Anti-MICA and / or MICB antibodies and uses thereof

Abstract

The present invention provides antibodies that specifically bind to human MICA / B and methods of using the same. In some aspects, the present invention relates to methods of treating cancer in a subject comprising administering to the subject an anti-MICA / B antibody.

Description

[0001] References to sequence lists submitted electronically via EFS-WEB

[0002] The contents of the sequence list submitted electronically in an ASCII text file (name: 3338_101PC02_Seqlisting_ST25.txt; size: 154,660 bytes; and creation date: March 18, 2019) filed together with this application are incorporated herein by reference in their entirety. Background of the Invention

[0004] Major histocompatibility complex class I chain-associated A and B (MICA / B) are highly polymorphic cell surface proteins associated with MHC class I glycoproteins. They contain three extracellular domains (α1, α2, and α3), a transmembrane region, and a cytoplasmic tail region, and act as ligands to stimulate the activating receptor NKG2D expressed on NK, γδT, CD8+αβT, and NKT cells. Normally, MICA / B is constitutively expressed at low levels on epithelial cells, endothelial cells, fibroblasts, and myeloid cells. However, it is upregulated or de novo expressed under stress conditions (such as during carcinogenesis, infection, and DNA damage responses) and in autoimmune diseases. Therefore, MICA / B expression acts as a signaling mechanism for cellular stress, and MICA / B binding to NKG2D triggers NK cells and co-stimulates T cells, leading to immune effector functions such as cytotoxicity and cytokine production. (Bauer, Science 1999; 285:727-9). Diefenbach et al., Nat Immunol. 2000; 1:119-26. Groh et al., Nat Immunol. 2001; 2:255-60. However, tumor cells possess a mechanism to inhibit NKG2D-mediated responses by cleaving MICA / B from the cell surface, resulting in decreased MICA / B surface density and the production of soluble MICA / B (sMICA / B), which downregulates NKG2D on cytotoxic effector cells. Groh et al., Nature 2002; 419:734-8. Salih, J. Immunol. 2002; 169:4098-4102. In tumor cells, the α3 domain of MICA / B interacts with a disulfide isomerase / molecular chaperone, endoplasmic reticulum protein 5 (ERp5), to induce a conformational change in MICA / B that can be cleaved by proteases, including integrin and metalloproteinases ADAM10 and ADAM17. Kaiser, Nature 2007; 447:482-6.

[0005] Currently, approximately 100 MICA alleles encoding 79 protein variants and 40 MICB alleles encoding 26 protein variants have been identified (EMBL-EBI Immunomorphism Database: https: / / www.ebi.ac.uk / ipd / imgt / hla / stats.html (accessed December 8, 2017)). MICA / B expression has been reported in a wide range of tumor types, with high expression associated with poor prognosis. Spear, Cancer Immunity 2013; 13. Ghadially et al., Br.J. Cancer 2017; 116:1208-17. Because long-term exposure to high concentrations of sMICA has been shown to reduce NKG2D activity on CD8 T and NK cells, sMICA / B production has been considered a mechanism for tumor immune escape. Groh et al., Nat. Immunol. 2001; 2:255-60. Salih, J. Immunol. 2002; 169:4098-4102. sMICA / B proteins have been identified in many human tumor types, including melanoma, lung, colon, ovarian, breast, prostate tumors, and myeloma. Zhang et al., Sci. Adv. 2017; 3:e1602133. sMICA levels are associated with poorer survival in patients with metastatic melanoma treated with the anti-CTLA-4 antibody ipilimumab. Koguchi, Cancer Res. 2015; 75:5084-92. Furthermore, melanoma patients responsive to ipilimumab have shown therapy-induced antiMICA / B antibodies; some of these patients have experienced decreased sMICA levels and exhibited antitumor activity. Zhang et al., Sci. Adv. 2017; 3:e1602133. Jinushi, PNAS. 2006; 103: 9190-9195.

[0006] The extent to which viruses and tumors devise mechanisms to evade NKG2D surveillance indicates the potential benefit of therapeutic strategies that restore or enhance NKG2D-dependent activation of NK and T cells. Therefore, there is a need to generate and select anti-MICA / B antibodies that bind to regions of MICA / B near the ERp5 binding site and / or retain MICA / B on the cell surface to enable efficient NKG2D binding, thereby eliciting enhanced antitumor activity in patients. Invention Overview

[0008] One aspect of the present invention relates to an antibody that specifically binds to human MHC class I peptide-associated sequence A (MICA) and / or human MHC class I peptide-associated sequence B (MICB), comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises VH complementarity-determining region (CDR) 1, VH-CDR2, and VH-CDR3, and the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence selected from SEQ ID NO: 7, 17, 27, 37, and 47. In some embodiments, VH-CDR2 comprises an amino acid sequence selected from SEQ ID NO: 6, 16, 26, 36, and 46. In some embodiments, VH-CDR1 comprises an amino acid sequence selected from SEQ ID NO: 5, 15, 25, 35, and 45. In some embodiments, VL-CDR1 comprises an amino acid sequence selected from SEQ ID NO: 8, 18, 28, 38, and 48. In some embodiments, VL-CDR2 comprises an amino acid sequence selected from SEQ ID NO: 9, 19, 29, 39, and 49. In some embodiments, VL-CDR3 comprises an amino acid sequence selected from SEQ ID NO: 10, 20, 30, 40, and 50.

[0009] In some embodiments, (a) VH-CDR1 comprises the amino acid sequence described in SEQ ID NO:5, VH-CDR2 comprises the amino acid sequence described in SEQ ID NO:6, VH-CDR3 comprises the amino acid sequence described in SEQ ID NO:7, VL-CDR1 comprises the amino acid sequence described in SEQ ID NO:8, VL-CDR2 comprises the amino acid sequence described in SEQ ID NO:9, and VL-CDR3 comprises the amino acid sequence described in SEQ ID NO:10; (b) VH-CDR1 comprises the amino acid sequence described in SEQ ID NO:15, VH-CDR2 comprises the amino acid sequence described in SEQ ID NO:16, VH-CDR3 comprises the amino acid sequence described in SEQ ID NO:17, VL-CDR1 comprises the amino acid sequence described in SEQ ID NO:18, VL-CDR2 comprises the amino acid sequence described in SEQ ID NO:19, and VL-CDR3 comprises the amino acid sequence described in SEQ ID NO:20; (c) VH-CDR1 comprises the amino acid sequence described in SEQ ID NO:5. The amino acid sequence described in NO:25, VH-CDR2 contains the amino acid sequence described in SEQ ID NO:26, VH-CDR3 contains the amino acid sequence described in SEQ ID NO:27, VL-CDR1 contains the amino acid sequence described in SEQ ID NO:28, VL-CDR2 contains the amino acid sequence described in SEQ ID NO:29, and VL-CDR3 contains the amino acid sequence described in SEQ ID NO:30; (d) VH-CDR1 contains the amino acid sequence described in SEQ ID NO:35, VH-CDR2 contains the amino acid sequence described in SEQ ID NO:36, VH-CDR3 contains the amino acid sequence described in SEQ ID NO:37, VL-CDR1 contains the amino acid sequence described in SEQ ID NO:38, VL-CDR2 contains the amino acid sequence described in SEQ ID NO:39, and VL-CDR3 contains the amino acid sequence described in SEQ ID NO:40; or (e) VH-CDR1 contains the amino acid sequence described in SEQ ID NO:45, VH-CDR2 contains the amino acid sequence described in SEQ ID NO:26, VH-CDR3 contains the amino acid sequence described in SEQ ID NO:27, VL-CDR1 contains the amino acid sequence described in SEQ ID NO:28, VL-CDR2 contains the amino acid sequence described in SEQ ID NO:29, and VL-CDR3 contains the amino acid sequence described in SEQ ID NO:40; The amino acid sequence described in NO:46, VH-CDR3 contains the amino acid sequence described in SEQ ID NO:47, VL-CDR1 contains the amino acid sequence described in SEQ ID NO:48, VL-CDR2 contains the amino acid sequence described in SEQ ID NO:49, and VL-CDR3 contains the amino acid sequence described in SEQ ID NO:50.

[0010] In some embodiments, the antibody has one or more properties selected from the following: (a) the antibody inhibits the shedding of MICA from tumor cells; (b) the antibody increases membrane-bound MICA on tumor cells; (c) the antibody reduces the level of soluble MICA in the patient's serum; (d) the antibody mediates enhanced ADCC and / or ADCP; (e) the antibody mediates enhanced antigen processing and / or cross-presentation on cells; (f) the antibody inhibits tumor growth and / or cancer metastasis; (g) the antibody reduces tumor volume; (h) the antibody increases progression-free survival; (i) the antibody increases overall survival; and (j) any combination thereof.

[0011] In some embodiments, VH comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:2, 12, 22, 32, and 42. In some embodiments, VH comprises an amino acid sequence selected from SEQ ID NO:2, 12, 22, 32, and 42.

[0012] In some embodiments, VL comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:4, 14, 24, 34, and 44. In some embodiments, VL comprises an amino acid sequence selected from SEQ ID NO:4, 14, 24, 34, and 44.

[0013] In some embodiments, (a) VH comprises the amino acid sequence described in SEQ ID NO:2 and VL comprises the amino acid sequence described in SEQ ID NO:4; (b) VH comprises the amino acid sequence described in SEQ ID NO:12 and VL comprises the amino acid sequence described in SEQ ID NO:14; (c) VH comprises the amino acid sequence described in SEQ ID NO:22 and VL comprises the amino acid sequence described in SEQ ID NO:24; (d) VH comprises the amino acid sequence described in SEQ ID NO:32 and VL comprises the amino acid sequence described in SEQ ID NO:34; or (e) VH comprises the amino acid sequence described in SEQ ID NO:42 and VL comprises the amino acid sequence described in SEQ ID NO:44.

[0014] In some embodiments, (a) the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:58 and a light chain comprising the amino acid sequence described in SEQ ID NO:60; (b) the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:130 and a light chain comprising the amino acid sequence described in SEQ ID NO:60; (c) the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:62 and a light chain comprising the amino acid sequence described in SEQ ID NO:64; (d) the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:66 and a light chain comprising the amino acid sequence described in SEQ ID NO:68; or (e) the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:70 and a light chain comprising the amino acid sequence described in SEQ ID NO:72.

[0015] In some embodiments, as determined by yeast surface display, the antibody binds to an epitope on the human MICA comprising one or more amino acid residues selected from the following in the human MICA: G254, D255, L257, Y264, W267, and any combination thereof corresponding to SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the antibody binds to an epitope on the human MICA comprising G254, D255, L257, Y264, and W267 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the antibody binds to an epitope on the human MICA comprising amino acid residues W150-M163 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the antibody binds to an epitope on the human MICA comprising amino acid residues Y231-T238 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the antibody binds to an epitope on the human MICA containing amino acid residues D255-Q265 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the epitope contains amino acid residues W253-W267 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the antibody binds to an epitope on the human MICA containing L201-N220 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the antibody binds to an epitope on the human MICA containing T238-Q252 corresponding to SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the antibody binds to epitopes on the human MICA, including L201-N220 and T238-Q252 corresponding to SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the antibody binds to epitopes on the human MICA, including R240, Q241, D242, V244, and R279 corresponding to SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the antibody binds to epitopes on the human MICA, including P258, G260, G262, and Y264 corresponding to SEQ ID NO:51.In some embodiments, such as those determined by X-ray cocrystallography, the antibody binds to an epitope on the human MICA that comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some implementations, such as those determined by X-ray cocrystallography, the antibody binds to an epitope on the human MICA that comprises at least two residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some implementations, such as those determined by X-ray cocrystallography, the antibody binds to an epitope on the human MICA containing at least four residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some implementations, such as those determined by X-ray cocrystallography, the antibody binds to an epitope on the human MICA containing at least six residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some implementations, such as those determined by X-ray cocrystallography, the antibody binds to epitopes on the human MICA, including T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269.

[0016] In some embodiments, the antibody is selected from IgG1, IgG2, IgG3, IgG4, or variants thereof. In some embodiments, the antibody is non-fucosylated. In some embodiments, the antibody includes a constant region in the heavy chain, wherein the constant region contains a G-to-A mutation at the position corresponding to residue 234 in SEQ ID NO:58.

[0017] Another aspect of the present invention relates to an antibody that specifically binds to human MHC class I peptide-associated sequence A (MICA) and / or human MHC class I peptide-associated sequence B (MICB), comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity-determining region (CDR) 1, VH-CDR2, and VH-CDR3, and the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; wherein the VH-CDR1 comprises the amino acid sequence described in SEQ ID NO:5, the VH-CDR2 comprises the amino acid sequence described in SEQ ID NO:6, the VH-CDR3 comprises the amino acid sequence described in SEQ ID NO:7, the VL-CDR1 comprises the amino acid sequence described in SEQ ID NO:8, the VL-CDR2 comprises the amino acid sequence described in SEQ ID NO:9, and the VL-CDR3 comprises the amino acid sequence described in SEQ ID NO:10; and wherein the antibody is non-fucosylated. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO:2, and VL comprises the amino acid sequence described in SEQ ID NO:4. In some embodiments, the antibody includes a constant region in the heavy chain, which contains a G-to-A mutation at position corresponding to residue 234 in SEQ ID NO:58. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:58 and a light chain comprising the amino acid sequence described in SEQ ID NO:60. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:130 and a light chain comprising the amino acid sequence described in SEQ ID NO:60. In some embodiments, the antibody is non-fucosylated. In some embodiments, the antibody is hypofucosylated.

