO, and open histograms from wild-type mice. The antibody used for anti-TREM 2 staining was clone 237920 from R & D Systems.
Figure 6 cell surface expression of TREM2 (open histogram) was significantly higher on TAMs compared to granulocytic or monocytic MDSCs within both MC38 and CT26 tumors. Lymphocytes do not express TREM 2. Isotype control staining is shown in gray filled histograms.
Figure 7 cell surface expression of TREM2 (open histogram) was significantly higher on CD 14-derived macrophages compared to any PBMC subset. Human PBMC or macrophages were surface stained for TREM2 (open histogram) or isotype control (grey histogram). A pre-validated multi-color FACS suite is used to distinguish PBMC subsets as neutrophils, monocytes or T cells.
Figure 8 cell surface expression of TREM2 (open histogram) was significantly higher on TAMs compared to other infiltrates or non-CD 45 positive cells. Single cell suspensions from human tumor tissue were surface stained for TREM2 (open histogram) or isotype control (grey histogram). The immune and non-immune subsets were distinguished using a pre-validated multi-color FACS set.
Figure 9A-figure 9f anti-TREM 2mAb afuc-PI7012 in combination with anti-PD-1 mAb resulted in significant anti-tumor activity in Panc-02 pancreatic tumor model. (A) Tumor volume was followed over time in female C57BL/6J mice implanted with Panc-02 tumor cells and treated with the indicated mabs. The Y-axis represents the mean +/-standard deviation of the mean tumor volume of 10 mice in each group. (B) Tumor volumes from individual animals treated with isotype control mAb. (C) Tumor volume from a single animal treated with anti-TREM 2mAb afuc-
FIG. 10 tumor-free BALB/c mice were re-challenged with CT26 tumor cells three months later (squares symbols) after treatment with anti-TREM 2mAb plus anti-PD-1 mAb. Age-matched treatment naive mice (circle symbols) received an equivalent number of CT26 cells and tumor growth was followed during the study period. Mice were not provided additional treatment during the study period.
Detailed Description
Definition of
For the purpose of interpreting the specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any of the definitions set forth below conflict with any document incorporated by reference herein, the set definition shall control.
It is to be understood that the aspects and embodiments of the invention described herein include "comprising," "consisting of … …," and "consisting essentially of … …" aspects and embodiments.
For all compositions described herein, and all methods of using the compositions described herein, a composition can comprise, or can "consist essentially of, the listed components or steps. When a composition is described as "consisting essentially of the listed components," the composition contains the listed components, and may contain other components that do not substantially affect the condition being treated, but does not contain any other components other than those explicitly listed that substantially affect the condition being treated; or if the composition does contain additional components other than those listed that substantially affect the condition being treated, the composition does not contain concentrations or amounts of the additional components sufficient to substantially affect the condition being treated. When a method is described as "consisting essentially of the listed steps," the method contains the listed steps and may contain other steps that do not substantially affect the condition being treated, but the method does not contain any other steps that do not substantially affect the condition being treated other than those steps explicitly listed. As a non-limiting specific example, when a composition is described as 'consisting essentially of a certain component', the composition can additionally contain any number of pharmaceutically acceptable carriers, vehicles, or diluents, as well as other such components, which do not materially affect the condition being treated.
The term "optionally" when used in succession is intended to include from one to all of the recited combinations and includes all sub-combinations.
As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic compound, such as an anti-TREM 2 antigen binding agent or an anti-TREM 2 antibody, administered to an individual in a single dose form or as part of a series of doses, alone or in combination with another therapeutic modality, effective to produce or promote the desired therapeutic effect. Examples of desired therapeutic effects are enhancement of the immune response, slowing or delaying tumor development; stabilizing the disease; ameliorating one or more symptoms. The effective amount may be administered in one or more doses.
The term "treating" as used herein refers to delaying or reversing the progression of a condition, such as cancer. The term "treatment" as used herein refers to the act of treating a condition such as cancer.
As used herein, "individual" or "subject" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse.
The term "modulate" refers to decreasing or inhibiting, or alternatively, activating or increasing, the recited variable.
The terms "increase" and "activation" refer to an increase in the recited variable of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more.
The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% reduction in the recited variable to 1/2, 1/3, 1/4, 1/5, 1/10, 1/20, 1/50, 1/100, or more.
The term "agonize" refers to the activation of receptor signaling to induce a biological response associated with the activation of the receptor. An "agonist" is an entity that binds to and agonizes a receptor.
The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with the activation of the receptor. An "antagonist" is an entity that binds to and antagonizes a receptor.
The term "about" as used herein refers to the usual range of error for the corresponding value that is readily known to those skilled in the art. An exemplary error range is plus or minus 5%. Reference herein to "about" a value or parameter includes (and describes) embodiments relating to that value or parameter per se.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
For any of the structural and functional features described herein, methods of determining these features are known in the art.
Antibodies
Structure of the product
The present application provides antibodies and compositions comprising antibodies that bind to TREM2 protein, including antibodies that disable non-stimulatory bone marrow cells.
The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that comprise one or more antigen binding domains that specifically bind an antigen or epitope. Antibodies specifically include whole antibodies (e.g., whole immunoglobulins), antibody fragments, and multispecific antibodies.
The recognized immunoglobulin genes include kappa, lambda, α, gamma, and mu constant region genes as well as tens of thousands of immunoglobulin variable region genes.A light chain is classified as either kappa or lambda.an antibody or "class" of an immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain2、IgG3、IgG4IgA1 and IgA2Heavy chain constant domains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.
An exemplary immunoglobulin (antibody) building block is composed of two pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50-70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains, respectively. The heavy chain of IgG1 comprises, from N-terminus to C-terminus, VH, CH1, CH2, and CH3 domains, respectively. The light chain comprises, from N-terminus to C-terminus, VL and CL domains. The IgG1 heavy chain comprises a hinge between the CH1 domain and the CH2 domain. In certain embodiments, the immunoglobulin construct comprises at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domain found in the antibodies provided herein is derived or derived from an immunoglobulin-based construct such as a minibifunctional antibody or a nanobody. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody, such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody such as a bovine antibody, a human antibody, a camelid antibody, a mouse antibody, or any chimeric antibody.
In some embodiments, an antibody provided herein comprises a heavy chain. In one embodiment, the heavy chain is an IgA heavy chain. In one embodiment, the heavy chain is an IgD heavy chain. In one embodiment, the heavy chain is an IgE heavy chain. In one embodiment, the heavy chain is an IgG heavy chain. In one embodiment, the heavy chain is an IgM heavy chain. In one embodiment, the heavy chain is an IgG1 heavy chain. In one embodiment, the heavy chain is an IgG2 heavy chain. In one embodiment, the heavy chain is an IgG3 heavy chain. In one embodiment, the heavy chain is an IgG4 heavy chain. In one embodiment, the heavy chain is an IgA1 heavy chain. In one embodiment, the heavy chain is an IgA2 heavy chain.
The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are highly variable in sequence and/or form structurally defined loops ("hypervariable loops"). Typically, a native four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from Complementarity Determining Regions (CDRs) that have the highest sequence variability and/or are involved in antigen recognition. In addition to CDR1 in VH, the CDRs typically comprise amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable regions that form the antigen-binding regions. This particular region has been described by Kabat et al, U.S. Dept. of Health and Human Services, sequence of Proteins of Immunological Interest (1983) and by Chothia et al, J Mol Biol 196:901-917(1987), wherein an overlap or subgroup comprising amino acid residues is defined when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact number of residues that encompass a particular CDR will vary depending on the sequence and size of the CDR. In view of the variable region amino acid sequence of an antibody, one skilled in the art can determine which residues constitute a particular CDR in a routine manner.
The amino acid sequence boundaries of the CDRs may be determined by one of skill in the art using any of a number of known numbering schemes, including by Kabat et al (supra) ("Kabat" numbering scheme); Al-Lazikani et Al, 1997, J.mol.biol.,273:927-948 ("Chothia" numbering scheme); MacCallum et al, 1996, J.mol.biol.262:732-745 ("contact numbering scheme"); lefranc et al, Dev. Comp. Immunol.,2003,27:55-77 ("IMGT" numbering scheme); and those described by Honegge and Pl ü ckthun, J.mol.biol.,2001,309:657-70 ("AHo" numbering scheme); each of these documents is incorporated by reference herein in its entirety.
Table A provides the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 positions as identified by Kabat and Chothia procedures. For CDR-H1, residue numbering using both the Kabat numbering scheme and the Chothia numbering scheme is provided.
CDRs can be obtained, for example, using antibody numbering software such as Abnum, available at www.bioinf.org.uk/abs/Abnum, and specified in Abnum described in Abhinandan and Martin, Immunology,2008,45: 3832-.
Residues in cdrs according to the Kabat numbering scheme and Chothia numbering scheme.
When numbered using the Kabat numbering convention, the C-terminus of CDR-H1 varies between H32 and H34 depending on the length of the CDR.
When referring to residues in the constant region of an antibody heavy chain, the "EU numbering scheme" is typically used (e.g. as reported in Kabat et al (supra)). Unless otherwise stated, EU numbering scheme is used to refer to residues in the antibody heavy chain constant region described herein.
As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In a particular such embodiment, in a single chain Fab molecule, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain. As described in more detail herein, an scFv has a variable domain of a light chain (VL) linked from its C-terminus by a polypeptide chain to the N-terminus of a variable domain of a heavy chain (VH). Alternatively, the scFv comprises a polypeptide chain in which the C-terminus of the VH is linked to the N-terminus of the VL by a polypeptide chain.
A "Fab fragment" (also known as an antigen binding fragment) contains the constant domain of the light Chain (CL) and the first constant domain of the heavy chain (CH1) and the variable domains VL and VH on the light and heavy chains, respectively. The variable domains comprise complementarity determining loops (CDRs, also known as hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
“F(ab’)2A "fragment contains two Fab' fragments joined by a disulfide bond near the hinge region. F (ab')2Fragments can be generated, for example, by recombinant methods or by pepsin digestion of intact antibodies F (ab') fragments can be dissociated, for example, by treatment with β -mercaptoethanol.
An "Fv" fragment comprises a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain.
"Single chain Fv" or "scFv" include the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the structure required for antigen binding. For an overview of scFv, see Pluckthun, the Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994). HER2 antibody scFv fragments are described in WO 93/16185; U.S. patent nos. 5,571,894; and U.S. Pat. No. 5,587,458.
The "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e.V)H-VLOr VL-VH) Rather, the Fc domain can be at VHOr VLAnd then. Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.
The term "single domain antibody" or "sdAb" refers to a molecule in which one variable domain of an antibody specifically binds an antigen in the absence of another variable domain. Single domain antibodies and fragments thereof are described in Arabi Ghahronoudi et al, FEBS Letters,1998,414:521-526 and Muylermans et al, Trends in biochem. Sci.,2001,26:230-245, each of which is incorporated herein by reference in its entirety. Single domain antibodies are also known as sdabs or nanobodies. Sdab is very stable and is readily expressed as a fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of peptide single-domain antibody fragments". Appl. Microbiol Biotechnol.77(1): 13-22).
The terms "full-length antibody," "intact antibody," and "full antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a naturally occurring antibody, as well as having a heavy chain comprising an Fc region. For example, when used to refer to an IgG molecule, a "full-length antibody" is an antibody comprising two heavy chains and two light chains.
The term "epitope" means that portion of an antigen that specifically binds to an antibody. Epitopes often consist of surface accessible amino acid residues and/or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, can be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in binding as well as other amino acid residues that are not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination, such as, for example, testing binding of an antibody to a TREM2 variant having different point mutations or to a chimeric TREM2 variant.
A "multispecific antibody" is an antibody comprising two or more different antigen binding domains that collectively specifically bind two or more different epitopes. The two or more different epitopes can be epitopes on the same antigen (e.g., a single TREM2 molecule expressed by a cell) or on different antigens (e.g., different TREM2 molecules, or a TREM2 molecule and a non-TREM 2 molecule expressed by the same cell). In some aspects, the multispecific antibody binds two different epitopes (i.e., "bispecific antibody"). In some aspects, a multispecific antibody binds three different epitopes (i.e., a "trispecific antibody").
A "monospecific antibody" is an antibody that comprises one or more binding sites that specifically bind a single epitope. An example of a monospecific antibody is a naturally occurring IgG molecule that, although bivalent (i.e., having two antigen binding domains), recognizes the same epitope at each of the two antigen binding domains. The binding specificity can be present at any suitable titer.