[0018] Some aspects of the present invention relate to an antibody that specifically binds to human MHC class I peptide-associated sequence A (MICA) and / or human MHC class I peptide-associated sequence B (MICB), comprising a heavy chain containing the amino acid sequence described in SEQ ID NO:58 and a light chain containing the amino acid sequence described in SEQ ID NO:60, wherein the antibody is non-fucosylated.

[0019] In some implementations, antibodies are produced by cell lines with reduced or eliminated expression of fucosyltransferase.

[0020] Some aspects of this invention relate to a polynucleotide or polynucleotide group encoding an antibody described herein. Other aspects of this invention relate to a vector or vector group comprising the polynucleotide or polynucleotide group. Still other aspects of this invention relate to a host cell comprising the antibody, polynucleotide or polynucleotide group, or vector.

[0021] Some aspects of this invention relate to an immunoconjugate comprising the antibody and a therapeutic agent. In some embodiments, the therapeutic agent is selected from the following: cytotoxins, non-cytotoxic drugs, radioactive agents, secondary antibodies, enzymes, anti-hypertrophic agents, and any combination thereof. In some embodiments, the cytotoxin is selected from the following: dolastatin, monomethylolpropionate E (MMAE), maytansine, duocarmycin, calicheamicin, pyrrolobenzodiazepine, duocarmycin, centanamycin, SN38, doxorubicin, derivatives thereof, synthetic analogs thereof, and any combination thereof. In some embodiments, the cytotoxin comprises cytotoxin A.

[0022] Some aspects of this invention relate to pharmaceutical compositions comprising the antibody, a polynucleotide or group of polynucleotides, a carrier or group of carriers or an immunoconjugate, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a second antibody. In some embodiments, the second antibody specifically binds to a protein selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-8, IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, and any combination thereof.

[0023] Some aspects of the present invention relate to methods of treating cancer in an individual in need, comprising administering to the subject an antibody, a polynucleotide or polynucleotide group, a vector or vector group, a host cell, an immune conjugate, or a pharmaceutical composition. In some embodiments, the cancer comprises a tumor. In some embodiments, the method comprises administering a second therapy to the subject. In some embodiments, the second therapy comprises an effective amount of an antibody that specifically binds to proteins selected from: inducible T cell costimulator (ICOS), CD137 (4-1BB), CD134 (OX40), NKG2A, CD27, CD96, glucocorticoid-induced TNFR-related protein (GITR) and herpesvirus entry mediator (HVEM), programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), CTLA-4, B and T lymphocyte attenuators (BTLA), T cell immunoglobulin and mucin domain-3 (TIM). -3), lymphocyte activation gene-3 (LAG-3), adenosine A2a receptor (A2aR), cytotoxic lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immune receptor with Ig and ITIM domains (TIGIT) and receptor for the V domain Ig repressor (VISTA) for T cell activation, KIR, TGFβ, IL-10, IL-8, IL-2, B7-H4, Fas ligand, CXCR4, mesothelin, CEACAM-1, CD52, HER2, and any combination thereof. In some embodiments, the second therapy comprises an effective amount of pegylated IL-10, IL-10-Fc, or pegylated IL-2. In some embodiments, the second therapy comprises a chemotherapy agent for cancer, radiation, an agent that activates innate immune cells, and / or an agent that enhances the survival of NK and / or CD8+ T cells.

[0024] Other features and advantages of the invention will become apparent from the following detailed description and embodiments, which should not be considered limiting. All references cited herein (including scientific articles, newspaper reports, GenBank entries, patents, and patent applications cited throughout this application) are expressly incorporated herein by reference. Brief description of the attached diagram

[0026] Figures 1A-1D For the anti-MICA / B antibody MICA.36(19G6) heavy chain variable region ( Figures 1A-1B Nucleotides: SEQ ID NO:1; Amino acids: SEQ ID NO:2) and light chain variable regions ( Figures 1C-1D Sequence alignment of nucleotide (SEQ ID NO:3); amino acid (SEQ ID NO:4). Figure 1A and Figure 1C Demonstrating the heavy chain variable region of MICA.36(19G6) Figure 1A ) and light chain variable region ( Figure 1C The nucleotide sequence and corresponding amino acid sequence of ). Figure 1B and Figure 1D Display the variable region of the heavy chain ( Figure 1B ) and light chain variable region ( Figure 1D The phylogenetic sequence relative to the MICA.36(19G6) (“input”) sequence. The complementarity-determining regions (CDRs) identified by the Kabat number are marked with horizontal lines and accordingly annotated. Figures 1A-1D ).

[0027] Figures 2A-2F For the anti-MICA / B antibody MICA.52(16A5) heavy chain variable region ( Figures 2A-2B Nucleotide: SEQ ID NO:11; Amino acid: SEQ ID NO:12), Light chain variable region 1 ( Figures 2C-2D ; Nucleotide SEQ ID NO:13; Amino acid: SEQ ID NO:14) and light chain variable region 2 ( Figures 2E-2F Sequence alignment of nucleotides (SEQ ID NO:127; amino acids (SEQ ID NO:128)). Figure 2A , Figure 2C and Figure 2E Displaying the 16A5 heavy chain variable region of the anti-MICA / B antibody ( Figure 2A ) and light chain variable regions 1 (MICA.52) and 2 (MICA.53) (respectively) Figure 2C and Figure 2E The nucleotide sequence and corresponding amino acid sequence of ). Figure 2B , Figure 2D and Figure 2F Display the variable region of the heavy chain ( Figure 2B ) and light chain variable regions 1 and 2 ( Figure 2D and Figure 2F Phylogenetic sequences relative to MICA.52 or MICA.53(16A5) (“input”) sequences. Complementarity-determining regions (CDRs) identified by Kabat numbering are marked with horizontal lines and annotated accordingly. Figures 2A-2F ).

[0028] Figures 3A-3D For the anti-MICA / B antibody MICA.54(24G11) heavy chain variable region ( Figures 3A-3B Nucleotides: SEQ ID NO:21; Amino acids: SEQ ID NO:22) and light chain variable regions ( Figures 3C-3D Sequence alignment of nucleotide (SEQ ID NO:23); amino acid (SEQ ID NO:24). Figure 3A and Figure 3C Demonstrates the heavy chain variable region of the anti-MICA / B antibody MICA.54(24G11). Figure 3A ) and light chain variable region ( Figure 3C The nucleotide sequence and corresponding amino acid sequence of ). Figure 3B and Figure 3D Display the variable region of the heavy chain ( Figure 3B ) and light chain variable region ( Figure 3D The phylogenetic sequence relative to the MICA.54(24G11) (“input”) sequence. The complementarity-determining regions (CDRs) identified by the Kabat number are marked with horizontal lines and accordingly annotated. Figures 3A-3D ).

[0029] Figures 4A-4D For the anti-MICA / B antibody MICA.2(3F5) heavy chain variable region ( Figures 4A-4B Nucleotides: SEQ ID NO:31; Amino acids: SEQ ID NO:32) and light chain variable regions ( Figures 4C-4D Sequence alignment of nucleotide (SEQ ID NO:33); amino acid (SEQ ID NO:34). Figure 4A and Figure 4C Demonstrating the MICA.2(3F5) heavy chain variable region of the anti-MICA / B antibody ( Figure 4A ) and light chain variable region ( Figure 4C The nucleotide sequence and corresponding amino acid sequence of ). Figure 4B and Figure 4D Display the variable region of the heavy chain ( Figure 4B ) and light chain variable region ( Figure 4D The phylogenetic sequence relative to the MICA.2(3F5) (“input”) sequence. The complementarity-determining regions (CDRs) identified by the Kabat number are marked with horizontal lines and accordingly annotated. Figures 4A-4D ).

[0030] Figures 5A-5D For the 71C2 heavy chain variable region of anti-MICA / B antibody ( Figures 5A-5B Nucleotides: SEQ ID NO:41; Amino acids: SEQ ID NO:42) and light chain variable regions ( Figures 5C-5D Sequence alignment of nucleotide (SEQ ID NO:43); amino acid (SEQ ID NO:44). Figure 5A and Figure 5C Displaying the 71C2 heavy chain variable region of the anti-MICA / B antibody ( Figure 5A ) and light chain variable region ( Figure 5C The nucleotide sequence and corresponding amino acid sequence of ). Figure 5B and Figure 5D Display the variable region of the heavy chain ( Figure 5B ) and light chain variable region ( Figure 5D The phylogenetic sequence relative to the 71C2 (“input”) sequence. The complementarity-determining regions (CDRs) identified by Kabat numbering are marked with horizontal lines and accordingly annotated. Figures 5A-5D ).

[0031] Figures 6A-6H As measured by surface plasmon resonance (SPR), the anti-MICA / B antibody MICA.36 ( Figures 6A-6D ) and MICA.38 ( Figures 6E-6H ) for the MICA allele MICA*002 ( Figure 6A and Figure 6E ), MICA*004 ( Figure 6B and Figure 6F ), MICA*008 ( Figure 6C and Figure 6G ) and MICA*009 ( Figure 6D and Figure 6H A graphical representation of the binding affinity dynamics of ).

[0032] Figures 6I-6L As measured using a Biacore 3000 instrument via SPR, the anti-MICA / B antibody MICA.2 ( Figure 6I ), 16A5 ( Figure 6J ), 19G6 Figure 6K ) and 71C2 ( Figure 6L Graphical representation of the binding affinity kinetics of MICA alleles MICA*002, MICA*004, MICA*008 and MICA*009.

[0033] Figures 7A-7G This is a graphical representation of the in vitro specific binding activity of MICA.36 to MICA / B antigen expressed by cells, as analyzed using a Scatchard plot. Figure 7A A standard curve is plotted for the titrated antibody concentration (x-axis; nM antibody) against the association count per minute (y-axis; CPM). Figures 7B-7F Demonstrating the interaction between MICA.36 and human MICA*008 overexpressed in Chinese hamster ovary (CHO) cells. Figure 7B ), Human MICB*005 ( Figure 7C ) and cynomolgus monkey MICA / B (clones #4 and #6) Figures 7D-7E ); and in 786-O cells ( Figure 7F ) and SW480 cells ( Figure 7G Specific binding, total binding, and non-specific binding (NSB) of endogenously expressed human MICA / B on the surface of the virus.

[0034] Figures 8A-8N This demonstrates the binding affinity of MICA / B antibodies to common MICA or MICB alleles expressed in various cell lines, obtained using fluorescence activated cell sorting (FACS). Figure 8A , Figure 8D , Figure 8F , Figure 8H , Figure 8J and Figure 8L The results show that at increasing antibody concentrations, 19G6(8A) and 24G11( Figure 8D ), 71C2 ( Figure 8F ), 16A5 ( Figure 8H and Figure 8J (two subclones) and MICA.2 ( Figure 8L It binds to the MICA / B antigens MICA*002 (triangle), MICA*004 (inverted triangle), MICA*008 (hollow rhombus), MICA*009 (solid square), MICA*010 (solid circle) and MICB (hollow square) expressed on the surface of CHO cells. Figure 8B , Figure 8E , Figure 8G , Figure 8I , Figure 8K and Figure 8M Showcase 19G6(8B), 24G11( Figure 8E ), 71C2 ( Figure 8G ), 16A5 ( Figure 8I and Figure 8K ) and MICA.2 ( Figure 8M The binding of MICA-expressing cell lines 786-O (square), A2058 (triangular), HeLa (inverted triangular) and RPMI-8226 (round). Figure 8C and Figure 8N Compared to the negative control (CHO), 19G6 ( Figure 8C ) and MICA.2 ( Figure 8N Cross-reactivity with cynomolgus monkey MICA / B expressed on the surface of CHO cell clones #4 and #6.

[0035] Figures 9A-9F The results of epitope localization of various anti-MICA / B antibodies using hydrogen / deuterium exchange (HDX) mass spectrometry (MS) are presented. Figures 9A-9E As measured using mass spectrometry, MICA is compared with mAb MICA.2 ( Figure 9A ), MICA.39 ( Figure 9B ), MICA.40 ( Figure 9C ), and non-fucosylated MICA.36 with Fc containing G236A. Figure 9D ) and MICA.36 ( Figure 9E Graphical representation of the differences after interaction in HDX. Figure 9F A band diagram of the crystal structure of human MICA bound to the natural killer cell receptor (NKG2D), highlighting the located MICA.36 epitope.

[0036] Figures 10A-10F The results of epitope localization of various anti-MICA / B antibodies using yeast are shown. Figures 10A-10C Showcase MICA.36 ( Figure 10A ), MICA.2 ( Figure 10B ) and 24G11 ( Figure 10C Each epitope of ). Figures 10D-10F Demonstration for MICA.2 ( Figure 10D ), MICA.36 Figure 10E ) and 24G11 ( Figure 10F The same epitopes are superimposed on the band diagram of the crystal structure of MICA bound to NKG2D.

[0037] Figures 11A-11D As measured by FACS, compared with exposure to isotype control (circle), after exposure to increasing concentrations of anti-MICA / B antibody MICA.36 (non-fucosylated, G236A variant; inverted triangle), the maximum fluorescence intensity was measured at ectopic expression of MICA (…). Figures 11A-11C ) or MICB ( Figure 11D LLC cells ( Figure 11A ), E.G7-Ova cells ( Figure 11B ) and B16 cells ( Figure 11C and Figure 11D A graphical representation of the surface MICA or MICB content measured on the surface.

[0038] Figures 12A-12D Compared to exposure to an isotype control (round), exposure to increasing concentrations of anti-MICA / B antibody MICA.36 (non-fucosylated, G236A variant; inverted triangle) showed the reduction in ectopic expression of MICA / B ( Figures 12A-12C ) or recombinant MICA*008 antigen ( Figure 12D LLC cells ( Figure 12A ), E.G7-Ova cells ( Figure 12B ) and B16 cells ( Figure 12C ) and CHO cells ( Figure 12D (e.g., a graphical representation of the concentration of soluble MICA / B in the supernatant of cell cultures, as measured by ELISA.)

[0039] Figures 13A-13EGraphical representation of surface MICA / B content on the surface of human cell lines endogenously expressing MICA / B. Compared to exposure to isotype control (circles), the content of surface MICA / B in SW480 cells after exposure to increasing concentrations of anti-MICA / B antibody MICA.36 (non-fucosylated, G236A variant; inverted triangles) was significantly higher in SW480 cells than in SW480 cells exposed to increasing concentrations of anti-MICA / B antibody MICA.36 (non-fucosylated, G236A variant; inverted triangles). Figure 13A ), HeLa cells ( Figure 13B HCT-116 cells ( Figure 13C ), 786-O cells ( Figure 13D ) and A2058 cells ( Figure 13E The surface localization of MICA / B is measured using FACS on the surface of the MICA / B by the maximum fluorescence intensity.

[0040] Figures 14A-14C Compared to exposure to the isotype control (round), exposure to increasing concentrations of anti-MICA / B antibody MICA.36 (non-fucosylated, G236A variant; inverted triangle) in cultured HeLa cells was compared by ELISA. Figure 14A ;Expressing MICA*008), HCT-116 cells ( Figure 14B ) and 786-O cells ( Figure 14C A graphical representation of the concentration of soluble MICA / B in the culture medium of human cell lines endogenously expressing MICA / B, as measured in the culture medium.

[0041] Figure 15A Graphical representation of antibody-dependent phagocytosis (ADCP) levels after co-culturing with increasing concentrations of MICA.36 IgG1 (round), MICA.36 IgG1 G236A (solid square), non-fucosylated MICA.36 IgG1 (NF; inverted triangle), and MICA.36-IgG1-NF-G236A (triangle) or isotype control (hIgG1, hollow square). Figure 15B This is a graphical representation of antigen cross-presentation as measured by the percentage of proliferating CD8 T cells after co-culturing with increasing concentrations of MICA.36 IgG1 (inverted triangle), MICA.36 IgG1 NF (circle), MICA.36 IgG1 G236A (solid square), MICA.36-IgG1-NF-G236A (triangle), or an allotype control (hollow square).

[0042] Figure 16A and 16BTo determine the expression of MICA / B in Raji cells after exposure to increasing concentrations of MICA.36 NF G236A (triangular), MICA.36 IgG1 (inverted triangular), MICA.36 IgG1 NF (round), MICA.36 IgG1 G236A (solid square), or an isotype control (hollow square), Figure 16A ) and A375 cells ( Figure 16B A graphical representation of the percentage of solubility.

[0043] Figure 17A and Figure 17B The effect of administered anti-MICA / B antibody MICA.36 NF G236A on the total tumor area in mice with induced lung tumors (…). Figure 17A ) and serum soluble MICA / B levels ( Figure 17B ) in vivo effects.

[0044] Figure 18A and Figure 18B The results of two dose-escalation studies in mice induced to develop lung tumors were presented, with total tumor size measured after administration of different doses (10 mg / kg (mpk), 3 mpk, 1 mpk, or 0.3 mpk) of the anti-MICA / B antibody MICA.36 NF G236A or an isotype control.

[0045] Figures 19A-19E The results are shown by administering anti-MICA / B antibodies, alone or in combination with anti-PD-1 antibodies, to MICA transgenic mice exhibiting EG7-MICA tumors. Figure 19A and Figure 19B The mean tumor volume in mice treated with the following methods ( Figure 19A ) and median tumor volume ( Figure 19B The graphic representations are as follows: allotype control (solid circle), MICA.36 mIgG2a and allotype control mIgG1 (square), MICA.36 mIgG1 D265A and allotype mIgG2a (triangle), anti-PD-1 mIgG1 D265A and allotype mIgG2a (inverted triangle), MICA.36 mIgG2a and anti-PD-1 mIgG1 D265A (diamond), or MICA.36 mIgG1 D265A and anti-PD-1 mIgG1 D265A (hollow circle). Figure 19CThe following is a graphical representation of the percentage of tumor growth inhibition (TGI%) calculated for each of the following treatment groups: 10 mg / kg MICA.36 mIgG2a and 10 mg / kg isotype mIgG1 (round), 10 mg / kg MICA.36 mIgG1 D265A and 10 mg / kg isotype mIgG2a (square), 10 mg / kg anti-PD-1 mIgG1 D265A and 10 mg / kg isotype mIgG2a (triangle), 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-PD-1 mIgG1 D265A (inverted triangle), and 10 mg / kg MICA.36 mIgG1 D265A and 10 mg / kg anti-PD-1 mIgG1 D265A (diamond). Figure 19D The progression-free survival (PFS%) of transgenic mice exhibiting EG7-MICA tumors after treatment with the following: dual controls of 10 mg / kg mIgG1 and 10 mg / kg mIgG2a (round), 10 mg / kg MICA.36 mIgG2a and 10 mg / kg mIgG1 (square), 10 mg / kg anti-PD-1 mIgG1 D265A and 10 mg / kg mIgG2a (inverted triangle), or 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-PD-1 mIgG1 D265A (diamond). Figure 19E The concentrations of soluble MICA in the serum of transgenic mice with EG7-MICA tumors are shown after treatment with the following: isotype control, MICA.36 mIgG2a, MICA.36 mIgG1 D265A, anti-PD-1 mIgG1 D265A, MICA.36 mIgG2a and anti-PD-1 mIgG1 D265A, or MICA.36 mIgG1 D265A and anti-PD-1 mIgG1 D265A.

[0046] Figures 20A-20D Results demonstrating the in vivo action of anti-MICA / B antibody in B6-MICA transgenic mice with EG7-MICA / B tumors. Figure 20A and Figure 20B The mean tumor volume in mice treated with the following methods ( Figure 20A ) and median tumor volume ( Figure 20BThe graphic representations are as follows: 20 mg / kg isotype mIgG2a (round), 10 mg / kg MICA.36 mIgG2a and 10 mg / kg isotype control mIgG2a (square), 10 mg / kg anti-CTLA-4 mIgG2a and 10 mg / kg isotype mIgG2a (triangle), or 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-CTLA-4 mIgG2a (inverted triangle). Figure 20C The percentage of tumor growth inhibition (TGI%) calculated for the following treatment groups is shown: 10 mg / kg MICA.36 mIgG2a and 10 mg / kg isotype mIgG2a (round), 10 mg / kg anti-CTLA-4 mIgG2a and 10 mg / kg isotype mIgG2a (square), and 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-CTLA-4 mIgG2a (triangle). Figure 20D The progression-free survival (PFS%) of transgenic mice with EG7-MICA / B tumors after treatment with the following: 20 mg / kg allotype mIgG2a (round), 10 mg / kg MICA.36 mIgG2a and 10 mg / kg allotype control mIgG2a (square), 10 mg / kg anti-CTLA-4 mIgG2a and 10 mg / kg allotype mIgG2a (triangle), or 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-CTLA-4 mIgG2a (inverted triangle).

[0047] Figures 21A-21D The results demonstrate the in vivo effects of the anti-MICA / B antibody in B6-MICA transgenic mice injected subcutaneously with EG7-MICA / B tumor cells. Figure 21A and Figure 21B The mean tumor volume in mice treated with the following methods ( Figure 21A ) and median tumor volume ( Figure 21B The graphic representations are as follows: 10 mg / kg allotype mIgG1 and 10 mg / kg allotype mIgG2a (round), 10 mg / kg MICA.36 mIgG2a and 10 mg / kg allotype mIgG1 (square), 10 mg / kg anti-PD-1 mIgG1 D265A and 10 mg / kg allotype mIgG2a (triangle), or 10 mg / kg MICA.36 mIgG2a and 10 mg / kg allotype mIgG2a anti-PD-1 mIgG1 (inverted triangle). Figure 21CThe following is a graphical representation of the percentage of tumor growth inhibition (TGI%) calculated for the following treatment groups: 10 mg / kg MICA.36 mIgG2a and 10 mg / kg allotype mIgG1 (round), 10 mg / kg anti-PD-1 mIgG1 D265A and 10 mg / kg allotype mIgG2a (square), and 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-PD-1 mIgG1 D265A (triangle). Figure 21D The progression-free survival (PFS%) of transgenic mice subcutaneously injected with EG7-MICA tumor cells after the following treatments are shown: 10 mg / kg allotype mIgG1 and 10 mg / kg allotype mIgG2a (round), 10 mg / kg MICA.36 mIgG2a and 10 mg / kg allotype mIgG1 (square), 10 mg / kg anti-PD-1 mIgG1 D265A and 10 mg / kg allotype mIgG2a (triangle), or 10 mg / kg MICA.36 mIgG2a and 10 mg / kg anti-PD-1 mIgG1 D265A (inverted triangle).

[0048] Figures 22A-22C The results of epitope localization using X-ray eutectic are presented. Figure 22A The surface representation of the α3 domain of MICA is shown, with epitope residues shown in black and the remaining surface residues shown in gray. Figure 22B and Figure 22C The diagram illustrates the relationship between MICA.36Fab (ribbon representation) and the α3 structural domain of MICA (surface representation, e.g.) Figure 22A (As shown) Two orientations of the complex. The MICA.36 epitope on the α3 domain of MICA is shown in black, while the remaining residues of the α3 domain of MICA are shown in gray.

[0049] Figures 23A-23B This demonstrates the binding affinity of the anti-MICA / B antibody obtained using FACS to common MICA alleles expressed on CHO cells. Figure 23B Display and Figure 23A The data are the same, but the Y-axis data of recombinant antibodies MICA.5, MICA.6, and MICA.7 and the isotype control are different.

[0050] Figures 24A-24B This demonstrates the binding affinity of the anti-MICA / B antibody obtained using FACS to MICA endogenously expressed on RPMI-8226 cells. Figure 24B Display and Figure 24AThe data were the same, but the Y-axis data for Ab2, Ab28, Ab29, the isotype control, and the supernatant (“supe”) from CHO cell lines expressing recombinant antibodies MICA.5, MICA.6, and MICA.7 were different.

[0051] Figure 25 Serum levels of soluble MICA / B in B6-MICA transgenic mice at 72 and 168 hours after administration of antibody MICA.36-mIgG2a at 1 mg / kg or 10 mg / kg, or after administration of an isotype control.

[0052] Figure 26 The results of PK analysis of sMICA in B6-MICA transgenic mice after administration of sMICA and antibody MICA.36-mIgG2a or isotype control are presented. Invention Details

[0054] This invention relates to antibodies and their antigen-binding fragments (“anti-MICA / B antibodies”) that specifically bind to MHC class I peptide-associated sequence A (MICA) and / or MHC class I peptide-associated sequence B (MICB). In some embodiments, MICA is human MICA. In some embodiments, MICB is human MICB. Other aspects of the invention relate to methods of treating an individual in need, comprising administering to the subject an antibody or its antigen-binding fragment that specifically binds to MICA and / or MICB. In some embodiments, the individual has cancer, and the anti-MICA / B antibody treats the cancer in the individual.

[0055] I. Terminology

[0056] To make the description of this invention easier to understand, certain terms are first defined. Additional definitions are set forth throughout the embodiments.

[0057] It should be noted that the term "a (or an)" refers to one or more of the same entity; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a (or an)," "one or more," and "at least one" are used interchangeably in this document.

[0058] Furthermore, the term “and / or” as used herein should be considered to specifically indicate that each of the two specified features or components has or does not have the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone) and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0059] It should be understood that whenever an aspect is described in this document using the language “contains”, similar aspects described in the terms “composed of” and / or “substantially composed of” are also provided.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide general dictionaries for those skilled in the art of the invention for many of the terms used herein.

[0061] Units, prefixes, and symbols are represented in their form accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right along the 5' to 3' direction. Amino acid sequences are written from left to right along the amino to carboxyl direction. The headings provided herein are not intended to limit the various aspects of the invention and are mentioned as a whole in the specification. Therefore, the terms defined below are defined more fully by reference to the entire specification.

[0062] The term “about” is used herein to mean approximately, roughly, around, or in the range of. When the term “about” is used in conjunction with a numerical range, its range is adjusted by extending the boundary upwards or downwards relative to the stated value. Generally, the term “about” can be adjusted upwards or downwards relative to the stated value by a difference of, for example, 10% (higher or lower).

[0063] As used herein, “MHC class I peptide-related sequence A” or “MICA” refers to a non-classical major histocompatibility complex (MHC) class I molecule. As used herein, “MHC class I peptide-related sequence B” or “MICB” refers to a non-classical major histocompatibility complex (MHC) class I molecule. As used herein, “MICA / B” refers to MICA and / or MICB.

[0064] MICA is a naturally expressed, glycosylated, and polymorphic protein typically anchored to the cell membrane. Membrane-bound MICA is cleaved by matrix metalloproteinases and ADAM proteases, releasing soluble forms of MICA into the extracellular matrix. MICA is a ligand for NKG2D, a receptor expressed on the surface of natural killer (NK) cells, CD8 T cells, and γδ T cells. MICA expression is induced in healthy cells during stress, and MICA acts as a signal to induce NK and T cell-mediated cell lysis in MICA-expressing cells. Effectively, MICA acts as a "kill me" signal for cells during stress. MICA is expressed by various tumor cell types.