The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and bind to one or more of the same epitopes, except for variants that may typically be produced during the production of the monoclonal antibody. The variants are usually present in only small amounts. Monoclonal antibodies are typically obtained by a method that includes selecting a single antibody from a plurality of antibodies. For example, the selection method can be to select unique clones from a pool of multiple clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody may be further altered, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
"Effector function" refers to those biological activities mediated by the Fc region of an antibody, which activities may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate Complement Dependent Cytotoxicity (CDC), Fc receptor binding, receptor ligand blocking, agonism, or antagonism to activate Antibody Dependent Cellular Cytotoxicity (ADCC) and Antibody Dependent Cellular Phagocytosis (ADCP).
anti-TREM 2 antibodies can include those described herein, such as the clones set forth in the tables. In some embodiments, the antibody comprises an alternative scaffold. In some embodiments, the antibody consists of an alternative scaffold. In some embodiments, the antibody consists essentially of an alternative scaffold. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the antibody consists of an antibody fragment. In some embodiments, the antibody consists essentially of an antibody fragment. A "TREM 2 antibody," "anti-TREM 2 antibody," or "TREM 2-specific antibody" is an antibody as provided herein that specifically binds to antigen TREM 2. In some embodiments, the antibody binds to the extracellular domain of TREM 2. In certain embodiments, the TREM2 antibodies provided herein bind an epitope of TREM2 that is conserved between TREM2 proteins from different species.
The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
A "humanized" form of a non-human antibody is a chimeric antibody that contains minimal sequences derived from the non-human antibody. A humanized antibody is a substantially human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs from a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody having the desired specificity, affinity, or biological effect, such as a mouse, rat, rabbit, chicken, or non-human primate antibody. When administered to a human subject, the humanized antibody is less likely to induce an immune response and/or induces an immune response of less severity than an antibody of a non-human species. In some cases, the selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues not found in the recipient antibody or the donor antibody. Such modifications can be made to further improve antibody function. Examples of how humanized antibodies can be prepared can be found in U.S. Pat. nos. 6,054,297, 5,886,152 and 5,877,293, each of which is incorporated herein by reference in its entirety. For additional details, see Jones et al, Nature,1986,321: 522-525; riechmann et al, Nature,1988,332: 323-329; and Presta, curr, Op, Structure, biol.,1992,2: 593-.
In one embodiment, one or more constant domains from a human antibody are fused to one or more variable domains of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered such that the potential immunogenicity of the non-human antibody is reduced when it is administered to a human subject, wherein the altered amino acid residues are not critical for immunospecific binding of the antibody to its antigen, or the alteration made to the amino acid sequence is a conservative alteration such that binding of the humanized antibody to the antigen is not significantly worse than binding of the non-human antibody to the antigen.
A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody lineage or human antibody coding sequence (e.g., obtained or redesigned from a human source). Human antibodies specifically exclude humanized antibodies. In one embodiment, the variable and constant domains are all derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared in a variety of ways, including by immunizing mice genetically modified to express antibodies produced by human heavy and/or light chain-encoding genes with an antigen of interest.
In some embodiments, the antibodies provided herein comprise antibody fragments. In some embodiments, the antibodies provided herein consist of antibody fragments. In some embodiments, an antibody provided herein consists essentially of an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is F (ab')2And (3) fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is a scfv (sfv) fragment. In some embodiments, the antibody fragment is a scFv-Fc fragment. In thatIn some embodiments, the antibody fragment is a fragment of a single domain antibody.
Sequences of TREM2 antibodies
VHStructural domains
In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs 1,3, 5, and 7HAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 1HAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 3HAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 5HAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 7HAnd (4) sequencing.
In some embodiments, the antibodies provided herein comprise an amino acid sequence identical to the illustrative V provided in SEQ ID NOs 1,3, 5, and 7HV having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity in sequenceHAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V provided as SEQ ID NOs 1,3, 5, and 7HA sequence having up to 1,2, 3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
VLStructural domains
In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs 2,4, 6, and 8LAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 2LAnd (4) sequencing. In some embodiments, the antibodies provided herein compriseV of SEQ ID NO 4LAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 6LAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 8LAnd (4) sequencing.
In some embodiments, the antibodies provided herein comprise an amino acid sequence identical to the illustrative V provided in SEQ ID NOs 2,4, 6, and 8LV having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity in sequenceLAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V provided as SEQ ID NOs 2,4, 6, and 8LA sequence having up to 1,2, 3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
VH-VLCombination of
In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs 1,3, 5, and 7HA sequence; and V selected from SEQ ID NO 2,4, 6 and 8LAnd (4) sequencing.
In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 1HSequence and V of SEQ ID NO 2LAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 3HSequence and V of SEQ ID NO 4LAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 5HSequence and V of SEQ ID NO 6LAnd (4) sequencing. In some embodiments, the antibodies provided herein comprise V of SEQ ID NO 7HSequence and V of SEQ ID NO 8LAnd (4) sequencing. At a certain pointIn some aspects, any of SEQ ID NOs 1,3, 5, and 7 can be combined with any of SEQ ID NOs 2,4, 6, and 8. For example, SEQ ID NO 1 can be combined with any of SEQ ID NO 2,4, 6 or 8. As another example, SEQ ID NO 2 can be combined with any one of SEQ ID NO 1,3, 5, or 7.
In some embodiments, the antibodies provided herein comprise an amino acid sequence identical to the illustrative V provided in SEQ ID NOs 1,3, 5, and 7HV having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity in sequenceHA sequence; and V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the illustrative VL sequences provided as SEQ ID Nos. 2,4, 6, and 8LAnd (4) sequencing. In some embodiments, antibodies provided herein comprise VH sequences provided in SEQ ID NOs 1,3, 5, and 7 with up to 1,2, 3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions and VL sequences provided in
CDR
In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs 1,3, 5, and 7HOne to three CDRs of a domain. In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs 1,3, 5, and 7HTwo to three CDRs of a domain. In some embodiments, the antibodies provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 1,3, 5, and7, three CDRs of the VH domain. In some aspects, the CDR is an exemplary CDR. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDR is a Chothia CDR. In some aspects, the CDR is an AbM CDR. In some aspects, the CDR is a contact CDR. In some aspects, the CDRs are IMGT CDRs.
In some embodiments, the CDR is a CDR having at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1, CDR-H2 or CDR-H3 of SEQ ID NOs: 1,3, 5 and 7. In some embodiments, CDR-H1 is a CDR-H1 of a VH domain selected from SEQ ID NOs 1,3, 5, and 7, having up to 1,2, 3, 4, or 5 amino acid substitutions. In some embodiments, CDR-H2 is a CDR-H2 selected from the VH domains of SEQ ID NOs 1,3, 5, and 7, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR-H3 is a CDR-H3 selected from the VH domains of SEQ ID NOs 1,3, 5, and 7, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise one to three CDRs of a VL domain selected from SEQ ID NOs 2,4, 6, and 8. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VL domain selected from SEQ ID NOs 2,4, 6, and 8. In some embodiments, the antibodies provided herein comprise three CDRs of the VL domain selected from SEQ ID NOs 2,4, 6, and 8. In some aspects, the CDR is an exemplary CDR. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDR is a Chothia CDR. In some aspects, the CDR is an AbM CDR. In some aspects, the CDR is a contact CDR. In some aspects, the CDRs are IMGT CDRs.
In some embodiments, the CDR is a CDR having at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1, CDR-L2 or CDR-L3 of SEQ ID NOS: 2,4, 6 and 8. In some embodiments, the CDR-L1 is a CDR-L1 of a VL domain selected from SEQ ID NOs 2,4, 6, and 8, having up to 1,2, 3, 4, or 5 amino acid substitutions. In some embodiments, CDR-L2 is a CDR-L2 selected from the VL domains of SEQ ID NOs 2,4, 6, and 8, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some embodiments, CDR-L3 is a CDR-L3 selected from the VL domains of SEQ ID NOs 2,4, 6, and 8, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NOs 1,3, 5, and 7 and one to three CDRs of a VL domain selected from SEQ ID NOs 2,4, 6, and 8. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NOs 1,3, 5, and 7 and two to three CDRs of a VL domain selected from SEQ ID NOs 2,4, 6, and 8. In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NOs 1,3, 5, and 7 and three CDRs of a VL domain selected from SEQ ID NOs 2,4, 6, and 8. In some aspects, the CDR is an exemplary CDR. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDR is a Chothia CDR. In some aspects, the CDR is an AbM CDR. In some aspects, the CDR is a contact CDR. In some aspects, the CDRs are IMGT CDRs.
In some embodiments, the antibodies provided herein comprise the selected CDR-H3 of SEQ ID NO. 11. In some aspects, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO. 11. In some embodiments, CDR-H3 is a selected CDR-H3 of SEQ ID NO. 11, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-H2 of SEQ ID NO. 10. In some aspects, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO. 10. In some embodiments, CDR-H2 is CDR-H2 of SEQ ID NO. 10, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-H1 of SEQ ID NO. 9. In some aspects, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO. 9. In some embodiments, CDR-H1 is CDR-H1 of SEQ ID NO. 9, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-H3 of SEQ ID NO. 11 and CDR-H2 of SEQ ID NO. 10. In some embodiments, the antibodies provided herein comprise CDR-H3 of SEQ ID NO. 11, CDR-H2 of SEQ ID NO. 10, and CDR-H1 of SEQ ID NO. 9. In some embodiments, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO. 11, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO. 10, and CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO. 9. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO. 11, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO. 10, having up to 1,2, 3, 4,5, 6, 7 or 8 amino acid substitutions; and CDR-H1 is CDR-H1 of SEQ ID NO. 9, having up to 1,2, 3, 4 or 5 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-L3 of SEQ ID NO. 14. In some aspects, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO. 14. In some embodiments, CDR-L3 is CDR-L3 of SEQ ID NO. 14, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-L2 of SEQ ID NO 13. In some aspects, CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L2 of SEQ ID NO. 13. In some embodiments, CDR-L2 is CDR-L2 of SEQ ID NO. 13, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-L1 of SEQ ID NO. 12. In some aspects, CDR-L1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L1 of SEQ ID NO. 12. In some embodiments, CDR-L1 is CDR-L1 of SEQ ID NO. 12, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-L3 of SEQ ID NO. 14 and CDR-L2 of SEQ ID NO. 13. In some embodiments, the antibodies provided herein comprise CDR-L3 of SEQ ID NO. 14, CDR-L2 of SEQ ID NO. 13, and CDR-L1 of SEQ ID NO. 12. In some embodiments, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO. 14, CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L2 of SEQ ID NO. 13, and CDR-L1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L1 of SEQ ID NO. 12. In some embodiments, CDR-L3 is CDR-L3 of SEQ ID NO. 14, having up to 1,2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO 13, having up to 1,2, 3 or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO. 12, having up to 1,2, 3, 4,5 or 6 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-H3 of SEQ ID NO. 11, CDR-H2 of SEQ ID NO. 10, CDR-H1 of SEQ ID NO. 9, CDR-L3 of SEQ ID NO. 14, CDR-L2 of SEQ ID NO. 13, and CDR-L1 of SEQ ID NO. 12. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H3 of SEQ ID NO. 11, CDR-H2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H2 of SEQ ID NO. 10, CDR-H1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-H1 of SEQ ID NO. 9, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L3 of SEQ ID NO. 14, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L2 of SEQ ID NO. 13, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90% or 95% identity to CDR-L1 of SEQ ID NO. 12, 75%, 80%, 85%, 90% or 95% identity. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO. 11, having up to 1,2, 3, 4,5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO. 10, having up to 1,2, 3, 4,5, 6, 7 or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO. 9, having up to 1,2, 3, 4 or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO. 14, having up to 1,2, 3, 4 or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO 13, having up to 1,2, 3 or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO. 12, having up to 1,2, 3, 4,5 or 6 amino acid substitutions. In some aspects, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants". In some embodiments, the variants are obtained from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, the variants are not obtained from the sequences provided herein and can be re-isolated, e.g., according to the methods provided herein for obtaining antibodies.
In some embodiments, the antibodies provided herein comprise CDR-H1 of SEQ ID No. 9, CDR-H2 of SEQ ID No. 10, CDR-H3 of SEQ ID No. 11, CDR-L1 of SEQ ID No. 12, CDR-L2 of SEQ ID No. 13, and CDR-L1 of SEQ ID No. 14.