[0065] The term "MICA" includes any variant or isoform of a MICA that is naturally expressed by cells including, but not limited to, tumor cells. Therefore, the antibodies described herein may cross-react with MICA from species other than humans (e.g., cynomolgus monkey MICA). Alternatively, the antibodies may be specific to human MICA without exhibiting any cross-reactivity with other species. MICA, or any variant or isoform thereof, may be isolated from cells or tissues that naturally express it or generated recombinantly using techniques well known in this art and / or those described herein.

[0066] Human MICA (SEQ ID NO:51) consists of 332 amino acids. At least two additional isoforms of human MICA have been identified. Isoform 1, designated MICA1 (UniProt ID No. Q29983-1; SEQ ID NO:109), consists of 383 amino acids. Isoform 2, designated MICA2 (UniProt ID No. Q29983-2; SEQ ID NO:88), consists of 287 amino acids. Compared to the amino acid sequence of MICA1, MICA2 lacks amino acid residues 109-204.

[0067] The following are the amino acid sequences of three known human MICA isoforms.

[0068] (A) Human MICA (SEQ ID NO:51):

[0069]

[0070] (B) Human MICA isoform 1 (UniProt ID No. Q29983-1; SEQ ID NO: 109):

[0071]

[0072] (C) Human MICA isoform 2 (UniProt ID No. Q29983-2; SEQ ID NO:88):

[0073]

[0074] The signal sequence of MICA corresponds to amino acids 1-23 (underlined). Therefore, the mature isoforms of MICA, MICA1, and MICA2 consist of amino acids 24-332, 383, or 287, respectively. The extracellular domain of the mature human MICA consists of amino acids 24-307 of SEQ ID NO:51 and has the following amino acid sequence:

[0075]

[0076] Several known MICA alleles exist. In some implementations, MICA is MICA*002 (also referred to herein as the MICA*002 allele), which has the following amino acid sequence:

[0077]

[0078] In some implementations, the MICA is MICA*004 (also referred to herein as the MICA*004 antigen), which has the following amino acid sequence:

[0079] In some implementations, the MICA is MICA*008 (also referred to herein as the MICA*008 antigen), which has the following amino acid sequence:

[0080] In some implementations, the MICA is MICA*009 (also referred to herein as the MICA*009 antigen), which has the following amino acid sequence:

[0081] In some implementations, the MICA is MICA*009 (also referred to herein as the MICA*009 antigen), which has the following amino acid sequence:

[0082] Many different alleles of human MICA exist as known in this art, including seventy-three or more MICA alleles. See Frigoul and Lefranc, Recent Res. Devel. Human Genet. 3:95-145 (2005). The antibodies disclosed in this specification can bind to one or more MICA alleles. In some embodiments, the anti-MICA / B antibody specifically binds to a single MICA allele. In some embodiments, the anti-MICA / B antibody binds to one or more MICA alleles with high affinity and to other MICA alleles with low affinity. In some embodiments, the anti-MICA / B antibody binds to one or more MICA alleles selected from: MICA*002, MICA*004, MICA*008, and MICA*009. In some implementations, the anti-MICA / B antibody binds to MICA*004, MICA*008, and MICA*009 with high affinity, and to MICA*002 with low affinity, depending on the situation.

[0083] The cynomolgus monkey MICA protein contains the following amino acid sequence (including the signal sequence):

[0084]

[0085] MICB is a naturally expressed, glycosylated, and polymorphic protein that is generally anchored to the cell membrane. Cleavage of membrane-bound MICB releases its soluble form into the extracellular matrix. MICB is a ligand for NKG2D and acts as a signal to induce NK and T cell-mediated cell lysis in cells expressing MICB. MICB acts as a “kill me” signal for cells during stress and is expressed by various tumor cells. The term “MICB” includes any variant or isoform of MICB that is naturally expressed by cells including, but not limited to, tumor cells.

[0086] The antibodies described herein may cross-react with MICBs from species other than humans (e.g., cynomolgus monkey MICBs). Alternatively, the antibodies may be specific to human MICBs without exhibiting any cross-reactivity with other species. MICBs or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them or generated recombinantly using techniques well known in this art and / or those described herein.

[0087] Human MICB (SEQ ID NO:132) consists of 383 amino acids (UniProt ID No. Q29980) and has the following amino acid sequence:

[0088] Additional isoforms of human MICB have been identified. Isoform 2 of MICB (UniProt ID No. Q29980-2) has the following amino acid sequence:

[0089]

[0090] In some implementations, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). In some antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region comprises a hinge and three domains CH1, CH2, and CH3. In some antibodies (e.g., naturally occurring IgG antibodies), each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region comprises a domain (abbreviated CL). The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including binding to various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine residue. Unless otherwise specified herein, amino acids in the variable regions are numbered using the Kabat numbering system, while those in the constant regions are numbered using the EU system.

[0091] In some implementations, such as the term "IgG antibody" as used herein, for example, human IgG1, IgG2, IgG3, and IgG4 antibodies have the structure of naturally occurring IgG antibodies, that is, they have the same number of heavy and light chains and disulfide bonds as naturally occurring IgG antibodies of the same subclass. For example, anti-MICA / B IgG1, IgG2, IgG3, or IgG4 antibodies consist of two heavy chains (HC) and two light chains (LC), wherein the two HCs and LCs are linked by the same number and position as those present in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies (unless the antibody has been mutated to modify the disulfide bonds).

[0092] Antibodies typically bind specifically to their corresponding antigens with high affinity, which is determined by the dissociation constant (K). D ) is 10 -5 Up to 10 -11 M or smaller. Greater than approximately 10. -4 M any K D Generally considered to indicate nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to that antigen and substantially the same antigen but not with high affinity to unrelated antigens; this high affinity means K. D 10-7 M or smaller, 10 -8 M or smaller, 5×10 -9 M or smaller or between 10 -8 M and 10 -10 M is between or smaller than M. If an antigen exhibits a high degree of sequence identity with a given antigen, for example, if the antigen exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with a given antigen, then the antigen is "substantially identical" to the given antigen. For example, in some embodiments, antibodies that specifically bind to human MICA / B may also have cross-reactivity with MICA / B antigens from certain primate species (e.g., cynomolgus monkey MICA / B), but not with MICA / B antigens from other species or with antigens other than MICA / B.

[0093] Immunoglobulins can be derived from any commonly known isotype, including (but not limited to) IgA, secretory IgA, IgG, and IgM. IgG isotypes are subdivided in some species: in humans, IgG1, IgG2, IgG3, and IgG4, and in mice, IgG1, IgG2a, IgG2b, and IgG3. In some embodiments, the anti-MICA / B antibody described herein has the IgG1 isotype. Immunoglobulins (e.g., IgG1) exist in several allotypes, which differ from each other by at most a few amino acids. For example, "antibody" includes both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0094] As used herein, the term "antigen-binding portion" of an antibody refers to one or more segments of an antibody that retain the ability to specifically bind to an antigen (e.g., human MICA / B). The antigen-binding function of antibodies has been demonstrated to be performed by segments of full-length antibodies. Examples of binding segments encompassed within the term "antigen-binding portion" of an antibody (e.g., the anti-MICA / B antibody described herein) include (i) segments formed by V L V H (i) Fab fragments consisting of LC and CH1 domains (fragments derived from papain cleavage) or similar monovalent fragments; (ii) F(ab')2 fragments (fragments derived from pepsin cleavage) or similar divalent fragments comprising two Fab fragments connected by disulfide bridging at the hinge region; (iii) fragments consisting of V H (iv) The Fd fragment composed of the CH1 domain; and the V of the single arm of the antibody. L and V H The Fv segment, composed of structural domains, (v) is composed of V HThe dAb fragment composed of structural domains (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) combinations of two or more separated CDRs that may be joined by synthetic joints, depending on the situation. Furthermore, although the two structural domains V of the Fv fragment... L and V H Encoded by a single gene, but which can be joined using recombination methods via a synthetic linker, the synthetic linker allows the domain to be prepared as a single protein chain, wherein V L and V H Regions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding moiety" of the antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for use in the same manner as intact antibodies. Antigen-binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0095] "Bispecific" or "bifunctional antibody" is an artificial hybrid antibody with two different pairs of heavy / light chains and two different binding sites. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0096] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies contained in the population are substantially similar and bind to the same epitopes (e.g., the antibody exhibits single binding specificity and affinity) except for possible variants that may arise during monoclonal antibody production (which are generally present in small amounts). The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. The term "human monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies exhibiting single binding specificity and having variable regions derived from human germline immunoglobulin sequences and, where applicable, constant regions. In some embodiments, the human monoclonal antibody system is generated from a hybridoma comprising B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with immortalized cells, having a genome containing human heavy chain transgenes and light chain transgenes.

[0097] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) or hybridomas prepared therefrom of the human immunoglobulin gene; (b) antibodies isolated from host cells transformed to express antibodies, such as antibodies isolated from transfected tumors; (c) antibodies isolated from recombinant, combined human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain specific human germline immunoglobulin sequences encoded by germline genes, but include variable and constant regions that subsequently undergo rearrangements and mutations, such as during antibody maturation. As is known in this art (see, for example, Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains antigen-binding domains encoded by various genes rearranged to form antibodies specific to foreign antigens. In addition to rearrangements, variable regions can be further modified by multiple single-amino acid changes (called somatic mutations or hypermutations) to increase the antibody's affinity for foreign antigens. Constant regions will further change in response to antigens (i.e., isotype switching). Therefore, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to antigens cannot have sequence identity with the original nucleic acid molecule, but will instead be substantially identical or similar (i.e., possessing at least 80% identity).

[0098] A “human” antibody (HuMAb) is an antibody having variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, that constant region is also derived from human germline immunoglobulin sequences. Anti-MICA / B antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which germline CDR sequences derived from another mammalian species (such as mice) have been grafted onto a human framework sequence. The term “human” antibody is used synonymously with “fully human” antibody.

[0099] "Humanized" antibodies are antibodies in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody have been replaced with corresponding amino acids derived from human immunoglobulins. In some embodiments of humanized antibodies, some, most, or all of the amino acids outside the CDR domain have been replaced with amino acids derived from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible, as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to that of the original antibody.

[0100] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0101] As used in this article, “isotype” refers to the antibody class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0102] "Allotype" refers to a naturally occurring variant within a specific isotype group that differs by a few amino acids (see, for example, Jefferis et al. (2009) mAbs 1:1). The anti-MICA / B antibodies described herein can have any allotype. As used herein, antibodies of the isotype "IgG1f", "IgG1.1f", or "IgG1.3f" are IgG1, non-response IgG1.1, and non-response IgG1.3 antibodies of the isotype "f", i.e., 214R, 356E, and 358M, according to the EU index in Kabat.

[0103] The phrases “antibody that recognizes antigen” and “antibody that is specific to antigen” are used interchangeably with the term “antibody that specifically binds to antigen” in this article.

[0104] As used in this article, "isolated antibody" is intended to refer to an antibody that is essentially free of other proteins and cellular material.

[0105] As used herein, “MICA / B-binding antibody” is intended to refer, for example, in binding assays using human MICA / B-transfected CHO cells or MICA / B-expressing tumor cells, to antibodies that interact with MICA / B, in methods generally accepted in this technique, such as in the FACS-based binding assays described herein, where their EC50... 50 The concentrations are approximately 25 μg / mL or less, approximately 23 μg / mL or less, approximately 20 μg / mL or less, approximately 15 μg / mL or less, approximately 10 μg / mL or less, approximately 5 μg / mL or less, approximately 3 μg / mL or less, approximately 2 μg / mL or less, approximately 1 μg / mL or less, approximately 0.5 μg / mL or less, approximately 0.45 μg / mL or less, approximately 0.4 μg / mL or less, approximately 0.35 μg / mL or less, or approximately 0.3 μg / mL or less. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50 For approximately 200 nM or less, approximately 175 nM or less, approximately 160 nM or less, approximately 150 nM or less, approximately 125 nM or less, approximately 110 nM or less, approximately 100 nM or less, approximately 80 nM or less, approximately 75 nM or less, approximately 60 nM or less, approximately 50 nM or less, approximately 40 nM or less, approximately 35 nM or less, approximately 30 nM or less, approximately 25 nM or less, approximately 20 nM or less, approximately 15 nM or less, approximately 10 nM or less, approximately 9 nM or less, approximately 8 nM or less, approximately 7 nM or less, approximately 6 nM or less, approximately 5 nM or less, approximately 4 nM or less, about 3 nM or less, about 2 nM or less, about 1.9 nM or less, about 1.8 nM or less, about 1.7 nM or less, about 1.6 nM or less, about 1.5 nM or less, about 1.4 nM or less, about 1.3 nM or less, about 1.2 nM or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, or about 0.1 nM or less. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50 50 Less than approximately 10 nM. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50Less than approximately 5 nM. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50 Less than approximately 1.5 nM. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50 It is approximately 1 nM or less. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50 It is approximately 0.5 nM or less. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50 It is approximately 0.3 nM or less. In some embodiments, the anti-MICA / B antibody binds to human MICA / B expressed on, for example, CHO cells, whose EC50... 50 It is approximately 0.2 nM or less.

[0106] As used herein, "inhibiting, preventing, or reducing" the shedding of MICA / B by cells (e.g., tumor cells) is intended to refer to antibodies that inhibit, prevent, or reduce the release of soluble MICA / B from the cell surface. Without being mechanistically specific, the anti-MICA / B antibodies disclosed herein reduce the amount of MICA / B released into the plasma, for example, through lysis in the form of soluble MICA / B. In some embodiments, the antibody increases membrane-bound MICA / B on the cell surface. In some embodiments, the anti-MICA / B antibody increases the retention of surface MICA / B on cells transfected with human MICA / B, where the MICA / B retains EC 50The values ​​are approximately 10 nM or less, approximately 5 nM or less, approximately 1 nM or less, approximately 0.85 nM or less, approximately 0.8 nM or less, approximately 0.75 nM or less, approximately 0.7 nM or less, approximately 0.65 nM or less, approximately 0.6 nM or less, approximately 0.55 nM or less, approximately 0.5 nM or less, approximately 0.45 nM or less, approximately 0.4 nM or less, approximately 0.35 nM or less, approximately 0.3 nM or less, approximately 0.25 nM or less, approximately 0.2 nM or less, approximately 0.15 nM or less, or approximately 0.1 nM or less. In some embodiments, the anti-MICA / B antibody increases the retention of surface MICA / B on cells transfected with human MICA / B by at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.75-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, or at least about 20-fold.

[0107] In some implementations, anti-MICA / B antibodies reduce the level of soluble MICA / B (sMICA / B) in patient serum. In some implementations, anti-MICA / B antibodies reduce soluble MICA / B shed from cells transfected with human MICA / B, with an IC50 value of [missing information]. 50Approximately 10 nM or less, approximately 5 nM or less, approximately 1 nM or less, approximately 0.7 nM or less, approximately 0.6 nM or less, approximately 0.5 nM or less, approximately 0.45 nM or less, approximately 0.4 nM or less, approximately 0.35 nM or less, approximately 0.3 nM or less, approximately 0.25 nM or less, approximately 0.2 nM or less, approximately 0.19 nM or less, approximately 0.18 nM or less, approximately 0.17 nM or less, approximately 0.16 nM or less. Approximately 0.15 nM or less, approximately 0.14 nM or less, approximately 0.13 nM or less, approximately 0.12 nM or less, approximately 0.11 nM or less, approximately 0.1 nM or less, approximately 0.09 nM or less, approximately 0.08 nM or less, approximately 0.07 nM or less, approximately 0.06 nM or less, approximately 0.05 nM or less, approximately 0.04 nM or less, approximately 0.03 nM or less, approximately 0.02 nM or less, or approximately 0.01 nM or less. In some implementations, the anti-MICA / B antibody reduces MICA / B shedding from cells transfected with human MICA by approximately 0.9-fold or less, approximately 0.85-fold or less, approximately 0.8-fold or less, approximately 0.75-fold or less, approximately 0.7-fold or less, approximately 0.65-fold or less, approximately 0.6-fold or less, approximately 0.55-fold or less, approximately 0.5-fold or less, approximately 0.45-fold or less, approximately 0.4-fold or less, approximately 0.35-fold or less, approximately 0.3-fold or less, approximately 0.25-fold or less, approximately 0.2-fold or less, approximately 0.15-fold or less, approximately 0.1-fold or less, or approximately 0.05-fold or less. In some embodiments, the anti-MICA / B antibody can reduce the soluble MICA / B in an individual's serum by, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0108] As used herein, the term "shedding" refers to the release of soluble MICA / B from the surface of MICA / B-expressing cells. MICA / B is expressed as a transmembrane protein localized to the cell surface. Cleavage within its extracellular domain releases a portion of MICA / B into the plasma, which is referred to herein as "soluble MICA / B" or "sMICA / B".

[0109] "Effective function" refers to the interaction between the antibody's Fc region and an Fc receptor or ligand, or the biochemical event resulting from such interaction. Exemplary "effective functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP), as well as downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require a combination of the Fc region and a binding domain (e.g., antibody variable domain).

[0110] "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs binding to IgG antibodies include receptors of the FcγR family, including allelic variants and alternative splice forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Various characteristics of human FcγRs are known in this art. Most innate effector cell types co-express one or more activating FcγRs and one inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitory FcγRIIB in either mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the type of activated Fc receptors it binds to.

[0111] The “Fc region” (fragment of the crystallizable region), or “Fc domain”, or simply “Fc”, refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) and to the first component (C1q) of the classical complement system. Therefore, the Fc region contains the constant regions of the antibody, excluding the first constant region immunoglobulin domains (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region contains immunoglobulin domains CH2 and CH3 and a hinge between the CH1 and CH2 domains. Although the definition of the boundary of the Fc region of the immunoglobulin heavy chain may vary, as defined herein, the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 for IgG1, from V222 for IgG2, from L221 for IgG3, and from P224 for IgG4 to the carboxyl terminus of the heavy chain, where the numbering is based on the EU index in Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, that is, it extends from amino acid 341 of IgG to amino acids 447 or 446 (if no C-terminal lysine residue) or amino acid 445 (if no C-terminal glycine and lysine residues). As used herein, the Fc region can be a native sequence Fc, including any allotype or variant Fc (e.g., non-naturally occurring Fc).

[0112] A “natural sequence Fc region” or “natural sequence Fc” contains an amino acid sequence identical to that of an Fc region found in nature. Natural sequence human Fc regions include the natural sequence human IgG1 Fc region; the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants. Natural sequence Fc includes various allotypes of Fc (see, for example, Jefferis et al. (2009) mAbs 1:1).

[0113] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., MICA) that an immunoglobulin or antibody specifically binds to, for example, as defined by a particular method used to identify it. Epitopes can be formed from adjacent amino acids (typically linear epitopes) or non-adjacent amino acids (typically conformational epitopes) adjacent to each other by ternary folding of a protein. Epitopes formed from adjacent amino acids are usually, but not always, retained after exposure to denaturing solvents, while epitopes formed by ternary folding are usually lost upon treatment with denaturing solvents. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitope binds to a given antibody (i.e., epitope localization) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which the reactivity of overlapping or adjacent peptides (e.g., from MICA) with a given antibody (e.g., anti-MICA / B antibody) is tested. Methods for determining the spatial conformation of epitopes include those techniques described in this art and those described herein, such as X-ray crystallography, X-ray cocrystalography, antigen mutation analysis, 2D nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, edited by GEMorris, (1996)).

[0114] The term "epitope localization" refers to a method for identifying molecular determinants used for antibody-antigen recognition.

[0115] The term "binding to the same epitope" when referring to two or more antibodies means, as determined by established methods, that the antibodies bind to the same segment of amino acid residues. Techniques used to determine whether an antibody binds to the "same epitope on the MICA" as described herein include, for example, epitope localization methods, such as X-ray analysis of crystals of antigen:antibody complexes and hydrogen / deuterium exchange mass spectrometry (HDX-MS) that provide atomic resolution of the epitope. Other methods monitor the binding of antibodies to antigen fragments or mutant variants of antigens, where binding loss due to modifications of amino acid residues within the antigen sequence is generally considered an indicator of epitope component. Additionally, computational combinatorial methods for epitope localization can be used. These methods rely on the ability of the antibody of interest to affinityly separate specific short peptides from a combinatorial phage-presented peptide library. Antibodies with identical VH and VL or identical CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0116] An antibody that "competes with another antibody to bind to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Whether two antibodies compete with each other to bind to a target, that is, whether one antibody inhibits the binding of another antibody to a target and the degree of inhibition, can be determined using known competition experiments, such as... Surface plasmon resonance (SPR) analysis is used to determine this. In some embodiments, an antibody competes with another antibody and inhibits the binding of that other antibody to the target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is the “blocking antibody” (i.e., a cold antibody that is first cultured with the target). Competition analysis can be performed as described below: for example, Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or Ed Harlow and David Lane, “Using Antibodies,” Chapter 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. If two antibodies bidirectionally block each other by at least 50%, that is, whether one antibody or the other first comes into contact with the antigen in the competition experiment, then the antibodies are “cross-competitive.”

[0117] Competitive binding assays used to determine whether two antibodies bind competitively or cross-competitively include: competition for binding to cells expressing MICA / B, such as by flow cytometry, as described in the examples. Other methods include: SPR (e.g., Solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition analysis (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct labeling analysis, solid-phase direct labeling sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology) 176:546(1990)); and directly labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77(1990)).

[0118] As used herein, the terms "specific binding," "selective binding," "selectively binding," and "specific binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibodies (i) bind to an epitope on a predetermined antigen using, for example, surface plasmon resonance (SPR) technology. When the 2000 instrument uses a predetermined antigen (e.g., recombinant human MICA or MICB) as an analyte and an antibody as a ligand for determination, or when determined by Scacha analysis of the binding of the antibody to antigen-positive cells, the equilibrium dissociation constant (K0) of the binding is... D (approximately less than 10) -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower, and (ii) the affinity for the predetermined antigen is at least twice that for non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. Therefore, an antibody that "specifically binds to human MICA / B" refers to an antibody with an affinity of 10... -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibodies that bind to soluble or cell-bound human MICA / B. Antibodies that "cross-react with cynomolgus monkey MICA / B" refer to those formulated at a concentration of 10... -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibodies that bind to cynomolgus monkey MICA / B. In some embodiments, such antibodies that do not cross-react with MICA / B from non-human species exhibit substantially undetectable binding to these proteins in standard binding assays.

[0119] As used in this article, the term "k" assoc "or "k a "Intended to refer to the association rate of a specific antibody-antigen interaction, while the term "k" as used herein..." dis "or "k d "Intended to refer to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term 'K'..." D "Intended to refer to the dissociation constant, which is derived from k..." d With k a The ratio (i.e., k) d / k a The antibody's Kc is obtained and expressed as molar concentration (M). D The value can be determined using methods well-established in this technique. Ka is available for use in the determination of antibodies.D Methods include surface plasmon resonance; biosensor systems, such as System; or flow cytometry and Scacha analysis.

[0120] As used in this article, the term "high affinity" for IgG antibodies refers to the antibody's affinity for the target antigen's K+. D 10 -8 M or smaller, 10 -9 M or smaller, or 10 -10 M or smaller. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for IgM isotypes refers to the antibody's Kg. D 10 -10 M or smaller, or 10 -8 M or smaller.

[0121] In cases of in vitro or in vivo analysis using antibodies or their antigen-binding fragments, the term "EC" is used. 50 "" refers to the concentration of the antibody or its antigen-binding moiety that induces 50% of the maximum response, which is half the response between the maximum response and the baseline.

[0122] As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in organisms (including viruses) that can be isolated from natural sources and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0123] A polypeptide is a chain containing at least two consecutively linked amino acid residues, with no upper limit on chain length. One or more amino acid residues in a protein may contain modifications such as (but not limited to) glycosylation, phosphorylation, or disulfide bond formation. A protein may contain one or more polypeptides.

[0124] As used in this article, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0125] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in this technology. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, the non-essential amino acid residues predicted in the anti-MICA / B antibody are substituted with another amino acid residue from the same side chain family. Methods for identifying nucleotides and amino acid substitutions that do not eliminate antigen binding are well known in this technique (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0126] For nucleic acids, the term "significant homology" indicates that, when optimal alignment and comparison are performed with appropriate nucleotide insertions or deletions, at least about 80%, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides of two nucleic acids or their designated sequences are identical. Alternatively, significant homology exists when a segment hybridizes with the complementary sequence of that segment under selective hybridization conditions.

[0127] For peptides, the term “significant homology” indicates that, when optimal alignment and comparison are performed with appropriate nucleotide insertions or deletions, at least about 80%, at least about 90% to 95%, or at least about 98% to 99.5% of amino acids are identical between two peptides or their designated sequences.

[0128] Taking into account the number of gaps that need to be introduced for optimal alignment of two sequences and the length of each gap, the percentage of identity between two sequences is a function of the number of common positions shared by the sequences (i.e., homology % = number of common positions / total number of positions × 100). As described in the non-limiting embodiments below, the comparison of sequences and the determination of the percentage of identity between two sequences can be implemented using mathematical algorithms.

[0129] The percentage of identity between two nucleotide sequences can be determined using the GAP procedure in the GCG software package (available at worldwideweb.gcg.com) with the NWSgapdna.CMP matrix and nick weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN procedure (version 2.0), with a PAM120 weighted residue table, a nick length penalty of 12, and a nick penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) algorithm in the GAP program, which is incorporated into the GCG software package (available at http: / / www.gcg.com), using a Blossum62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0130] The nucleic acid and protein sequences described herein can be further used as “query sequences” to perform searches against public databases, for example, to identify relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. For gap alignments to be obtained for comparative purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic AcidsRes.25(17):3389-3402. When using BLAST and gapped BLAST programs, the preset parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0131] Nucleic acids can be present in whole cells, cell lysates, or in partially purified or substantially pure forms. A nucleic acid is considered "isolated" or "substantially pure" when purified to separate it from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by means of standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in this field. See F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0132] Nucleic acids (e.g., cDNA) can be mutated using standard techniques to provide gene sequences. For coding sequences, these mutations may affect the amino acid sequence as needed. Specifically, consider DNA sequences that are substantially homologous to or derived from the natural V, D, J, constant, transform, and other such sequences described herein (where "derived from" means the sequence is identical to or derived from another sequence).