Fc region
The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Except as used hereinIt is further specified herein that the numbering of amino acid residues in the Fc region or constant region is otherwise according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD, 1991. As used herein, an "Fc polypeptide" of a dimeric Fc refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising a C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc polypeptide of dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences. The Fc may have classes IgA, IgD, IgE, IgG and IgM, and several of these classes may be further divided into subclasses (isotypes), e.g. IgG1、IgG2、IgG3、IgG4、IgA1And IgA2。
The terms "Fc receptor" and "FcR" are used to describe a receptor that binds the Fc region of an antibody. For example, the FcR may be a native sequence human FcR. Typically, an FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor") that have similar amino acid sequences, differing primarily in their cytoplasmic domains. Immunoglobulins of other isotypes may also be bound by certain FcRs (see, e.g., Janeway et al, immune Biology: the immune system in health and disease, (Elsevier science Ltd., NY) (4 th edition, 1999)). The activating receptor Fc γ RIIA contains in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (reviewed inAnnu.Rev.Immunol.15:203-234 (1997). FcR reviewed in ravatch and Kinet, annu. rev. immunol 9:457-92 (1991); capel et al, immunolmethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). Other FcRs, including those to be identified in the future, are identified by the term herein"FcR" encompasses. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587 (1976); and Kim et al, J.Immunol.24:249 (1994)).
In some embodiments, the antibody is an IgG1 antibody.
In some embodiments, the antibody is an IgG3 antibody.
In some embodiments, the antibody is an IgG2 antibody.
In some embodiments, the antibody is an IgG4 antibody.
Modifications in the CH2 domain may affect FcR binding to Fc. Many amino acid modifications in the Fc region for selectively altering the affinity of Fc for different Fc γ (Fc-gamma) receptors are known in the art. In one embodiment, the Fc comprises one or more modifications to facilitate selective binding to an fey receptor.
Exemplary mutations that alter FcR binding to Fc are as follows:
S298A/E333A/K334A, S298A/E333A/K334A/K326A (JImmunal methods, 2011, 2, 28 days Lu Y, Vernes JM, Chiang N, etc.; 365(1-2): 132-41);
F243L/R292P/Y300L/V305I/P396L, F243L/R292P/Y300L/L235V/P396L (Stavenhagen JB, Gorlatov S, Tuaillon N, etc. Cancer Res.2007, 9.15.9.67 (67) (8818) 8882-90; Nordstrom JL, Gorlatov S, Zhang W, etc. Breast Cancer Res.2011, 11.30.11.13 (6): R123);
F243L (Stewart R, Thom G, Protein Eng Des Sel.2011 9/24 (9):671-8.), S298A/E333A/K334A (Shields RL, Namenuk AK, Hong K et al J Biol chem.2001 3/2; 276(9): 6591-;
S239D/I332E/A330L, S239D/I332E (Lazar GA, Dang W, Karki S et al Proc Natl Acadsi USA.2006, 3, 14, 3(11): 4005-10);
S239D/S267E, S267E/L328F (Chu SY, Vostinar I, Karki S, et al Mol Immunol.2008.9 months; 45(15): 3926-33);
S239D/D265S/S298A/I332E, S239E/S298A/K326A/a327H, G237F/S298A/a330L/I332E, S239D/I332E/S298A, S239D/K326E/a330L/I332E/S298A, G236A/S239D/D270L 70 2/I332E, S239E/S267E/H268D, L234F/S267E/N325L, G237L/V266L/S267L and other mutations listed in WO 2011/L and WO 2011/L, which are incorporated herein by reference. The mutations are listed on page 283 in Therapeutic antibody engineering (by William r.strohl and Lila m.strohl, edited by Woodhead biomedical publication series (Woodhead Publishing series in Biomedicine) 11, ISBN 1907568379,2012, month 10).
In some embodiments, an antibody described herein comprises a modification to improve its ability to mediate effector function. Such modifications are known in the art and include no fucosylation, or engineering the affinity of Fc for the activating receptor, primarily FCGR3a (for ADCC) and for C1q (for CDC). Table B below summarizes the various designs reported in the literature for performing effector function engineering.
In certain embodiments, the antibodies provided herein comprise an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at one or more of positions 298, 333, and 334 of the Fc region. In some embodiments, the antibodies provided herein comprise an Fc region having one or more amino acid substitutions at positions 239, 332 and 330, as described in Lazar et al, proc. natl. acad. sci. usa,2006,103: 4005-.
In some embodiments, the antibodies provided herein comprise one or more alterations that improve or attenuate C1q binding and/or CDC. See U.S. Pat. nos. 6,194,551; WO 99/51642; and Idusogene et al, J.Immunol.,2000,164: 4178-; each of which is incorporated herein by reference in its entirety.
Thus, in one embodiment, an antibody described herein may comprise a dimeric Fc comprising one or more amino acid modifications as indicated in table B that confer improved effector function. In another embodiment, the antibody may be made free of fucosylation to improve effector function.
Table B: CH2 Domain and Effect functional engineering
Fc modifications that reduce Fc γ R and/or complement binding and/or effector function are known in the art. Recent publications describe strategies that have been used to engineer antibodies with reduced or silenced effector activity (see Strohl, WR (2009), Curr Opin Biotech 20: 685-. These strategies include reducing effector function by altering glycosylation, using the IgG2/IgG4 backbone, or introducing mutations in the hinge or CH2 region of the Fc. For example, U.S. patent publication No. 2011/0212087(Strohl), international patent publication No. WO 2006/105338 (xenocor), U.S. patent publication No. 2012/0225058 (xenocor), U.S. patent publication No. 2012/0251531(Genentech), and shop et al ((2012) j.mol.biol.420:204-219) describe specific modifications to reduce Fc γ R or complement to Fc binding.
Specific non-limiting examples of known amino acid modifications to reduce Fc γ R or complement binding to Fc include those identified in table C below:
table C: modifications to reduce Fc γ R or complement binding to Fc
Methods of producing antibodies with little or no fucose at the Fc glycosylation site (Asn 297, EU numbering) without altering the amino acid sequence are well known in the art.The technique (ProBioGen AG) is based on the introduction of a gene for an enzyme that deflects the cellular trehalose biosynthetic pathway into cells for antibody production this prevents the addition of sugar "trehalose" by antibody-producing cells to the N-linked antibody carbohydrate moiety (von Horsten et al (2010) glycobiology, 12.2010; 20(12): 1607-18.) examples of cell lines capable of producing deglycosylated antibodies include CHO-DG44 (see Henning von Horsten et al, Glycobiol 2010,20:1607- > D-lysu-4-hexulose Reductase (RMD) stably overexpressing the bacterial oxidoreductase or Lec13 CHO cells defective in the protein glycosylation direction (see Ripka et al, Arch. Biophys.,1986,249: 533; U.S. publication No. 2003/0157108; WO 2003/0157108; and WO 2004/056312. and WO 76. refer to the methods cited in the text incorporated by reference in their entirety for antibody production and for the methods described herein incorporated by the encystein et al, Biotech et al, Bioglycosylating methods for antibody production in the entire aspects of the invention, such as CHO-WO 3976, WO 76, the methods cited in the entire sections, incorporated by Biotech et al, for antibody production, Biotech et al, Biotech, for antibody production, and for antibody production of low levels, for each of the methods cited patent lines, such as Biotech, Biotech et al, Biotech et al.
The antibodies can be completely non-trehalose-glycosylated (meaning that they do not contain detectable trehalose), or they can be partially non-trehalose-glycosylated, meaning that the isolated antibodies contain an amount of trehalose that is less than 95%, less than 85%, less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, less than 25%, less than 15%, or less than 5% of the amount of trehalose normally detected for similar antibodies produced by a mammalian expression system.
In some aspects, the antibodies provided herein comprise an IgG1 domain having reduced fucose content at position Asn 297, as compared to the naturally occurring IgG1 domain. The Fc domain is known to have improved ADCC. See, shiplds et al, j.biol.chem.,2002,277:26733-26740, which is incorporated herein by reference in its entirety. In some aspects, the antibody does not comprise any trehalose at position Asn 297. The amount of trehalose may be determined using any suitable method, for example as described in WO 2008/077546, which is herein incorporated by reference in its entirety.
In some embodiments, the antibodies provided herein comprise bisected oligosaccharides, such as biantennary oligosaccharides bisected by GlcNAc that are attached to the Fc region of the antibody. The antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; U.S. Pat. nos. 6,602,684; and U.S. patent publication No. 2005/0123546; each of these patents is incorporated by reference herein in its entirety.
Other illustrative glycosylation variants that can be incorporated into the antibodies provided herein are described, for example, in U.S. patent publication nos. 2003/0157108, 2004/0093621, 2003/0157108, 2003/0115614, 2002/0164328, 2004/0093621, 2004/0132140, 2004/0110704, 2004/0110282, 2004/0109865; international patent publication nos. 2000/61739, 2001/29246, 2003/085119, 2003/084570, 2005/035586, 2005/035778; 2005/053742, 2002/031140; okazaki et al, J.mol.biol.,2004,336: 1239-1249; and Yamane-Ohnuki et al, Biotech.Bioeng, 2004,87:614-622, each of which is incorporated herein by reference in its entirety.
In some embodiments, the antibodies provided herein comprise an Fc region having at least one galactose residue in an oligosaccharide attached to the Fc region. The antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and in WO 1999/22764; each of these patents is incorporated by reference herein in its entirety.
Examples of cell lines capable of producing deglycosylated antibodies include CHO-DG44 stably overexpressing the bacterial oxidoreductase GDP-6-deoxy-D-lysu-4-hexulose Reductase (RMD) (see Henning von Horsten et al, Glycobiol 2010,20: 1607-osa 1618) or Lec13 CHO cells deficient in protein fucosylation (see Ripka et al, Arch. biochem. Biophys.,1986,249: 533-545; U.S. Pat. Pub. No. 2003/0157108; WO 2004/056312; each of which is incorporated herein by reference in its entirety), and knockout cell lines such as the alpha-1, 6-fucosyltransferase gene or FUT8 knockout CHO cells (see Yamane-Ohnuki et al, Biotech. Bioeng.,2004,87: 614-622; Kanda et al, Biohnol. Biotech. 2006, 680: 94; and WO 2003/085107; each of which is incorporated herein by reference in its entirety) .
In some embodiments, the antibody has antibody-dependent cellular phagocytosis (ADCP) activity. ADCP can occur when an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell. Phagocytic cells, including monocytes and macrophages, which carry Fc receptors on their cell surface, recognize and bind the Fc region of antibodies that bind to target cells. After the Fc receptor binds to the antibody-bound target cell, phagocytosis of the target cell can be initiated. ADCP can be considered a form of ADCC.
In some embodiments, the antibody is capable of forming an immune complex. For example, the immune complex can be a tumor cell covered by an antibody.
In some aspects, the anti-TREM 2 antibody does not substantially bind bone marrow cells present outside of the cancer tissue. In some aspects, the anti-TREM 2 antibody does not substantially bind stimulatory bone marrow cells present in the cancer tissue.
In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is a polyclonal antibody.
In some embodiments, the antibody is produced by a hybridoma. In other embodiments, the antibody is produced by a recombinant cell engineered to express the desired variable and constant domains.
In some embodiments, the antibody may be a single chain antibody or other antibody derivative that retains the antigen specificity and lower hinge region or variant thereof.
In some embodiments, the antibody can be a multifunctional antibody, a recombinant antibody, a human antibody, a humanized antibody, a fragment thereof, or a variant thereof. In a particular embodiment, the antibody fragment or derivative thereof is selected from the group consisting of a Fab fragment, a Fab' 2 fragment, a CDR, and an ScFv.
In some embodiments, the antibody is specific for a surface antigen such as a TREM2 protein. In some embodiments, the therapeutic antibody is specific for a tumor antigen (e.g., a molecule specifically expressed by a tumor cell). In particular embodiments, the therapeutic antibody may have a human or non-human primate IgG1 or IgG3 Fc portion.
Bonding of
With respect to binding of an antibody to a target molecule, the terms "bind" to, specifically binds to, or selectively binds to a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen, "is specific for, selectively binds to, or selectively interacts with (e.g., interacts with) a non-specific or non-selective interaction to a measurable degree (e.g., a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.
"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). As used herein, "affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope), unless otherwise indicated. The affinity of molecule X for its partner Y can be determined by the dissociation equilibrium constant (K)D) And (4) showing. The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity canMeasured by common methods known in the art, including those described herein, such as Surface Plasmon Resonance (SPR) techniques (e.g., Surface Plasmon Resonance (SPR))) Or bio-layer interferometry (e.g. of
The term "k" as used hereind”(s-1) Refers to the off-rate constant for a particular antibody-antigen interaction. This value is also referred to as kDissociationThe value is obtained.
The term "k" as used hereina”(M-1×s-1) Refers to the association rate constant for a particular antibody-antigen interaction. This value is also referred to as kAssociation ofThe value is obtained.
The term "K" as used hereinD"(M) refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. KD＝kd/ka. In some embodiments, the affinity of an antibody is as K for the interaction between the antibody and its antigenDA description is given. For clarity, as is known in the art, a smaller KDValues indicate higher affinity interactions, while larger KsDValues indicate lower affinity interactions.
The term "K" as used hereinA”(M-1) Refers to the association equilibrium constant for a particular antibody-antigen interaction. KA＝ka/kd。
When used herein in the context of two or more antibodies, the term "competes with … …" or "cross-competes with … …" indicates that the two or more antibodies compete for binding to an antigen (e.g., TREM 2). In one exemplary assay, TREM2 is coated on a surface and contacted with a first TREM2 antibody, after which a second TREM2 antibody is added. In another exemplary assay, a first TREM2 antibody is coated on a surface and its TREM2 is contacted, followed by the addition of a second TREM2 antibody. If in either assay the presence of the first TREM2 antibody reduces the binding of the second TREM2 antibody, then the antibodies compete with each other. The term "competes with … …" also includes combinations of antibodies wherein one antibody decreases the binding of the other antibody, but wherein no competition is observed when the antibodies are added in the reverse order. However, in some embodiments, the first and second antibodies inhibit binding to each other regardless of the order in which they are added. In some embodiments, an antibody reduces binding of another antibody to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. The skilled artisan can select the concentration of antibody to use in a competition assay based on the affinity of the antibody to TREM2 and the titer of the antibody. The assays described in this definition are illustrative, and the skilled artisan can use any suitable assay to determine whether antibodies compete with each other. Suitable assays are described, for example, in Cox et al, "Immunoassay Methods", Assay guide Manual [ Internet ], 24-day updates 12/2014 (www.ncbi.nlm.nih.gov/books/NBK 92434/; 29-day visits 9/2015); simman et al, Cytometry,2001,44: 30-37; and Finco et al, J.pharm.biomed.anal.,2011,54: 351-; each of these documents is incorporated by reference herein in its entirety.
In some embodiments, the antibodies provided herein are administered at less than or equal to about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 3, 4,5, 6, 7,8, 9, or 10x10-9K of MDBinding to human TREM2 as measured by Biacore assay. In some embodiments, the K of an antibody provided hereinDAt about 0.001-0.01, 0.01-0.1, 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-1, 0.25-0.75, 0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-5, 2-7, 3-8, 3-5, 4-6, 5-7, 6-8, 7-9, 7-10, or 5-10x10-9M as measured by Biacore assay.
In some casesIn embodiments, the antibodies provided herein are administered at less than or equal to about 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, or 1.4x10-9K of M or lessDBinding to human TREM2 as measured by Biacore assay. In some embodiments, the antibodies provided herein are administered at 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, or 1.9-1.5x10-9K between MDBinding to human TREM2 as measured by Biacore assay. In some embodiments, the antibodies provided herein are administered at less than or equal to about 10, 9.56, 9.5, 9.0, 8.88, 8.84, 8.5, 8, 7.5, 7.32, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1x10-4K of (1/s) or lessdBinding to human TREM2 as measured by Biacore assay. In some embodiments, the antibodies provided herein are administered at 7-10, 7-8, 8-9, 9-10, 7-7.5, 7.5-8, 8.-8.5, 8.5-9, 9-9,5, or 9.5-10x10-4K between (1/s)dBinding to human TREM2 as measured by Biacore assay. In some embodiments, the antibodies provided herein are administered at greater than or equal to about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 45, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 7,8, 9, or 10x105K of (1/Ms) or greateraBinding to human TREM2 as measured by Biacore assay. In some embodiments, the antibodies provided herein are administered at 4-7, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, or 6.5-7, 7-8, 8-9, or 9-10x105K between (1/Ms)aBinding to human TREM2 as measured by Biacore assay.
In some embodiments, the antibodies provided herein bind to human TREM2 with an EC50 of less than or equal to 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1nM as measured by flow cytometry. In some embodiments, the antibody binds human TREM2 with an EC50 of between 0.6-1.4nM as measured by flow cytometry. In some embodiments, the antibody binds human TREM2 with an EC50 of about 0.5, 0.6, 0.9, 1.1, 1.2, 1.3, 1.4, or 1.5nM as measured by flow cytometry.
In some embodiments, an antibody provided herein binds mouse TREM2 with an EC50 of less than or equal to 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1nM as measured by flow cytometry. In some embodiments, the antibody binds mouse TREM2 with an EC50 of between 0.6-1.4nM as measured by flow cytometry. In some embodiments, the antibody binds mouse TREM2 with an EC50 of about 0.5, 0.6, 0.9, 1.1, 1.2, 1.3, 1.4, or 1.5nM as measured by flow cytometry.
In some embodiments, the antibodies provided herein do not bind human TREM2 with an EC50 of greater than or equal to 20nM or greater, as measured by flow cytometry. In some embodiments, the antibodies provided herein do not bind to mouse TREM2 with an EC50 of greater than or equal to 3nM or greater as measured by flow cytometry.
To screen for Antibodies that bind to an epitope on a target antigen (e.g., TREM2) bound by an antibody of interest, a conventional cross-blocking assay can be performed, such as the assay described in Antibodies, a Laboratory Manual, Cold Spring harbor Laboratory, Harlow, and David Lane, eds (1988). Alternatively or additionally, epitope mapping can be performed by methods known in the art.
Competition between antibodies can be determined by assays in which the antibodies tested inhibit or block specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al, Cancer Res.50:1495,1990; Fendly et al, Cancer Research 50: 1550-. A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits or blocks binding of the reference antibody by, e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay. Antibodies identified by competition assays (competitive antibodies) include antibodies that bind the same epitope as the reference antibody and/or antibodies that bind a neighboring epitope sufficiently adjacent to the epitope bound by the reference antibody to achieve steric hindrance. For example, a second competitive antibody that competes for binding to TREM2 with the first antibody described herein can be identified. In certain instances, the second antibody can block or inhibit binding of the first antibody, e.g., by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, as measured in a competitive binding assay. In certain instances, the second antibody can displace greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the first antibody.
Function(s)
In some embodiments, the antibody has antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC can occur when an antibody binds to an antigen on the surface of a pathogenic or tumorigenic target cell. Effector cells that carry Fc γ receptors (Fc γ R or FCGR) on their cell surface, including cytotoxic T cells, Natural Killer (NK) cells, macrophages, neutrophils, eosinophils, dendritic cells, or monocytes, recognize and bind to the Fc region of antibodies that bind to target cells. The binding may trigger activation of intracellular signaling pathways, leading to cell death. In particular embodiments, the immunoglobulin Fc region subtype (isotype) of an antibody includes human IgG1 and IgG 3. As used herein, ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize antibodies that bind to a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu.Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in U.S. patent No. 5,500,362 or 5,821,337 may be performed. Effector cells useful in the assay include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al, Proc. Natl. Acad. Sci. (USA)95: 652-.
In some embodiments, the antibody has Complement Dependent Cytotoxicity (CDC) activity. Antibody-induced CDC is mediated by proteins of the classical complement cascade and is triggered by the binding of complement proteins Clq to antibodies. Binding of the Fc region of the antibody to Clq induces activation of the complement cascade. In particular embodiments, the immunoglobulin Fc region subtype (isotype) of an antibody includes human IgG1 and IgG 3. As used herein, CDC refers to the ability of a molecule to solubilize a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., a polypeptide (e.g., an antibody)) that is complexed to a homologous antigen. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996).
In some embodiments, the antibody is an agonistic antibody. An agonistic antibody may induce (e.g., increase) one or more activities or functions of an NSM upon binding of the antibody to a TREM2 protein expressed on a cell. Agonistic antibodies can bind to and activate NSM, resulting in changes in cell proliferation or altered antigen presentation capacity. Agonistic antibodies can bind to and activate NSM, thereby triggering an intracellular signaling pathway that leads to altered cell growth or apoptosis.
In some embodiments, the antibody is an antagonist antibody. An antagonistic antibody can block (e.g., decrease) one or more activities or functions of an NSM after the antibody binds to a TREM2 protein expressed on a cell. For example, an antagonist antibody can bind to one or more NSM proteins and block the binding of a ligand to one or more NSM proteins, thereby preventing differentiation and proliferation of the cell or altering antigen presentation capacity. The antagonist antibody can bind to TREM2 protein and prevent activation of TREM2 protein by a ligand of TREM2 protein, thereby altering the intracellular signaling pathway that promotes cell growth and survival.
In some embodiments, the antibody is a subtractive antibody. A depleting antibody is an antibody that, upon contact, will kill non-stimulatory myeloid cells through the interaction of the antibody with other immune cells having various molecules. For example, when bound to a cell carrying a TREM2 protein, the antibody can engage a complement protein and induce complement-dependent cytolysis. When bound to cells carrying a TREM2 protein, the antibody can also trigger adjacent cells carrying Fc receptors to kill them by antibody-dependent cellular cytotoxicity (ADCC).
In some embodiments, the antibody is a neutralizing antibody, and the antibody neutralizes one or more biological activities of the NSM. In some embodiments, the TREM2 protein is expressed on the surface of non-stimulatory bone marrow cells and the antibody recognizes the extracellular domain of the TREM2 protein.
In some embodiments, the antibody is selective for NSM (preferentially binds TREM 2). In certain embodiments, an antibody that selectively binds NSM has a dissociation constant (Kd) in the range of 0.0001nM to 1 μ M. In certain embodiments, the antibody specifically binds to an epitope on the TREM2 protein that is conserved between proteins from different species. In another embodiment, selective binding includes, but does not require, exclusive binding.
In one embodiment, the anti-TREM 2 antibody bound to its target is responsible for causing depletion of the non-stimulatory myeloid cells to which it binds in vivo. In some embodiments, the effector protein induced by the clustered antibody may trigger multiple responses, including release of inflammatory cytokines, modulation of antigen production, endocytosis, or cell killing. In one embodiment, the antibody is capable of recruiting and activating complement in vivo or mediating antibody-dependent cellular cytotoxicity (ADCC), or mediating phagocytosis in vivo by binding to Fc receptors. The antibody may also deplete the non-stimulatory myeloid cells upon binding by inducing apoptosis or necrosis of the non-stimulatory myeloid cells.
In some embodiments, incapacitation of non-stimulatory myeloid cells is achieved in vitro and is achieved by: a) killing non-irritating bone marrow cells; b) magnetic bead depletion is carried out on the non-stimulated bone marrow cells; or c) sorting the non-stimulated bone marrow cells using Fluorescence Activated Cell Sorting (FACS).
In some embodiments, the antibody is bound or conjugated to an effector molecule. In particular embodiments, the antibody is conjugated to at least one therapeutic agent selected from the group consisting of: radionuclides, cytotoxins, chemotherapeutic agents, drugs, prodrugs, toxins, enzymes, immunomodulators, anti-angiogenic agents, pro-apoptotic agents, cytokines, hormones, oligonucleotides, antisense molecules, siRNA, secondary antibodies and secondary antibody fragments.
In certain embodiments, the antibody is conjugated to a drug, such as a toxin, a chemotherapeutic agent, an immunomodulatory agent, or a radioisotope. Several methods of preparing ADCs (antibody drug conjugates) are known in the art and are described, for example, in U.S. patents 8,624,003 (pot), 8,163,888 (one step), and 5,208,020 (two step). The antibody or antigen-binding fragment thereof can be conjugated to at least one agent, including a radionuclide, cytotoxin, chemotherapeutic agent, drug, prodrug, toxin, enzyme, immunomodulator, anti-angiogenic agent, pro-apoptotic agent, cytokine, hormone, oligonucleotide, antisense molecule, siRNA, second antibody, and a second antibody fragment having antigen binding properties.