[0133] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, in which the additional DNA segment can be attached to a viral genome. Some vectors can replicate autonomously in a host cell in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) can integrate into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. In addition, some vectors can guide the expression of genes to which they are operatively attached. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally, expression vectors used in recombinant DNA technology are often in plasmid form. In this specification, since plasmids are the most common form of vector, "plasmid" and "vector" are used interchangeably. However, other forms of expression vectors that provide equivalent functionality are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0134] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acids that are not naturally present in that cell, and may be a cell in which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to a specific individual cell, but also to the offspring of such cells. Because certain modifications may appear in subsequent generations due to mutations or environmental influences, such offspring may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0135] "Immune response," as understood in this art, generally refers to a biological response in vertebrates against foreign agents or abnormalities (e.g., cancer cells), protecting the organism from the effects of these agents and the diseases they cause. Immune responses are mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver. This action results in the selective targeting, binding, damage, destruction, and / or elimination from vertebrates of invasive pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation. + Cells, CD8 + The activation or suppression of T cells (or Treg cells), or the activation or suppression of any other cells of the immune system (such as NK cells).

[0136] "Immunomotor" or "immunomodulatory agent" refers to an agent that may involve regulating, modulating, or altering an immune response, such as an agent that targets components of a signaling pathway. "Regulating," "modulating," or "altering" an immune response refers to any alteration in the cells of the immune system or in the activity of such cells (e.g., effector T cells, such as Th1 cells). Such regulation includes stimulating or suppressing the immune system, which may manifest as an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have an enhancing function in the tumor microenvironment. In some embodiments, immunomodulators target molecules on the surface of T cells. "Immunomodulatory target" or "immunomodulatory target" is a molecule that is targeted by the binding of a substance, agent, part, compound, or molecule, and whose activity is altered by the binding of the substance, agent, part, compound, or molecule, such as cell surface molecules. Immunomodulatory targets include, for example, receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands") on the cell surface.

[0137] "Immunotherapy" refers to the treatment of individuals who have a disease or are at risk of contracting a disease or experiencing a relapse of a disease by means of methods that involve inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

[0138] "Immunostimulatory therapy" or "immunostimulatory treatment" refers to therapies that induce (induce or enhance) an individual's immune response for purposes such as treating cancer.

[0139] "Enhancing the endogenous immune response" means increasing the effectiveness or efficacy of the existing immune response in an individual. This increase in effectiveness and efficacy can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0140] "T effector cells" eff "Cells" refers to T cells with cytolytic activity (e.g., CD4+). + and CD8 + T cells and T helper (Th) cells (e.g., Th1 cells), which secrete cytokines and activate and guide other immune cells, but do not include regulatory T cells (Treg cells). Certain anti-MICA / B antibodies described herein enable T cells to secrete cytokines and activate and guide other immune cells. eff Cell activation, such as CD4 activation + and CD8 + T eff Cell and Th1 cell activation.

[0141] An increased capacity to stimulate an immune response or immune system can be caused by enhanced agonistic activity of T cell co-stimulatory receptors and / or enhanced antagonistic activity of inhibitory receptors. This increased capacity can also be measured by ECGs in analyses that measure the immune response. 50 The increase in the maximum activity level may reflect changes such as changes in cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly by detecting CD107a or granzyme), and proliferation. The ability to stimulate an immune response or immune system activity may be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

[0142] As used herein, the term "linkage" refers to the combination of two or more molecules. Linkages can be covalent or non-covalent. Linkages can also be gene linkages (i.e., fusion via recombination). Such linkages can be achieved using a variety of techniques recognized in this field, such as chemical binding and recombinant protein production.

[0143] As used herein, “administration” means the physical introduction of a composition containing a therapeutic agent into an individual using any of the various methods and delivery systems known to those skilled in the art. Different routes of administration of the anti-MICA / B antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” means a mode of administration other than gastrointestinal and local administration, typically by injection, and includes (but is not limited to) intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, spinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via non-parenteral routes, such as local, transdermal, or transmucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration. The giving can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0144] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8+). + Cells) and helper T cells (e.g., CD4 cells) + T cell-mediated responses. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0145] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ cells. + T cell mediated.

[0146] As used herein, the phrase “inhibit tumor growth” includes any measurable reduction in tumor growth, such as inhibiting tumor growth by at least about 10%, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%. In some embodiments, the inhibition of tumor growth is measured as a tumor growth inhibition percentage (TGI%). The TGI% can be determined by calculating the TGI at date “t” for all treated animals according to the following formula: [1-((T t / T0) / (C t / C0))] / [(C t -C0) / C t ]×100[Equation 1], where T t = Individual tumor size of the treated animal at time "t", T0 = Individual tumor size of the treated animal at the time of the first measurement, C t= Median tumor size of the control animal at time "t", C0 = Median tumor size of the control animal at the time of the first measurement.

[0147] As used herein, “cancer” refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to malignant tumors or cell formation that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0148] As used herein, the term "treat / treating / treatment" refers to any type of intervention or method performed on an individual or administration of an active agent to a subject with the aim of reversing, alleviating, improving, suppressing, slowing, or preventing the worsening, development, severity, or relapse of symptoms, complications, symptom, or biochemical markers associated with a disease, or enhancing overall survival. Treatment can be for individuals who have a disease or individuals who do not have a disease (e.g., for prevention).

[0149] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. A "therapeutic effective dose" or "therapeutic dosage" of a drug or therapeutic agent is any amount of a drug, when used alone or in combination with another therapeutic agent, that promotes disease regression demonstrated by: a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, an increase in overall survival (the length of time a patient diagnosed with the disease, such as cancer, has survived since the date of diagnosis or the start of initiation of treatment for the disease), or prevention of injury or disability resulting from the disease. A therapeutic effective dose or dosage of a drug includes a "preventive effective dose" or "preventive dosage," which is any amount of a drug, when administered alone or in combination with another therapeutic agent to a subject at risk of developing or relapsing into the disease, that inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease remission or inhibit disease progression or recurrence can be assessed using a variety of methods known to those skilled in the art, such as in human individuals during clinical trials, in animal model systems that predict efficacy in humans, or by analyzing the activity of the agent in in vitro assays.

[0150] For example, an anticancer agent is a drug that promotes cancer regression in an individual. In some implementations, a therapeutically effective amount of the drug promotes cancer regression to the point of elimination. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anti-regenerative agent, causes in a patient a slowing of tumor growth or a reduction in size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of asymptomatic periods, prevention of damage or disability due to the disease, or other ways to improve disease symptoms. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity caused by the administration of the drug, or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level.

[0151] For the treatment of tumors, for example, a therapeutically effective amount or dose of the drug inhibits cell or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to an untreated individual. In some embodiments, a therapeutically effective amount or dose of the drug completely inhibits cell or tumor growth, i.e., inhibits 100% of cell or tumor growth. The ability of the compound to inhibit tumor growth can be assessed using the analyses described below. Alternatively, this property of the composition can be assessed by examining the compound's ability to inhibit cell growth, such inhibition being measured in vitro using analyses known to a skilled practitioner. In some embodiments described herein, tumor regression can be observed for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0152] The term "patient" includes human and other mammalian individuals who receive preventative or therapeutic treatment.

[0153] As used herein, the term "individual" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat an individual suffering from cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0154] As mentioned in this article, the term "weight-based" dosage or administration means that the dose administered to a patient is calculated based on the patient's weight. For example, when a patient weighing 60 kg requires 3 mg / kg of anti-MICA / B antibody, the appropriate amount of anti-MICA / B antibody to be administered (i.e., 180 mg) can be calculated and used.

[0155] The term "fixed dose" as used in connection with the combination described herein means that two or more different antibodies (e.g., an anti-MICA / B antibody and a secondary antibody, such as a PD-1 or PD-L1 antibody) are present in the composition in a specific (fixed) amount or ratio to each other, or are administered separately. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In some embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg / ml). In some implementations, the ratio of the two antibodies (e.g., anti-MICA / B and anti-PD1 or anti-PD-L1) is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, or about 1:1. The ratio of the number of milligrams of the first antibody (e.g., anti-MICA / B antibody) to the number of milligrams of the second antibody is approximately 80, 1:200, 200:1, 180:1, 160:1, 140:1, 120:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. For example, a 2:1 ratio of anti-MICA / B antibody to anti-PD-1 antibody (such as nivolumab) means that a vial or injection may contain approximately 480 mg of anti-MICA / B antibody and 240 mg of anti-PD-1 antibody, or approximately 2 mg / ml of anti-MICA / B antibody and 1 mg / ml of anti-PD-1 antibody.

[0156] Regarding the methods and dosages described herein, the term "flat dose" refers to the dose administered to a patient without regard to the patient's weight or body surface area (BSA). Therefore, the flat dose is not provided in mg / kg form, but rather in the absolute amount of the drug (e.g., anti-MICA / B antibody). For example, a 60kg individual and a 100kg individual would receive the same dose of antibody (e.g., 480 mg of anti-MICA / B antibody).

[0157] As used in this article, the terms “ug” and “uM” are interchangeable with “μg” and “μM”, respectively.

[0158] The various aspects described herein are further described in detail in the following subsections.

[0159] II. Anti-human MICA / B antibody

[0160] This document describes antibodies, such as fully human antibodies, that specifically bind to MHC class I peptide-associated sequences A / B (MICA / B) and inhibit, prevent, or reduce the shedding of MICA / B from tumor cells, thereby increasing the retention of MICA / B on the surface of tumor cells. For example, the antibody specifically binds to human MICA / B, and more specifically, to a specific domain (e.g., the α3 domain) within the extracellular domain of human MICA. In some embodiments, the antibody specifically binds to MICA / B, thereby reducing the level of MICA / B in serum, while the antibody increases the level of membrane-bound MICA / B. In some embodiments, the anti-MICA / B antibody cross-reacts with MICA / B from one or more non-human primates (such as cynomolgus monkey MICA / B). In some embodiments, the antibody specifically binds to the α3 domain of both human MICA / B and the α3 domain of cynomolgus monkey MICA / B. In some embodiments, the antibody binds to human MICA / B with high affinity.

[0161] The anti-MICA / B antibodies described in this article exhibit one or more of the following functional properties:

[0162] (a) Binds to membrane-bound human MICA / B;

[0163] (b) Binding to membrane-bound MICA / B in cynomolgus monkeys;

[0164] (c) Reduce the level of soluble MICA / B in individual serum;

[0165] (d) Increases membrane-bound MICA / B on tumor cells;

[0166] (e) Inhibit, prevent or reduce the shedding of MICA / B from tumor cells;

[0167] (f) Mediating enhanced antigen processing and / or cross-presentation in cells;

[0168] (g) Mediating ADCC and / or ADCP enhancement

[0169] (h) Inhibit tumor growth and / or cancer metastasis;

[0170] (i) Reduce tumor volume;

[0171] (j) Increased survival without deterioration;

[0172] (k) Increase total lifespan; and

[0173] (l) any combination thereof.

[0174] In some implementations, the anti-MICA / B antibodies described herein bind to human MICA / B with high affinity, for example, at 10... -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, or 10 -9 M to 10 -7 M of K D In some implementations, such as by means of surface plasmon resonance, for example using BIACORE. TM (For example, as described in the examples) the anti-MICA / B antibody was measured at 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or smaller, 10 -10 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, or 10 -8 M to 10 -7 M of K D Binding to human MICA / B. In some implementations, such as by means of an ELISA (e.g., a MICA / B ELISA kit, such as ABCAM ab100592), anti-MICA / B antibodies are detected via EC. 50EC at concentrations of 100 nM or less, 10 nM or less, 1 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, or 10 ug / mL or less, 5 ug / mL or less, 1 ug / mL or less, 0.9 ug / mL or less, 0.8 ug / mL or less, 0.7 ug / mL or less, 0.6 ug / mL or less, 0.5 ug / mL or less, 0.4 ug / mL or less, 0.3 ug / mL or less, 0.2 ug / mL or less, 0.1 ug / mL or less, 0.05 ug / mL or less, or 0.01 ug / mL or less 50 Binding to human sMICA / B. In some embodiments, the anti-MICA / B antibody described herein is, for example, in a concentration of 10... -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, or 10 -9 M to 10 -7 M of K D Combined with MICA / B, a cynomolgus monkey. In some implementations, such as via BIACORE. TM (For example, as described in the examples) the anti-MICA / B antibody was measured at 10 -6 M or smaller, 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or smaller, 10 -10 M or smaller, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, or 10 -8 M to 10 -7 M of K DThe antibody binds to soluble cynomolgus monkey MICA / B. For example, as measured by ELISA (e.g., as described in the examples), the anti-MICA / B antibody can be expressed in ECMO concentrations of 100 nM or less, 10 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM. 50 Combined with sMICA / B, a cynomolgus monkey.