Non-stimulatory myeloid cells (NSM)
Provided herein are methods and compositions for incapacitating and/or detecting non-stimulatory bone marrow cells (NSMs), including the use of anti-TREM 2 antibodies. Also provided herein are methods and compositions for targeting and/or detecting non-stimulatory myeloid cells that express NSM protein.
Also provided herein are methods and compositions for incapacitating and/or detecting non-stimulatory myeloid cells, including the use of antibodies directed against non-human homologs of human NSM proteins in that non-human individual.
As used herein, a non-stimulatory myeloid cell is a myeloid cell that is not sufficiently effective to stimulate an immune response (e.g., differently effective to stimulate an anti-tumor response in a tumor microenvironment compared to a stimulatory myeloid cell). In some embodiments, non-stimulatory myeloid cells are not as effective at presenting an antigen (e.g., a tumor antigen) to a T cell, or are not as effective at stimulating a tumor-specific T cell response, as stimulatory myeloid cells. In some embodiments, the non-stimulatory myeloid cells may exhibit a reduced ability to take up, process, and/or present tumor-associated antigens to T cells as compared to stimulatory myeloid cells. Non-stimulatory myeloid cells may or may not contain reduced or no capacity to restensitize cytotoxic T lymphocytes, or in some cases, may not stimulate effective tumor cell killing. Non-stimulatory myeloid cells may exhibit lower expression of genes and cell surface markers involved in antigen processing, antigen presentation, and/or antigen co-stimulation, including but not limited to CD80, CD86, MHCI, and MHCII, as compared to stimulatory myeloid cells.
Non-stimulatory bone marrow cells may exhibit lower expression of genes associated with cross-presentation, co-stimulation, and/or stimulatory cytokines when compared to stimulatory bone marrow cells, including, without limitation, any one or more of TAP1, TAP2, PSMB8, PSMB9, TAPBP, PSME2, CD24a, CD274, BTLA, CD40, CD244, ICOSL, ICAM1, TIM3, PDL2, RANK, FLT3, CSF2RB, CSF2RB2, CSF2RA, IL12b, XCR1, CCR7, CCR2, CCL22, CXCL9, and CCL 5; and increased expression of the anti-inflammatory cytokine IL-10. In some embodiments, the non-stimulatory myeloid cells are dependent on the transcription factor IRF4 and the cytokine GM-CSF or CSF-1 for differentiation and survival. In some embodiments, non-stimulatory myeloid cells can promote tumor angiogenesis by secreting Vascular Endothelial Growth Factor (VEGF) and Nitric Oxide Synthase (NOS), and support tumor growth by secreting Epidermal Growth Factor (EGF).
In some embodiments, the non-stimulatory myeloid cells are tumor-associated macrophages (TAMs), neutrophils, monocytes, or Dendritic Cells (DCs). In some embodiments, the non-stimulatory myeloid cells are not Dendritic Cells (DCs). In some embodiments, the non-stimulatory myeloid cells are neutrophils.
In some embodiments, the non-stimulatory myeloid cells are Tumor Associated Macrophages (TAMs). TAMs are macrophages that are present near or within cancerous tumors and are derived from circulating monocytes or resident tissue macrophages.
In some embodiments, the non-stimulatory myeloid cells and stimulatory myeloid cells are differentiated based on their expressed markers or their selectively expressed markers. Expression of cell surface markers can be described as '+' or 'positive'. The absence of cell surface markers may be described as '-' or 'negative'. Expression of cell surface markers can be further described as 'high' (cells expressing high levels of markers) or 'low' (cells expressing low levels of markers). The level of the marker can be determined by various methods known in the art, such as immunostaining and FACS analysis, or gel electrophoresis and Western blotting.
In some embodiments, the non-stimulatory myeloid cells are Dendritic Cells (DCs). In some embodiments, dendritic cells can be differentiated by spike or dendritic morphology. In one embodiment, the non-stimulatory dendritic cells are at least CD45+, HLA-DR +, CD14-, CD11c +, and BDCA1+ (also referred to as DC1 cells). In one embodiment, the non-stimulatory dendritic cells are not CD45+, HLA-DR +, CD14-, CD11c +, and BDCA3+ (also referred to as DC2 cells). In one embodiment, the dendritic cells that are CD45+, HLA-DR +, CD14-, CD11c + and BDCA3+ are stimulatory myeloid cells.
In some embodiments, the non-stimulatory myeloid cells are tumor-associated macrophages. In some embodiments, e.g., in humans, the non-stimulatory tumor-associated macrophages are at least CD45+, HLA-DR +, CD14 +. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、CD11b+. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、CD11c+. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、BDCA3-. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、BDCA3-、CD11c+. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、CD11b+And CD11c+. In some embodiments, the non-stimulatory tumor-associated macrophages are at least CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+And CD11c+。
In some embodiments, the methods and compositions of the invention can be used to target TAMs and DCs in other mammals, for example, in mice. In the embodiment, mouse TAMs and DCs are contacted with a TREM2 antibody. In one embodiment, for example in a mouse, the tumor-associated macrophage is at least CD45+, HLA-DR +, CD14+, CD11bHeight ofAnd CD11cIs low in(also known as TAM 1). In one embodiment, for example in a mouse, the tumor-associated macrophage is at least CD45+, HLA-DR +, CD14+, CD11bIs low inAnd CD11cHeight of(also known as TAM 2). The term "CD 11b as used hereinHeight ofMacrophages "are macrophages that express high levels of CD11 b. The term "CD 11b as used hereinIs low inMacrophages "are involved in expressing on their surface in comparison to CD11bHeight ofMacrophage CD11b level, and macrophages with substantially lower levels of CD11 b. The term "CD 11c as used hereinHeight of"relates to macrophages expressing high levels of CD11 c. The term "CD 11c as used hereinIs low inMacrophages "are involved in their expression on the surface in comparison to Cd11cHeight ofMacrophage CD11c level, and macrophages with substantially lower levels of CD11 c.
In some embodiments, the non-stimulatory myeloid cells of the invention comprise one or more of TAM and DC1 cells.
In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cells of the invention comprise one or more of TAM1, TAM2, and DC1 cells. In such embodiments, the non-stimulatory bone marrow cells of the invention are contacted with a TREM2 antibody.
In some embodiments, the non-stimulatory myeloid cells are myeloid cells within a tumor.
In some embodiments, the non-stimulatory myeloid cells are located within the margins of the tumor lesion or in the transformed tumor duct where they come into contact with cognate T cells. In one embodiment, the localization of the non-stimulatory myeloid cells is altered such that the cells are no longer localized at the tumor margin or are no longer in contact with T cells.
In some embodiments, the non-stimulatory myeloid cells are in an immune cell population comprising stimulatory myeloid cells and non-stimulatory myeloid cells. In some embodiments, the non-stimulatory myeloid cells are in a population of immune cells that comprises only non-stimulatory myeloid cells. The immune cell population of the invention can be pure, homogeneous, heterogeneous, derived from a variety of sources (e.g., diseased tissue, tumor tissue, healthy tissue, cell banks), maintained in primary cell culture, maintained in immortalized culture, and/or maintained in ex vivo culture.
In some embodiments, the non-stimulatory myeloid cells are tumor-associated macrophages.
In some embodiments, the non-stimulatory myeloid cells are dendritic cells.
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14-、CD11c+And BDCA1+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14-、CD11c+And BDCA1+The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14-、CD11c+And BDCA1+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14-、CD11c+And BDCA1+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、BDCA3-The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、BDCA3-The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11b+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、CD11b+The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11b+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、CD11b+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11c+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、CD11c+The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11c+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、CD11c+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-And CD11c+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、BDCA3-And CD11c+The cell of (1). In some embodiments, the non-irritating boneMarrow cells are characterized by CD45+、HLA-DR+、CD14+、BDCA3-And CD11c+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、BDCA3-And CD11c+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11b+And CD11c+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、CD11b+And CD11c+The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11b+And CD11c+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、CD11b+And CD11c+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+And CD11c+. In some embodiments, the non-stimulatory myeloid cell comprises CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+And CD11c+The cell of (1). In some embodiments, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+And CD11c+The cell composition of (a). In some embodiments, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、BDCA3-、CD11b+And CD11c+The cell composition of (a).
In some embodiments, the non-stimulatory myeloid cells are not CD45+、HLA-DR+、CD14-、CD11c+And BDCA3+. In some embodiments, the non-stimulatory myeloid cells comprise a non-CD 45+、HLA-DR+、CD14-、CD11c+And BDCA3+The cell of (1).
In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11bHeight ofAnd CD11cIs low in. In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cell inclusion is CD45+、HLA-DR+、CD14+、CD11bHeight ofAnd CD11cIs low inThe cell of (1). In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11bHeight ofAnd CD11cIs low inThe cell composition of (a). In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、CD11bHeight ofAnd CD11cIs low inThe cell composition of (a). In such embodiments, non-stimulatory mouse bone marrow cells are contacted with a TREM2 antibody.
In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11bIs low inAnd CD11cHeight of. In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cell inclusion is CD45+、HLA-DR+、CD14+、CD11bIs low inAnd CD11cHeight ofThe cell of (1). In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cell is CD45+、HLA-DR+、CD14+、CD11bIs low inAnd CD11cHeight ofThe cell composition of (a). In some embodiments, e.g., in a mouse, the non-stimulatory myeloid cells consist essentially of CD45+、HLA-DR+、CD14+、CD11bIs low inAnd CD11cHeight ofThe cell composition of (a). In such embodiments, non-stimulatory mouse bone marrow cells are contacted with a TREM2 antibody.
In some embodiments, the non-stimulatory myeloid cells are in a cancer tissue.
In some embodiments, the immune cell population is in a cancer tissue.
In some embodiments, the non-stimulatory cells and the stimulatory myeloid cells are in a cancer tissue.
In some embodiments, the biological sample comprises a population of immune cells comprising non-stimulatory myeloid cells and stimulatory myeloid cells.
NSM cells may collectively refer to DC1, TAM1, and TAM2 cells that are present in tumor tissue and can be distinguished from other cell types based on their NSM cell marker expression. For example, genes and related proteins expressed or translated in greater abundance in NSM cells may serve as NSM markers compared to genes and related proteins for SDC. An exemplary NSM marker is CD11 b. Additional exemplary NSM markers are listed in table a. NSM cells can express TREM2, MS4A7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1/CD206, SIGLEC1, STAB1, TMEM37, MERRTK, and TMEM119 on their cell surface. In some aspects, the NSM cell does not express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR 1.
In one embodiment, the NSM cells express one or more of the NSM marker genes listed in table a. In another embodiment, the NSM cell expresses 2, 3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more of the NSM markers listed in table D. In another embodiment, the NSM cells express most or all of the NSM markers listed in table D. In another embodiment, the NSM cell is identified as expressing MRC1, MS4a7, C1QC, APOE, C1QB, C1QA, and C5AR 1.
Table D
Stimulatory myeloid cells
As used herein, a stimulatory myeloid cell (also referred to as SDC in some aspects) is a myeloid cell that is effective to stimulate an immune response (e.g., to more effectively stimulate an anti-tumor response in a tumor microenvironment compared to a non-stimulatory myeloid cell). In some embodiments, the stimulatory myeloid cells effectively present an antigen (e.g., a tumor antigen) to T cells, or effectively stimulate a tumor-specific T cell response, as compared to non-stimulatory myeloid cells. In some embodiments, the stimulatory myeloid cells may exhibit increased ability to take up, process and/or present tumor-associated antigens to T cells compared to non-stimulatory myeloid cells. Stimulatory myeloid cells may have an increased capacity to restensitize cytotoxic T lymphocytes relative to non-stimulatory myeloid cells, or in some cases, may stimulate effective tumor cell killing. Stimulated bone marrow cells may exhibit higher expression of genes and cell surface markers involved in antigen processing, antigen presentation, and/or antigen co-stimulation, including but not limited to CD80, CD86, MHCI, and MHCII, as compared to non-stimulated bone marrow cells.
Exemplary stimulatory myeloid cell markers are listed in table a. For example, in human SDC, the expression of Xcr1, Clec9a, and BDCA3(CD141) are markers for the identity of the SDC. It should be noted that CD103 can also be used as a strong marker of SDC identity in mice, but it is not expressed in human SDC.
In one embodiment, the SDC is a tumor-infiltrating myeloid cell that has dendritic cell identity and also expresses one or more of the SDC markers listed in table a. In another embodiment, the SDC is a tumor-infiltrating myeloid cell that has dendritic cell identity and also expresses two, three, four, five, six, seven, eight, nine, or all of the SDC markers listed in table a. In another embodiment, the SDC is identified as a tumor-infiltrating myeloid dendritic cell that expresses BDCA3, KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, XCR1, and CLEC 9A. SDC cells may express at least one of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR 1. In some embodiments, SDCs do not substantially express TREM2, MS4a7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1/CD206, SIGLEC1, STAB1, TMEM37, merk, and/or TMEM119 on their cell surface. In some embodiments, the SDC does not substantially express C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, merk, C1QC, TMEM119, MS4a7, APOE, CYP4F18, TREM2, TLR7, and/or LILRB 4. Flow cytometry and PCR, as well as other art-recognized assays, can be used to assess the expression of the markers disclosed herein.
The stimulatory myeloid cells may be CD45+、HLA-DR+、CD14-、CD11c+And BDCA3+. The stimulatory myeloid cells may be CD45+、HLA-DR+And BDCA3+. The stimulatory myeloid cells may be CD45+、HLA-DR+、CD14-And BDCA3+. The stimulatory myeloid cells may be CD45+、HLA-DR+、CD11c+And BDCA3+。
Proteins, nucleotides and homologues
Provided herein are methods and compositions for incapacitating and/or detecting non-stimulatory human bone marrow cells that express an NSM protein. In some embodiments, the present invention relates to incapacitating and/or detecting non-stimulatory myeloid cells from non-human mammalian cells that express a homolog of the NSM protein. For example, NSM protein in mice may exhibit a defined expression pattern similar to its human homologue. Accordingly, in one embodiment, provided herein are methods and compositions for incapacitating and/or detecting non-stimulatory mouse bone marrow cells that express an NSM protein. Also provided herein are similar methods and compositions for incapacitating and/or detecting non-stimulatory cells from any individual expressing a homolog of an NSM protein with a similar expression pattern, said cells exhibiting an expression pattern similar to that of said NSM protein.
The NSM protein or nucleotide may include at least one or more of C5AR1, LYVE1, ABCC3, MRC1, SIGLEC1, STAB1, C1QB, C1QA, TMEM37, merk, C1QC, TMEM119, MS4a7, APOE, CYP4F18, TREM2, TLR7, and LILRB4 and their homologs. SDC proteins or nucleotides may include at least one or more of KIT, CCR7, BATF3, FLT3, ZBTB46, IRF8, BTLA, MYCL1, CLEC9A, BDCA3, and XCR1, and homologs thereof. The cell surface NSM protein may comprise at least one or more of TREM2, MS4a7, C5AR1, LYVE1, ABCC3, LILRB4, MRC1/CD206, SIGLEC1, STAB1, TMEM37, merk, and TMEM 119. The cell surface NSM protein may be comprised of one or more anti-TREM 2 antibodies, alone or in combination. Typically, NSM is NSM protein or nucleotide positive and SDC protein or nucleotide negative; in contrast, SDC is typically positive for SDC protein or nucleotide and negative for NSM protein or nucleotide.
The antibodies described herein comprise at least one polypeptide, but they typically comprise a dimer of HC/LC, i.e., four polypeptides. Also described are polynucleotides encoding the polypeptides described herein. Antibodies are typically isolated.
As used herein, "isolated" means that an agent (e.g., a polypeptide or polynucleotide) has been identified and separated from and/or recovered from a component of its native cell culture environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolated also means that the agent has been produced synthetically, e.g., by human intervention.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description relating to the polypeptide applies equally to the description of the peptide and to the description of the protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.
The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. The naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refers to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., the presence of the a carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfide
Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
Also included in the invention are polynucleotides encoding polypeptides of the antibodies. The term "polynucleotide" or "nucleotide sequence" is intended to indicate a contiguous segment of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combinations thereof.
The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more (or all) of the selected codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260:2605-2608 (1985); Rossolini et al, mol.cell.Probes 8:91-98 (1994)).
"conservatively modified variants" applies to both amino acid sequences and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or when the nucleic acids do not encode an amino acid sequence, to substantially identical sequences. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. The nucleic acid variation is a "silent variation" which is a species of conservatively modified variation. Each nucleic acid sequence herein that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each such sequence.
With respect to amino acid sequences, one of ordinary skill in the art will recognize that a single substitution, deletion, or addition of a single amino acid or a small percentage of amino acids in a coding sequence that is altered, added, or deleted from a nucleic acid, peptide, polypeptide, or protein sequence is a "conservatively modified variant" in which the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The conservatively modified variants are those additional to, and do not exclude, polymorphic variants, interspecies homologs, and alleles described herein.
Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (a), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and [0139]8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins: Structures and molecular Properties (W H Freeman & Co., 2 nd edition (12 months 1993)).
The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, means that the two or more sequences or subsequences are the same. Sequences are "substantially identical" if they have a percentage of identical amino acid residues or nucleotides (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region) when compared and aligned for maximum correspondence over a window of comparison or specified region, as measured using one of the following sequence comparison algorithms (or other algorithms available to those of ordinary skill in the art) or by manual alignment and visual inspection. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. This definition also relates to the complement of the test sequence. Identity may be over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75-100 amino acids or nucleotides in length, or when not specified, across the entire sequence of a polynucleotide or polypeptide. Polynucleotides encoding polypeptides of the invention (including homologues from species other than human) may be obtained by a method comprising the steps of: screening the library under stringent hybridization conditions with a labeled probe having a polynucleotide sequence as described herein or a fragment thereof, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan.
For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are entered into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.
As used herein, a "comparison window" includes reference to a segment having any one of a number of consecutive positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein after optimally aligning two sequences, the sequences can be compared to a reference sequence having the same number of consecutive positions. Methods of aligning sequences for comparison are known to those of ordinary skill in the art. The optimal alignment of sequences for comparison can include, but is not limited to, the local homology algorithm by Smith and Waterman (1970) adv.Appl.Math.2:482c, the homology alignment algorithm by Needleman and Wunsch (1970) J.mol.biol.48:443, the similarity search method by Pearson and Lipman (1988) Proc.Nat' l.Acad.Sci.USA 85:2444, the computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package (Genetics computer group,575Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al, Current Protocols in Molecular Biology (1995)).
One example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms described in Altschul et al (1997) Nuc. acids Res.25:3389-3402 and Altschul et al (1990) J.mol.biol.215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National center for biotechnology Information (National center for biotechnology Information) available on the world wide web at ncbi. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the word length (W)11, the expect value (E)10, M-5, N-4, and compares the two chains as defaults. For amino acid sequences, the BLASTP program uses word length 3 and expected value (E)10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) proc. natl. acad. sci. usa 89:10915) alignment values (B)50, expected values (E)10, M-5, N-4, and comparing both strands as defaults. The BLAST algorithm is typically performed with the "low complexity" filter turned off.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-. One similarity measure provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability with which a match will occur by chance between two nucleotide or amino acid sequences. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.
The phrase "selectively (or specifically) hybridizes to" refers to a molecule that binds, duplexes, or hybridizes to a particular nucleotide sequence only under stringent hybridization conditions when that sequence is present in a complex mixture (including, but not limited to, total cell or library DNA or RNA).
The phrase "stringent hybridization conditions" refers to sequences of DNA, RNA, or other nucleic acids, or combinations thereof, that hybridize under conditions of low ionic strength and high temperature as is known in the art. Typically, under stringent conditions, a probe will hybridize to its target subsequence in a complex mixture of nucleic acids (including but not limited to total cell or library DNA or RNA) but not to other sequences in the complex mixture. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to Nucleic acid Hybridization is found in Tijssen, Laboratory Techniques in biochemistry and Molecular Biology- -Hybridization with Nucleic Probes, "Overview of principles of Hybridization and the protocol of Nucleic acids analysis" (1993).
As used herein, the term "engineered" is considered to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, changes to the glycosylation pattern, or modifications to the side chain groups of individual amino acids, as well as combinations of these methods. The engineered proteins are expressed and produced by standard molecular biology techniques.
By "isolated nucleic acid molecule or polynucleotide" is meant a nucleic acid molecule, i.e., DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell, or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule that is normally contained in a cell that contains the polynucleotide molecule, but which is present extrachromosomally, or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include RNA transcripts in vivo or in vitro, as well as both positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids described herein also include such molecules produced synthetically, e.g., by PCR or chemical synthesis. In addition, in certain embodiments, the polynucleotide or nucleic acid includes regulatory elements such as a promoter, ribosome binding site, or transcription terminator.
The term "polymerase chain reaction" or "PCR" generally refers to a method for amplifying a desired nucleotide sequence in vitro, as described, for example, in U.S. patent No. 4,683,195. In general, PCR methods involve repeated cycles of primer extension synthesis using oligonucleotide primers that preferentially hybridize to a template nucleic acid.
To the extent that a nucleic acid or polynucleotide has a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention, this means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, with the exception that the polynucleotide sequence may include up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence, or in one or more contiguous groups within the reference sequence. Indeed, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be routinely determined using known computer programs, such as those discussed above with respect to polypeptides (e.g., ALIGN-2).
A derivative or variant of a polypeptide is said to share "homology" or be "homologous" with the peptide if its amino acid sequence has at least 50% identity with a sequence of 100 amino acids from the original peptide. In certain embodiments, the derivative or variant is at least 75% identical to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 85% identical to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative is at least 90% identical to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% identical to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% identical to a peptide or fragment of a peptide having the same number of amino acid residues as the derivative.
The term "modification" as used herein refers to any change made to a given polypeptide, such as a change in the length, amino acid sequence, chemical structure of the polypeptide, a co-translational modification or a post-translational modification of the polypeptide. The term "(modified)" form means that the polypeptide in question is optionally modified, i.e. the polypeptide in question may be modified or unmodified.
In some aspects, a polypeptide comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a related (e.g., polypeptide and/or antibody) amino acid sequence set forth in one or more tables disclosed herein or set forth in one or more accession numbers disclosed herein, or a fragment thereof. In some aspects, an isolated antibody or protein disclosed herein comprises an amino acid sequence encoded by a polynucleotide that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a related nucleotide sequence set forth in one or more tables disclosed herein or set forth in one or more accession numbers disclosed herein, or a fragment thereof. In some aspects, a nucleotide sequence comprises a nucleotide sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleotide sequence disclosed herein, such as those set forth in one or more tables disclosed herein or set forth in one or more accession numbers disclosed herein.
Pharmaceutical composition
The present application provides compositions comprising antibodies, including pharmaceutical compositions comprising any one or more of the antibodies described herein and one or more pharmaceutically acceptable excipients. In some embodiments, the composition is sterile. The pharmaceutical compositions generally comprise an effective amount of the antibody.
In addition to one or more of the antibodies disclosed herein, these compositions may further comprise pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances well known to those skilled in the art. The substance should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance may depend on the route of administration, e.g. oral, intravenous, transdermal or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may include solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.
For intravenous, transdermal or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art are fully enabled to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired.
Whether a polypeptide, antibody (e.g., an anti-TREM 2 antibody), nucleic acid, small molecule, or other pharmaceutically useful compound is administered to an individual, administration is preferably in a "therapeutically effective amount" or a "prophylactically effective amount" (as appropriate, although prophylaxis may be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. The prescription of treatment, e.g., decisions regarding dosages, etc., is within the responsibility of the ordinary practitioner and other physicians, and generally takes into account the condition to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. (eds.), 1980.
Depending on the condition to be treated, the compositions may be administered alone, or in combination with other treatments, simultaneously or sequentially.
Method of producing a composite material
Preparation method
The antibodies described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567.
In one embodiment, isolated nucleic acids encoding the antibodies described herein are provided. The nucleic acid may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g. a light chain and/or a heavy chain of an antibody) or an amino acid sequence comprising a VHH of a single domain antibody. In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acids are provided. In one embodiment, the nucleic acid is provided in a polycistronic vector. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antigen-binding polypeptide construct, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antigen-binding polypeptide construct and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antigen-binding polypeptide construct. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of antibodies, nucleic acids encoding, for example, antibodies as described above are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).