[0175] In some implementations, the anti-MICA / B antibody is present at approximately 5 × 10⁻⁶. -4 M or smaller, approximately 1×10 -4 M or smaller, 5×10 -5 M or smaller, approximately 1×10 -5 M or smaller, approximately 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller or approximately 1×10 -8 M or smaller K D Specifically binds to human MICA / B, where K D Measurements were performed using surface plasmon resonance (Biacore) analysis. In some implementations, the anti-MICA / B antibody was measured at approximately 1 × 10⁻⁶. -4 M or smaller, approximately 1×10 -5 M or smaller, 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller or approximately 1×10 -8 M or smaller K D Specifically binds to the human MICA allele MICA*002, where K D Measurements were performed using surface plasmon resonance (Biacore) analysis. In some implementations, the anti-MICA / B antibody was measured at approximately 1 × 10⁻⁶. -4 M or smaller, approximately 1×10 -5 M or smaller, 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller or approximately 1×10 -8 M or smaller K D Specifically binds to the human MICA allele MICA*004, where K D Measurements were performed using surface plasmon resonance (Biacore) analysis. In some implementations, the anti-MICA / B antibody was measured at approximately 1 × 10⁻⁶. -4 M or smaller, approximately 1×10 -5 M or smaller, 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller or approximately 1×10 -8 M or smaller K DSpecifically binds to the human MICA allele MICA*008, where K D Measurements were performed using surface plasmon resonance (Biacore) analysis. In some implementations, the anti-MICA / B antibody was measured at approximately 1 × 10⁻⁶. -4 M or smaller, approximately 1×10 -5 M or smaller, 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller or approximately 1×10 -8 M or smaller K D Specifically binds to the human MICA allele MICA*009, where K D Measurements were performed using surface plasmon resonance (Biacore) analysis. In some implementations, the anti-MICA / B antibody was measured at approximately 1 × 10⁻⁶. -4 M or smaller, approximately 1×10 -5 M or smaller, 1×10 -6 M or smaller, approximately 1×10 -7 M or smaller or approximately 1×10 -8 M or smaller K D Specifically binds to the human MICB allele MICB*005, where K D Measurements were performed using surface plasmon resonance (Biacore) analysis.

[0176] In some implementations, the anti-MICA / B antibody is at least about 1×10 3 ms -1 At least approximately 5×10 3 ms -1 At least approximately 1×10 4 ms -1 At least approximately 5×10 4 ms -1 At least approximately 1×10 5 ms -1 At least approximately 5×10 5 ms -1 Or at least about 1×10 6 ms -1 association constant (k) a Rate-specific binding to human MICA / B, where k a Measurements were performed using surface plasmon resonance (Biacore) analysis.

[0177] In some implementations, the anti-MICA / B antibody is delivered at approximately 0.1s... -1 Or even smaller, 0.05s -1 Or even smaller, 0.01s -1 Or smaller, 5×10 -3s -1 or smaller, 1×10 -3 s -1 Or smaller, 5×10 -4 s -1 or smaller, 1×10 -4 s -1 Or smaller, 5×10 -5 s -1 or smaller or 1×10 -5 s -1 or a smaller dissociation constant (k) d Rate-specific binding to human MICA / B, where k d Measurements were performed using surface plasmon resonance (Biacore) analysis.

[0178] In some embodiments, the anti-MICA / B antibody specifically binds to MICA / B. In some embodiments, the anti-MICA / B antibody binds to both MICA and MICB. In some embodiments, the anti-MICA / B antibody specifically binds to the MICA*002 allele. In some embodiments, the anti-MICA / B antibody specifically binds to the MICA*004 allele. In some embodiments, the anti-MICA / B antibody specifically binds to the MICA*008 allele. In some embodiments, the anti-MICA / B antibody specifically binds to the MICA*009 allele. In some embodiments, the anti-MICA / B antibody specifically binds to the MICA*002, MICA*004, MICA*008, and MICA*009 alleles. In some embodiments, the anti-MICA / B antibody has a higher affinity for the MICA*002 allele than for the MICA*004, MICA*008, or MICA*009 alleles. In some embodiments, the anti-MICA / B antibody has a higher affinity for MICA*004 than for the MICA*002, MICA*008, or MICA*009 alleles. In some embodiments, the anti-MICA / B antibody has a higher affinity for the MICA*008 allele than for the MICA*002, MICA*004, or MICA*009 alleles. In some embodiments, the anti-MICA / B antibody has a higher affinity for the MICA*009 allele than for the MICA*002, MICA*004, or MICA*008 alleles.

[0179] In some embodiments, the anti-MICA / B antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises VH complementarity-determining region (CDR)1 (VH-CDR1), VH-CDR2, and VH-CDR3, and the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; wherein the VH-CDR3 comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:7, 17, 27, 37, and 47. In some embodiments, the anti-MICA / B antibody comprises VH-CDR3, which comprises an amino acid sequence selected from SEQ ID NO:7, 17, 27, 37, and 47. In one embodiment, the anti-MICA / B antibody comprises VH-CDR3 comprising the amino acid sequence described in SEQ ID NO:7.

[0180] In some embodiments, the anti-MICA / B antibody comprises VH-CDR2, which comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 6, 16, 26, 36, and 46. In one embodiment, the anti-MICA / B antibody comprises VH-CDR2 comprising the amino acid sequence described in SEQ ID NO: 6.

[0181] In some embodiments, the anti-MICA / B antibody comprises VH-CDR1, which comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:5, 15, 25, 35, and 45. In one embodiment, the anti-MICA / B antibody comprises VH-CDR1 comprising the amino acid sequence described in SEQ ID NO:5.

[0182] In some embodiments, the anti-MICA / B antibody comprises VL-CDR1, which contains an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8, 18, 28, 38, and 48. In one embodiment, the anti-MICA / B antibody comprises VL-CDR1 containing the amino acid sequence as described in SEQ ID NO: 8.

[0183] In some embodiments, the anti-MICA / B antibody comprises VL-CDR2, which contains an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 9, 19, 29, 39, and 49. In one embodiment, the anti-MICA / B antibody comprises VL-CDR2 containing the amino acid sequence as described in SEQ ID NO: 9.

[0184] In some embodiments, the anti-MICA / B antibody comprises VL-CDR3, which contains an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 10, 20, 30, 40, and 50. In one embodiment, the anti-MICA / B antibody comprises VL-CDR3 containing the amino acid sequence as described in SEQ ID NO: 10.

[0185] In some embodiments, the anti-MICA / B antibody comprises VH-CDR1 containing the amino acid sequence described in SEQ ID NO:5, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:6, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:7, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:8, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:9, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:10. In some embodiments, the anti-MICA / B antibody is non-fucosylated. In some embodiments, the anti-MICA / B antibody is hypofucosylated. In some embodiments, the anti-MICA / B antibody cross-competitively binds to human MICA / B with a reference antibody comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO:5, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:6, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:7, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:8, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:9, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:10. In some embodiments, the anti-MICA / B antibody binds to the same epitope as a reference antibody, which comprises VH-CDR1 containing the amino acid sequence described in SEQ ID NO:5, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:6, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:7, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:8, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:9, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:10.

[0186] In some embodiments, the anti-MICA / B antibody comprises VH-CDR1 containing the amino acid sequence described in SEQ ID NO:15, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:16, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:17, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:18, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:19, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:20. In some embodiments, the anti-MICA / B antibody is non-fucosylated. In some embodiments, the anti-MICA / B antibody is hypofucosylated. In some embodiments, the anti-MICA / B antibody cross-competitively binds to human MICA with a reference antibody comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO:15, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:16, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:17, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:18, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:19, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:20.

[0187] In some embodiments, the anti-MICA / B antibody comprises VH-CDR1 containing the amino acid sequence described in SEQ ID NO:25, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:26, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:27, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:28, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:29, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:30. In some embodiments, the anti-MICA / B antibody is non-fucosylated. In some embodiments, the anti-MICA / B antibody is hypofucosylated. In some embodiments, the anti-MICA / B antibody cross-competitively binds to human MICA with a reference antibody comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO:25, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:26, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:27, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:28, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:29, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:30.

[0188] In some embodiments, the anti-MICA / B antibody comprises VH-CDR1 containing the amino acid sequence described in SEQ ID NO:35, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:36, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:37, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:38, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:39, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:40. In some embodiments, the anti-MICA / B antibody is non-fucosylated. In some embodiments, the anti-MICA / B antibody is hypofucosylated. In some embodiments, the anti-MICA / B antibody cross-competitively binds to human MICA with a reference antibody comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO:35, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:36, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:37, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:38, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:39, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:40.

[0189] In some embodiments, the anti-MICA / B antibody comprises VH-CDR1 containing the amino acid sequence described in SEQ ID NO:45, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:46, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:47, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:48, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:49, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:50. In some embodiments, the anti-MICA / B antibody is non-fucosylated. In some embodiments, the anti-MICA / B antibody is hypofucosylated. In some embodiments, the anti-MICA / B antibody cross-competitively binds to human MICA with a reference antibody comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO:45, VH-CDR2 containing the amino acid sequence described in SEQ ID NO:46, VH-CDR3 containing the amino acid sequence described in SEQ ID NO:47, VL-CDR1 containing the amino acid sequence described in SEQ ID NO:48, VL-CDR2 containing the amino acid sequence described in SEQ ID NO:49, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO:50.

[0190] In some embodiments, the anti-MICA / B antibody comprises a VH containing an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:2, 12, 22, 32, and 42. In some embodiments, the VH contains an amino acid sequence selected from SEQ ID NO:2, 12, 22, 32, and 42.

[0191] In some embodiments, the anti-MICA / B antibody comprises a VL containing an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:4, 14, 24, 34, and 44. In some embodiments, the VL contains an amino acid sequence selected from SEQ ID NO:4, 14, 24, 34, and 44.

[0192] In some embodiments, the anti-MICA / B antibody comprises VH, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:2. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO:2. In some embodiments, the anti-MICA / B antibody comprises VL, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:4. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:4. In one embodiment, VH comprises the amino acid sequence described in SEQ ID NO:2, and VL comprises the amino acid sequence described in SEQ ID NO:4. In another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising VH containing the amino acid sequence described in SEQ ID NO:2 and VL containing the amino acid sequence described in SEQ ID NO:4. In another embodiment, VH comprises the amino acid sequence described in SEQ ID NO:2, and VL comprises the amino acid sequence described in SEQ ID NO:4, wherein the antibody is non-fucosylated. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a G-to-A mutation (also referred to as "G236A" according to EU designation) at position corresponding to residue 234 in SEQ ID NO:58.

[0193] In some embodiments, the anti-MICA / B antibody comprises VH, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:12. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO:12. In some embodiments, the anti-MICA / B antibody comprises VL, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:14. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:14. In another embodiment, VH comprises the amino acid sequence described in SEQ ID NO:12, and VL comprises the amino acid sequence described in SEQ ID NO:14. In another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising VH containing the amino acid sequence described in SEQ ID NO:12 and VL containing the amino acid sequence described in SEQ ID NO:14. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a G-to-A mutation at position corresponding to residue 234 in SEQ ID NO:58.

[0194] In some embodiments, the anti-MICA / B antibody comprises VH, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:22. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO:22. In some embodiments, the anti-MICA / B antibody comprises VL, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:24. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:24. In another embodiment, VH comprises the amino acid sequence described in SEQ ID NO:22, and VL comprises the amino acid sequence described in SEQ ID NO:24. In another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising VH containing the amino acid sequence described in SEQ ID NO:22 and VL containing the amino acid sequence described in SEQ ID NO:24. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a G-to-A mutation at position corresponding to residue 234 in SEQ ID NO:58.

[0195] In some embodiments, the anti-MICA / B antibody comprises VH, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:32. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO:32. In some embodiments, the anti-MICA / B antibody comprises VL, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:34. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:34. In another embodiment, VH comprises the amino acid sequence described in SEQ ID NO:32, and VL comprises the amino acid sequence described in SEQ ID NO:34. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:34 with a Ser residue insertion between positions 95 and 96 of SEQ ID NO:34. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:34 with an Ala residue insertion between positions 95 and 96 of SEQ ID NO:34. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:34 with a Gly residue insertion between positions 95 and 96 of SEQ ID NO:34. In another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising VH comprising the amino acid sequence described in SEQ ID NO:32 and VL comprising the amino acid sequence described in SEQ ID NO:34. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a G-to-A mutation at position corresponding to residue 234 in SEQ ID NO:58.

[0196] In some embodiments, the anti-MICA / B antibody comprises VH, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:42. In some embodiments, VH comprises the amino acid sequence described in SEQ ID NO:42. In some embodiments, the anti-MICA / B antibody comprises VL, which contains an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO:44. In some embodiments, VL comprises the amino acid sequence described in SEQ ID NO:44. In another embodiment, VH comprises the amino acid sequence described in SEQ ID NO:42, and VL comprises the amino acid sequence described in SEQ ID NO:44.

[0197] In another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising a VH containing the amino acid sequence described in SEQ ID NO:42 and a VL containing the amino acid sequence described in SEQ ID NO:44. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a mutation changing G to A at position corresponding to residue 234 in SEQ ID NO:58. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO:58, 62, 66, 70, and 74. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing an amino acid sequence selected from SEQ ID NO: 58, 130, 62, 66, 70, and 74. In some embodiments, the heavy chain amino acid sequence contains one or more deletions, substitutions, or mutations within the immunoglobulin constant region, such as within the CH1 domain, CH2 domain, CH3 domain, or hinge region.

[0198] In some embodiments, the anti-MICA / B antibody comprises a light chain having an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 60, 64, 68, 72, and 76. In some embodiments, the anti-MICA / B antibody comprises a light chain having an amino acid sequence selected from SEQ ID NO: 60, 64, 68, 72, and 76.

[0199] In some embodiments, the anti-MICA / B antibody comprises a heavy chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:58. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:130. In some embodiments, the anti-MICA / B antibody comprises a light chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO: 60. In some embodiments, the anti-MICA / B antibody comprises a light chain containing the amino acid sequence described in SEQ ID NO: 60. In one embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 58 and a light chain containing the amino acid sequence described in SEQ ID NO: 60. In one embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 130 and a light chain containing the amino acid sequence described in SEQ ID NO: 60. In another embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:58 and a light chain containing the amino acid sequence described in SEQ ID NO:60, wherein the antibody is non-fucosylated. In another embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:130 and a light chain containing the amino acid sequence described in SEQ ID NO:60, wherein the antibody is non-fucosylated. In another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising a heavy chain containing the amino acid sequence described in SEQ ID NO:58 and a light chain containing the amino acid sequence described in SEQ ID NO:60.