The term "substantially purified" means that the construct described herein, or variants thereof, may be substantially or essentially free of components normally associated with or interacting with proteins as found in its naturally occurring environment, i.e., native cells or host cells in the case of recombinantly produced heteromultimers, and in certain embodiments, the construct described herein, or variants thereof, is substantially free of cellular material, including preparations of proteins having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) contaminating protein. When the heteromultimer or variant thereof is recombinantly produced by a host cell, in certain embodiments, the protein is present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell. When the heteromultimer or variant thereof is recombinantly produced by a host cell, in certain embodiments, the protein is present in the culture medium at a dry cell weight of about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less. In certain embodiments, a "substantially purified" heteromultimer produced by the methods described herein has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, particularly, at least about 75%, 80%, 85%, and more particularly, at least about 90%, at least about 95%, at least about 99%, or greater, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.
"recombinant host cell" or "host cell" refers to a cell that includes an exogenous polynucleotide regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art to create a recombinant host cell. The exogenous polynucleotide may be maintained in a non-integrating vector, e.g., a plasmid, or may be integrated into the host genome. The host cell may comprise CHO, a CHO derivative, NS0, Sp2O, CV-1, VERO-76, HeLa, HepG2, Per.C6 or BHK.
As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain eukaryotic domain, such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, and the like.
As used herein, the term "prokaryote" refers to a prokaryotic organism. For example, the non-eukaryotic organism may belong to the eubacteria (including but not limited to Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain or archaea (including but not limited to methanococcus jannaschii, Methanobacterium thermoautotrophicum, halobacter salina, such as halobacter halophilus (halox volcanii) and halobacter species NRC-1), archaea (archaoglucoides), Pyrococcus excitans (Pyrococcus aureus), Pyrococcus fuliginosus (Pyrococcus fuliginosus), pyrus fuliginosus (Pyrococcus fuliginosus), pyrenococcus rhodochrous (pyrenococcus rhodochrous), pyrenococcus rhodochrous (pyrum) phylum, etc.).
For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, Methods in Molecular Biology, Vol.248 (compiled by B.K.C.Lo, Humana Press, Totowa, N.J.,2003), pp.245-254, which describes the expression of antibody fragments in E.coli.) after expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.
Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.
Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plantsTMA technique).
Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 strain transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as described, e.g., in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al, AnnalsN.Y.Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful feedsThe mammalian host cell line includes Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, N.J.), pp.255-268 (2003).
In one embodiment, the antibodies described herein are produced in stable mammalian cells by a method comprising: transfecting at least one stable mammalian cell with a nucleic acid encoding an antibody at a predetermined ratio; and expressing the nucleic acid in at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of input nucleic acids that results in the highest percentage of antibodies in the expressed product.
In some embodiments is a method of producing an antibody in a stable mammalian cell as described herein, wherein the expression product of at least one stable mammalian cell comprises a greater percentage of desired glycosylated antibody compared to monomeric heavy or light chain polypeptides or other antibodies.
In some embodiments is a method of producing a glycosylated antibody in a stable mammalian cell as described herein, the method comprising identifying and purifying the desired glycosylated antibody. In some embodiments, the identifying is achieved by one or both of liquid chromatography and mass spectrometry.
If desired, the antibody may be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques including ion exchange, hydrophobic interaction, affinity, size fractionation or gel filtration and reverse phase chromatographic techniques, performed at atmospheric pressure or at elevated pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunization, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques are also useful in conjunction with protein concentration. As is well known in the art, a variety of native protein knotsFc and antibody are synthesized, and these proteins can be used in the present invention for purifying antibodies. For example, bacterial protein a and protein G bind to the Fc region. Likewise, bacterial protein L binds the Fab region of some antibodies. Purification can often be achieved by specific fusion partners. For example, if a GST fusion is employed, the antibody can be purified using glutathione resin, and if a His tag is employed, Ni can be used+2The antibody is purified by affinity chromatography, or if a flag tag is used, an immobilized anti-flag antibody can be used. For general guidance in suitable Purification techniques, see, e.g., Protein Purification: Principles and Practice, 3 rd edition, Scopes, Springer-Verlag, NY,1994, incorporated by reference in its entirety. The degree of purification necessary will vary depending on the use of the antibody. In some cases, purification is not necessary.
In certain embodiments, the antibody is purified using anion exchange chromatography including, but not limited to: Q-Sepharose, DEAE-Sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE column chromatography.
In particular embodiments, the proteins described herein are purified using cation exchange chromatography including, but not limited to: SP-Sepharose, CM Sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns, and equivalents and analogs thereof.
In addition, the antibodies described herein can be chemically synthesized using techniques known in the art (see, e.g., Creighton,1983, Proteins: Structures and Molecular Principles, W.H.Freeman & Co., N.Y. and Hunkapiller et al, Nature,310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide can be synthesized by using a peptide synthesizer. In addition, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions, if desired. Non-canonical amino acids include, but are not limited to, the D-isomer of a common amino acid, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alanine, fluoroamino acids, design amino acids such as methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general. In addition, the amino acid may be a D (dextrorotatory) or L (levorotatory) amino acid.
Application method
In one aspect, the present application provides a method of contacting a non-stimulatory myeloid cell with an anti-TREM 2 antibody, such as a human antibody, which results in incapacitation of the non-stimulatory myeloid cell.
In another aspect, the present application provides a method of contacting a non-stimulatory myeloid cell with a mouse antibody to TREM2, said contacting resulting in incapacitation of said non-stimulatory myeloid cell.
In some embodiments, the non-stimulatory cell is one or more of a DC1 cell and a TAM cell.
In some embodiments, the present application provides a method of incapacitating a non-stimulatory myeloid cell, the method comprising contacting the non-stimulatory myeloid cell with a TREM2 antibody, thereby killing the non-stimulatory myeloid cell. Disabling … … refers to rendering the cell partially or completely non-functional. In some embodiments, incapacitation of non-stimulatory myeloid cells results in induction of growth arrest in the cells. In some embodiments, incapacitation of non-stimulatory myeloid cells results in apoptosis of the cells. In some embodiments, incapacitation of non-stimulatory cells results in lysis of the cells as, for example, by Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Cytotoxicity (ADCC). In some embodiments, incapacitation of non-stimulatory myeloid cells results in necrosis of the cells. In some embodiments, incapacitation of non-stimulatory myeloid cells results in induction of growth arrest in the cells. In some embodiments, disabling the non-stimulatory myeloid cells results in inactivation of the cells. In some embodiments, incapacitation of the non-stimulatory bone marrow cells results in neutralization of TREM2 protein activity in the cells. In some embodiments, incapacitation of non-stimulatory myeloid cells results in decreased proliferation of the cells. In some embodiments, incapacitation of non-stimulatory myeloid cells results in differentiation of the cells. In some embodiments, incapacitation of non-stimulatory myeloid cells results in a decrease in the ability of the cells to function as inhibitory antigen presenting cells, or an increase in the ability of the cells to function as activating antigen presenting cells. In some embodiments, incapacitation of non-stimulatory myeloid cells results in mislocalization of the tumor tissue or cells within the Tumor Microenvironment (TME). In some embodiments, incapacitation of non-stimulatory myeloid cells results in spatial histological alteration of cells within the tumor tissue or tumor microenvironment. In some embodiments, incapacitation of non-stimulatory myeloid cells results in altered temporal expression of cells within the tumor tissue or TME. In some embodiments, the method further comprises removing non-stimulatory myeloid cells.
In any and all aspects of incapacitating non-stimulatory myeloid cells as described herein, any increase or decrease or alteration in one or more characteristics or in an aspect of one or more functions is compared to cells not contacted with an anti-TREM 2 antibody.
In another aspect, the present application provides a method of contacting a non-stimulatory myeloid cell with an anti-TREM 2 antibody, said contacting resulting in modulation of the function of said non-stimulatory myeloid cell. The adjustment may be any one or more of the following. In some embodiments, the non-stimulatory cell is one or more of a DC1 cell, a TAM1 cell, and a TAM2 cell. In some embodiments, modulation of function results in incapacitation of non-stimulatory myeloid cells. In some embodiments, modulation of the function of the non-stimulatory myeloid cells results in an increased ability of the cells to stimulate both native CD8+ T cells and activated CD8+ T cells, e.g., by cross-presenting the non-stimulatory cells to naive CD8+ T cells with a tumor antigen on an mhc i molecule. In some embodiments, modulation increases T cell stimulation function of the non-stimulatory myeloid cells, including, for example, the ability of the cells to trigger T Cell Receptor (TCR) signaling, T cell proliferation, or T cell cytokine production. In one embodiment, the survival of the non-stimulatory cells is reduced, or proliferation of the non-stimulatory cells is reduced. In one embodiment, the ratio of stimulatory to non-stimulatory myeloid cells is increased.
In any and all aspects that reduce the function of non-stimulatory myeloid cells as described herein, any increase or decrease or alteration in one or more characteristics or in an aspect of one or more functions is compared to cells not contacted with an anti-TREM 2 antibody.
In some embodiments, the present application provides a method of killing (also referred to as inducing cell death) non-stimulatory bone marrow cells, the method comprising contacting the non-stimulatory bone marrow cells with an anti-TREM 2 antibody, thereby killing the non-stimulatory bone marrow cells. In some embodiments, killing is increased relative to non-stimulatory bone marrow cells that have not been contacted with an anti-TREM 2 antibody. In some embodiments, the contacting induces apoptosis of the non-stimulatory myeloid cells. In some embodiments, the contacting induces apoptosis of the non-stimulatory myeloid cells. In some embodiments, the non-stimulatory myeloid cells are in an immune cell population comprising both non-stimulatory myeloid cells and stimulatory myeloid cells. In some embodiments, the method further comprises removing non-stimulatory myeloid cells. In some embodiments, 10% to 80% of the cells are killed. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the cells are killed.
In some embodiments, the present application provides a method of increasing the ratio of stimulatory to non-stimulatory bone marrow cells in a population of immune cells comprising stimulatory bone marrow cells and non-stimulatory bone marrow cells, the method comprising contacting the population of immune cells with an anti-TREM 2 antibody. In some embodiments, the ratio is increased relative to a population of cells that have not been contacted with an anti-TREM 2 antibody. In some embodiments, the ratio of DC2 cells to DC1 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1 cells is increased. In some embodiments, the ratio of DC2 cells to TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1+ TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to TAM1+ DC1 cells is increased. In some embodiments, the ratio of DC2 cells to DC1+ TAM2 cells is increased. In some embodiments, the ratio of DC2 cells to DC1+ TAM1+ TAM2 cells is increased. In some embodiments, at least, the ratio is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
In some embodiments, the ratio of stimulatory myeloid cells to non-stimulatory myeloid cells prior to contacting is in the range of 0.001:1 to 0.1: 1. In some embodiments, the ratio of stimulatory myeloid cells to non-stimulatory myeloid cells after contacting is in the range of 0.1:1 to 100: 1.
In some embodiments, the non-stimulatory myeloid cells are reduced in number. In some embodiments, the stimulatory myeloid cells are DC2 cells. In some embodiments, the non-stimulatory myeloid cells are killed, for example, due to necrosis or apoptosis. In some embodiments, the non-stimulatory myeloid cells are induced to undergo growth arrest. In some embodiments, the non-stimulatory myeloid cells no longer proliferate. In some embodiments, the spatial localization of non-stimulatory myeloid cells is altered and the ratio in a specific region of the TME is increased. In some embodiments, the temporal expression of non-stimulatory myeloid cells is altered, and the ratio during a particular time period during tumor development is increased.
In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo. In some particular embodiments, the contacting is performed in vivo in a human. In some embodiments, the contacting is effected by administering an anti-TREM 2 antibody. In some embodiments, the individual (such as a human) receiving the antibody has cancer.
In another aspect, the invention provides a method of treating an immune related disorder (e.g., cancer) in an individual, comprising administering to the individual an effective amount of a composition comprising an anti-TREM 2 antibody. In another aspect, the invention provides a method of enhancing an immune response in an individual, comprising administering to the individual an effective amount of a composition comprising an anti-TREM 2 antibody. In some embodiments, these methods are also provided in combination with other co-therapies, such as PDL blocking therapy, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, CTLA4 blocking therapy, anti-CTLA-4 antibodies, broad checkpoint blocking therapy in which inhibitory molecules on T cells are blocked, adoptive T cell therapy, CAR T cell therapy, dendritic cell or other cell therapy, and conventional chemotherapy.
In some embodiments, the method further comprises determining the expression level of TREM2 protein in a biological sample from the individual. In some embodiments, the biological sample includes, but is not limited to, a bodily fluid, a tissue sample, an organ sample, urine, stool, blood, saliva, CSF, and any combination thereof. In some embodiments, the biological sample is derived from tumor tissue. In some embodiments, the expression level comprises mRNA expression level of mRNA encoding a TREM2 protein. In some embodiments, the expression level of TREM2 protein includes the protein expression level of NSM. In some embodiments, the expression level of TREM2 protein in the sample is detected using a method selected from the group consisting of: FACS, Western blot, ELISA, immunoprecipitation, immunohistochemical analysis, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques and FISH and combinations thereof.
In another aspect, the present application provides a method for determining the presence or absence of non-stimulatory myeloid cells in general, or for determining the presence or absence of specific non-stimulatory myeloid cells (e.g., DC1 cells, TAM1 cells, and/or TAM2 cells), the method comprising: contacting a cell population comprising non-stimulatory bone marrow cells with an anti-TREM 2 antibody; and quantifying the number of non-stimulatory myeloid cells. In another aspect, the present application provides a method for determining the presence or absence of non-stimulatory myeloid cells, the method comprising: contacting a population of immune cells comprising non-stimulatory myeloid cells and stimulatory myeloid cells with an anti-TREM 2 antibody; detecting the complex or moiety indicative of binding of the antibody to the cell, and optionally, quantifying the number of non-stimulatory myeloid cells in the population. In another aspect, a method of determining the relative ratio of non-stimulatory myeloid cells to stimulatory myeloid cells is provided, the method comprising: contacting a population of immune cells comprising non-stimulatory myeloid cells and stimulatory myeloid cells with an anti-TREM 2 antibody; quantifying the number of stimulatory myeloid cells and the number of non-stimulatory myeloid cells; and determining the relative ratio of non-stimulatory myeloid cells to stimulatory myeloid cells.
In embodiments described herein for detection and/or quantification, an anti-TREM 2 antibody binds to a TREM2 protein, but does not necessarily have to affect a biological response such as ADCC, although it may have an effect on a biological response.
In another aspect, the invention provides a method for identifying an individual who is responsive to immunotherapy (e.g., with an anti-TREM 2 antibody) for treating an immune-related disorder (e.g., cancer), the method comprising: detecting the expression level of TREM2 protein in a biological sample from the individual; and determining whether the individual can respond to immunotherapy based on the expression level of TREM2 protein, wherein an elevated level of TREM2 protein in the individual relative to the level of TREM2 protein in a healthy individual is indicative that the individual can respond to immunotherapy. In some embodiments, the methods are also useful for diagnosing an immune related disorder (e.g., cancer) in an individual and are based on the expression level of TREM2 protein, wherein an elevated level of TREM2 protein in the individual relative to the level of TREM2 protein in a healthy individual is indicative of the individual suffering from cancer. In some embodiments, the expression level comprises mRNA expression level of mRNA encoding a TREM2 protein. In other embodiments, the expression level of a TREM2 protein comprises the protein expression level of a TREM2 protein. In some embodiments, the expression level of TREM2 protein in the sample is detected using a method selected from the group consisting of: FACS, Western blot, ELISA, immunoprecipitation, immunohistochemical analysis, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques and FISH and combinations thereof. In these embodiments, an anti-TREM 2 antibody binds to a TREM2 protein, but does not necessarily have to affect a biological response such as ADCC. In some embodiments, the biological sample is derived from tumor tissue. In some embodiments, the biological sample includes, but is not limited to, a bodily fluid, a tissue sample, an organ sample, urine, stool, blood, saliva, CSF, and any combination thereof.
Also disclosed herein is a method of enhancing the immune response of a subject to a tumor or enhancing the efficacy of an immunotherapy treatment. In general, treatment that increases the abundance of SDC will improve subject outcomes, such as relapse-free survival time, and will enhance the efficacy of cancer immunotherapy treatment. Treatment can increase the relative or absolute abundance of SDC cells in a tumor in a subject. The treatment may result in a decrease in the relative or absolute abundance of NSM cells in the tumor of the subject.
An exemplary method of general therapeutic strategy includes increasing the number of SDC by systemic introduction of Flt 3L. Another approach is to treat autologous bone marrow or blood cells of the subject with Flt3L while blocking CSF 1. Expression of SDC transcription factors such as IRF8, Mycl1 or BATF3 or ZBTB46 in a population of bone marrow or blood progenitor cells, for example, by retroviruses, may also be used to drive SDC development. Another therapeutic strategy involves the systemic elimination of NSM cells, with selective privilege of SDC. This can produce an overall advantageous change in the ratio of these populations. Elimination of NSM cells can be achieved by any means, including administration (systemic administration or localized administration to the tumor) of an antibody against a TREM2 surface protein.
In some embodiments, a treatment that enhances SDC is applied as a therapeutic treatment to better enable the subject's innate immune system to control or eradicate the cancer. In another embodiment, treatment of enhanced SDC of the invention is applied in combination with a therapeutic treatment, such as an immunotherapy treatment (either prior to, concurrent with, or subsequent to the immunotherapy treatment), wherein the treatment of enhanced SDC serves as an adjunct or adjuvant treatment to increase the efficacy of the therapeutic treatment.
Application method
In some embodiments, the methods provided herein can be used to treat an immune related disorder in an individual. In one embodiment, the individual is a human and the antibody is a TREM2 antibody. In another embodiment, the individual is a mouse and the antibody is a TREM2 antibody.
In some embodiments, for in vivo administration of an anti-TREM 2 antibody described herein, the normal dose may vary from about 10ng per kg body weight of the individual per day up to about 100mg or more, preferably from about 1 mg/kg/day to 10 mg/kg/day, depending on the route of administration. For repeated administrations over several days or longer, depending on the severity of the disease or condition to be treated, treatment is continued until the desired suppression of symptoms is achieved. An exemplary dosing regimen includes administration of an initial dose of about 2mg/kg of anti-TREM 2 antibody followed by a weekly maintenance dose of about 1mg/kg every other week. Other dosage regimens may be useful depending on the pharmacokinetic decay pattern that the physician wishes to achieve. For example, once-a-week to twenty-once dosing of a subject is contemplated herein. In certain embodiments, administrations in the range of about 3 μ g/kg to about 2mg/kg (such as about 3 μ g/kg, about 10 μ g/kg, about 30 μ g/kg, about 100 μ g/kg, about 300 μ g/kg, about 1mg/kg, and about 2mg/kg) may be used. In certain embodiments, the frequency of administration is three times daily, twice daily, once every other day, once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or more. The progress of therapy is readily monitored by conventional techniques and assays. The dosing regimen, including the administration of the anti-TREM 2 antibody, can vary over time independently of the dose used.
In some embodiments, the methods provided herein (such as methods of enhancing an immune response or effecting a loss of ability of non-stimulatory bone marrow cells) can be used to treat cancer, and thus, an individual receiving an anti-TREM 2 antibody or an anti-TREM 2 antibody has cancer.
Any suitable cancer may be treated with the antibodies provided herein. The cancer may be any carcinoma, adenocarcinoma, soft tissue carcinoma, sarcoma, teratoma, melanoma, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or brain cancer known in the medical arts. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is immune-evasive. In some embodiments, the cancer is immune-responsive. In some embodiments, the cancer is melanoma, renal cancer, liver and gall bladder cancer, Head and Neck Squamous Carcinoma (HNSC), pancreatic cancer, colon cancer, bladder cancer, glioblastoma, prostate cancer, lung cancer, breast (breast) cancer, ovarian cancer, gastric cancer, kidney cancer, bladder cancer, esophageal cancer, kidney cancer, melanoma, leukemia, lymphoma, or mesothelioma. In some embodiments, the cancer is colon, pancreatic, or breast cancer.
In some embodiments, the immune-related disorder is an immune-related disorder associated with expression of a TREM2 protein on non-stimulatory bone marrow cells (in humans) or expression of a homolog of a TREM2 protein in a non-human species. In some embodiments, the immune-related disorder is an immune-related disorder associated with overexpression of TREM2 protein on non-stimulatory bone marrow cells as compared to stimulatory bone marrow cells. In some embodiments, the overexpression of TREM2 mRNA or TREM2 protein is about at least 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, or 100-fold higher compared to stimulatory bone marrow cells.
In some embodiments, the treatment enhances the immune response in the subject. In some embodiments, the enhanced immune response is an adaptive immune response. In some embodiments, the enhanced immune response is an innate immune response.
In some embodiments, the antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of an anti-TREM 2 antibody can be administered to treat cancer. An appropriate dosage of anti-TREM 2 antibody can be determined based on the type of cancer to be treated, the type of anti-TREM 2 antibody, the severity and course of the cancer, the clinical status of the individual, the clinical history and response to treatment of the individual, and the judgment of the attending physician.
Combination therapy
In some embodiments, the antibodies provided herein are administered with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered with the antibodies provided herein. In some embodiments, the immunotherapy is selected from checkpoint inhibitors; checkpoint inhibitors of T cells; an anti-PD 1 antibody; anti-PDL 1 antibody; anti-CTLA 4 antibodies; adoptive T cell therapy; CAR-T cell therapy; a dendritic cell vaccine; a monocyte vaccine; an antigen binding protein that binds both T cells and antigen presenting cells; a BiTE double antigen binding protein; a toll-like receptor ligand; a cytokine; (ii) cytotoxic therapy; chemotherapy; a cytostatic agent; radiotherapy; a small molecule inhibitor; a small molecule agonist; an immunomodulator; and epigenetic modulators, combinations thereof.
In some embodiments, the additional therapeutic agent is an antibody. In some embodiments, the additional therapeutic agent is an antibody that binds to one or more proteins on the surface of the tumor cell.
For the treatment of cancer, an anti-TREM 2 antibody can be combined with one or more antibodies that inhibit an immune checkpoint protein. Of particular interest are immune checkpoint proteins displayed on the surface of tumor cells. The immune checkpoint receptors that have been most actively studied in the context of clinical cancer immunotherapy, namely cytotoxic T lymphocyte-associated antigen 4(CTLA 4; also known as CD152) and programmed cell death protein 1(PD 1; also known as CD279), are both inhibitory receptors. The clinical activity of antibodies blocking any of these receptors suggests that anti-tumor immunity can be enhanced at multiple levels, and combinatorial strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.
Two ligands for PD-1 are PD-1 ligand 1 (PD-L1; also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD 273). PD-L1 is expressed on cancer cells and by binding to its receptor on T cells PD-1, it inhibits T cell activation/function. Inhibitors that block the interaction of PD-1 with its cognate ligands PD-L1 and PD-L2 on cancer cells can result in an increase in both T cell activation and function and prevent cancer cells from evading the immune system.
In some embodiments, the immunotherapy is an agent that interferes with the binding of PD-1 and PD-L1 or PD-L2. In some embodiments, the immunotherapy is an anti-PD 1 antibody. In some embodiments, the immunotherapy is an anti-PD-L1 antibody. In some embodiments, the immunotherapy is an anti-PD-L2 antibody.
Various PD-1, PD-L1 and PD-L2 antibodies are known in the art. In some embodiments, the additional therapeutic agent is at least one of: atelizumab (Atezolizumab) (PD-L1), aviluzumab (aveluumab) (PD-L1), dolacizumab (Durvalumab) (PD-L1), Nivolumab (Nivolumab) (PD-1), pamlizumab (Pembrolizumab) (PD-1), cimiraprizumab (cemipimab) (PD-1), yiprizumab (Ipilimumab) (CTLA-4), Tremelimumab (Tremelimumab) (CTLA-4), or any combination thereof.
The additional therapeutic agent may be administered by any suitable means. In some embodiments, the antibody provided herein and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, the antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions.
In embodiments where the antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can occur prior to, concurrently with, and/or after administration of the additional therapeutic agent. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent occurs within about one month of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent occurs within about one week of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent occurs within about one day of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent occurs within about twelve hours of each other. In some embodiments, administration of the antibody provided herein and the additional therapeutic agent occurs within about one hour of each other.
Kit and article
The present application provides kits comprising any one or more of the antibody compositions described herein. In some embodiments, the kit further comprises a component selected from any one of a secondary antibody, a reagent for immunohistochemical analysis, a pharmaceutically acceptable excipient, and an instruction manual, and any combination thereof. In a particular embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein and one or more pharmaceutically acceptable excipients.
The present application also provides an article of manufacture comprising any of the antibody compositions or kits described herein. Examples of articles of manufacture include vials (including sealed vials).
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