[0200] In some embodiments, the anti-MICA / B antibody comprises a heavy chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:62. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:62. In some embodiments, the anti-MICA / B antibody comprises a light chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:64. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a G-to-A mutation at position corresponding to residue 234 in SEQ ID NO:58. In some embodiments, the anti-MICA / B antibody comprises a light chain containing the amino acid sequence described in SEQ ID NO:64. In another embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:62 and a light chain containing the amino acid sequence described in SEQ ID NO:64. In yet another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody containing both a heavy chain containing the amino acid sequence described in SEQ ID NO:62 and a light chain containing the amino acid sequence described in SEQ ID NO:64.

[0201] In some embodiments, the anti-MICA / B antibody comprises a heavy chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:66. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:66. In some embodiments, the anti-MICA / B antibody comprises a light chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:68. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a G-to-A mutation at position corresponding to residue 234 in SEQ ID NO:58. In some embodiments, the anti-MICA / B antibody comprises a light chain containing the amino acid sequence described in SEQ ID NO:68. In another embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:66 and a light chain containing the amino acid sequence described in SEQ ID NO:68. In yet another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody containing a heavy chain containing the amino acid sequence described in SEQ ID NO:66 and a light chain containing the amino acid sequence described in SEQ ID NO:68.

[0202] In some embodiments, the anti-MICA / B antibody comprises a heavy chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO: 70. In some embodiments, the anti-MICA / B antibody comprises a heavy chain constant region containing a mutation changing G to A at position corresponding to residue 234 in SEQ ID NO: 58. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 70. In some embodiments, the anti-MICA / B antibody comprises a light chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:72. In some embodiments, the anti-MICA / B antibody comprises a light chain containing the amino acid sequence described in SEQ ID NO:72. In another embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:70 and a light chain containing the amino acid sequence described in SEQ ID NO:72. In yet another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising a heavy chain containing the amino acid sequence described in SEQ ID NO:70 and a light chain containing the amino acid sequence described in SEQ ID NO:72.

[0203] In some embodiments, the anti-MICA / B antibody comprises a heavy chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:74. In some embodiments, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:74. In some embodiments, the anti-MICA / B antibody comprises a light chain having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence described in SEQ ID NO:76. In some embodiments, the anti-MICA / B antibody comprises a light chain containing the amino acid sequence described in SEQ ID NO:76. In another embodiment, the anti-MICA / B antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO:74 and a light chain containing the amino acid sequence described in SEQ ID NO:76. In yet another embodiment, the anti-MICA / B antibody cross-competitively binds to human MICA / B with an antibody comprising a heavy chain containing the amino acid sequence described in SEQ ID NO:74 and a light chain containing the amino acid sequence described in SEQ ID NO:76.

[0204] In some embodiments, the anti-MICA / B antibody described herein is selected from IgG1, IgG2, IgG3, IgG4, or variants thereof. In one embodiment, the anti-MICA / B antibody described herein is an IgG1 antibody. In some embodiments, the anti-MICA / B antibody described herein is unfucosylated. In some embodiments, the anti-MICA / B antibody described herein is hypofucosylated.

[0205] In some embodiments, the anti-MICA / B antibody described herein is a human antibody. In some embodiments, the anti-MICA / B antibody described herein is a humanized antibody. In some embodiments, the anti-MICA / B antibody described herein is a chimeric antibody.

[0206] In some embodiments, the anti-MICA / B antibody described herein comprises a heavy chain constant region containing a mutation that changes G to A at the position corresponding to residue 234 in SEQ ID NO:58.

[0207] A. Human MICA / B epitopes

[0208] In some embodiments, anti-MICA / B antibodies bind to specific epitopes on human MICA / B. As used herein, the term "epitaph" includes any determinant capable of binding to an antigen-binding protein, such as an antibody. An epitope is a region of an antigen that binds to an antigen-binding protein targeting that antigen, and when the antigen is a protein, the epitope includes specific amino acids that directly contact the antigen-binding protein. Epitope determinants may include chemically active surface groups of a molecule (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups) and may have specific three-dimensional structural features and / or specific charge features. Generally, antibodies specific to a particular target antigen will preferentially recognize epitopes on that target antigen in complex mixtures of proteins and / or macromolecules.

[0209] In some embodiments, the epitope comprises at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids. Where the epitope comprises more than one amino acid, this more than one amino acid may be consecutive, such as G254 and D255; or separated by more than one amino acid that does not directly interact with or is not required for binding to anti-MICA / B antibodies, such as D255 and L257. In some embodiments, the epitope comprises two consecutive amino acids, three consecutive amino acids, four consecutive amino acids, five consecutive amino acids, six consecutive amino acids, seven consecutive amino acids, eight consecutive amino acids, nine consecutive amino acids, ten consecutive amino acids, or more than ten consecutive amino acids. In some embodiments, the epitope comprises at least two amino acids, which are not consecutive positions but are close in their three-dimensional conformation when human MICA / B is present.

[0210] Epitopes on antigens, such as those on human MICA / B that bind to anti-MICA / B antibodies, can be identified using any method known in this art. In some embodiments, epitopes are identified using yeast surface display. In some embodiments, epitopes are identified by hydrogen / deuterium exchange mass spectrometry (HDX-MS).

[0211] In some embodiments, the anti-MICA / B antibody binds to an epitope on the surface of human MICA. In some embodiments, the anti-MICA / B antibody binds to an epitope within the α3 domain of human MICA.

[0212] In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 190 and 230 of SEQ ID NO:51. In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 195 and 225 of SEQ ID NO:51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 200 and 221 of SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the anti-MICA / B antibody binds to an epitope on human MICA containing L201-N220 corresponding to SEQ ID NO:51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA containing SEQ ID NO:56.

[0213] In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 230 and 260 of SEQ ID NO:51. In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 235 and 255 of SEQ ID NO:51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 237 and 253 of SEQ ID NO:51. In some embodiments, as determined by hydrogen / deuterium exchange mass spectrometry (HDX-MS), the anti-MICA / B antibody binds to an epitope on human MICA containing T238-Q252 corresponding to SEQ ID NO:51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA containing SEQ ID NO:57.

[0214] In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to epitopes on human MICA that include L201-N220 and T238-Q252 corresponding to SEQ ID NO:51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to epitopes on human MICA that include SEQ ID NO:56 and 57.

[0215] In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 140 and 170 of SEQ ID NO: 51. In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 145 and 165 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 149 and 164 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA that includes SEQ ID NO: 52.

[0216] In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 220 and 250 of SEQ ID NO: 51. In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 225 and 245 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 230 and 239 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA containing SEQ ID NO: 53. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA containing N234 and / or L237 corresponding to SEQ ID NO: 51.

[0217] In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 245 and 275 of SEQ ID NO: 51. In some embodiments, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 250 and 270 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 252 and 268 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA containing SEQ ID NO: 55. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 254 and 266 of SEQ ID NO: 51. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA that comprises SEQ ID NO:54. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to an epitope on human MICA that comprises amino acids D255-Q265 and W267 corresponding to SEQ ID NO:51.

[0218] In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to epitopes on human MICA, including SEQ ID NO: 52-54. In some embodiments, as determined by HDX-MS, the anti-MICA / B antibody binds to epitopes on human MICA, including SEQ ID NO: 52, 53, and 55.

[0219] In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 230 and 290 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 235 and 285 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 239 and 280 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 230 and 250 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 235 and 250 of SEQ ID NO:51. In some implementations, such as those identified by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 239 and 245 of SEQ ID NO: 51.

[0220] In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 245 and 275 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 250 and 270 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 253 and 268 of SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to an epitope on human MICA located between amino acids 257 and 265 of SEQ ID NO:51.

[0221] In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to the following epitopes, which comprise one or more amino acids selected from the group consisting of R240, Q241, D242, V244, G254, D255, L257, P258, G260, G262, Y264, W267, and R279 corresponding to SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to the following epitopes, which comprise one or more amino acids selected from the group consisting of G254, D255, L257, Y264, and W267 corresponding to SEQ ID NO:51. In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to the following epitopes, which comprise amino acids G254, D255, L257, Y264, and W267 corresponding to SEQ ID NO:51.

[0222] In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to the following epitope, which comprises one or more amino acids selected from the group consisting of R240, Q241, D242, V244, and R279 corresponding to SEQ ID NO:51.

[0223] In some embodiments, as determined by yeast surface display, the anti-MICA / B antibody binds to epitopes comprising one or more amino acids selected from SEQ ID NO:51, specifically P258, G260, G262, and Y264. In some embodiments, as determined by yeast surface display, the antibody binds to epitopes comprising P258, G260, G262, and Y264, specifically SEQ ID NO:51.

[0224] In some embodiments, when the anti-MICA / B antibody binds to the human MICA, as determined by crystallography, for example using the conditions described herein, the antibody is located at a distance of about 30 Å or less, 25 Å or less, 30 Å or less, about 15 Å or less, about 10 Å or less, about 9 Å or less, about 8 Å or less, about 7 Å or less, about 6 Å or less, about 5 Å or less, about 4 Å or less, about 3 Å or less, or about 2 Å or less from at least one of the following residues in the human MICA: R240, Q241, D242, V244, G254, D255, L257, P258, G260, G262, Y264, W267, and R279 corresponding to SEQ ID NO:51. In some embodiments, when the anti-MICA / B antibody binds to the human MICA, as determined by crystallography, for example using the conditions described herein, the antibody is located at a distance of about 10 angstroms or less from at least one of the following residues in the human MICA: R240, Q241, D242, V244, G254, D255, L257, P258, G260, G262, Y264, W267, and R279 corresponding to SEQ ID NO:51. In some embodiments, when the anti-MICA / B antibody binds to the human MICA, as determined by crystallography, for example using the conditions described herein, the antibody is located at a distance of about 10 angstroms or less from at least one of the following residues in the human MICA: G254, D255, L257, Y264, and W267 corresponding to SEQ ID NO:51. In some embodiments, when the anti-MICA / B antibody binds to the human MICA, as determined by crystallography, for example using the conditions described herein, the antibody is located at a distance of about 10 angstroms or less from at least one of the following residues in the human MICA: R240, Q241, D242, V244, and R279 corresponding to SEQ ID NO:51. In some embodiments, when the anti-MICA / B antibody binds to the human MICA, as determined by crystallography, for example using the conditions described herein, the antibody is located at a distance of about 10 angstroms or less from at least one of the following residues in the human MICA: P258, G260, G262, and Y264 corresponding to SEQ ID NO:51.In some embodiments, when the anti-MICA / B antibody binds to human MICA, as determined by X-ray cocrystallography, for example using the conditions described herein, the antibody is located at a distance of about 30 Å or less, 25 Å or less, 20 Å or less, about 15 Å or less, about 10 Å or less, about 9 Å or less, about 8 Å or less, about 7 Å or less, about 6 Å or less, about 5 Å or less, about 4 Å or less, about 3 Å or less, or about 2 Å or less from at least one of the following residues in human MICA: R240, Q241, D242, V244, G254, D255, L257, P258, G260, G262, Y264, W267, and R279 corresponding to SEQ ID NO:51.

[0225] In some embodiments, such as those determined by X-ray cocrystallography, the anti-MICA / B antibody binds to an epitope on human MICA comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some embodiments, such as those determined by X-ray cocrystallography, the anti-MICA / B antibody binds to an epitope on human MICA containing at least two residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some implementations, such as those determined by X-ray cocrystallography, the anti-MICA / B antibody binds to an epitope on human MICA containing at least four residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some embodiments, such as those determined by X-ray cocrystallography, the anti-MICA / B antibody binds to an epitope on human MICA containing at least six residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269. In some implementations, such as those determined by X-ray cocrystallography, the anti-MICA / B antibody binds to epitopes on human MICA, including T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269.

[0226] The VH domain or one or more of its CDRs described herein can be attached to a constant domain to form a heavy chain, such as a full-length heavy chain. Similarly, the VL domain or one or more of its CDRs described herein can be attached to a constant domain to form a light chain, such as a full-length light chain. The full-length heavy chain (which may be absent, depending on the case, except for C-terminal lysine (K) or except for C-terminal glycine and lysine (GK)) and the full-length light chain can be combined to form a full-length antibody.

[0227] The VH domains described herein can be fused with constant domains of naturally occurring or modified human IgG (e.g., IgG1, IgG2, IgG3, or IgG4), for example, as further described herein. For instance, a VH domain may comprise an amino acid sequence of any VH domain described herein fused with a constant region of human IgG (e.g., IgG1), such as the following amino acid sequence of a wild-type human IgG1 constant domain:

[0228]

[0229] Or it may be fused with the amino acid sequence of an allotype of SEQ ID NO:91 and have the following amino acid sequence:

[0230] (SEQ ID NO:92; allotype-specific amino acid residues are in bold and underlined).

[0231] The VH domain of an anti-MICA / B antibody may contain an amino acid sequence of any VH domain described herein fused with a no-effect constant region, such as the following no-effect human IgG1 constant domain amino acid sequence:

[0232] (SEQ ID NO:93; "IgG1.1f", comprising substitutions L234A, L235E, G237A, A330S, and P331S, wherein the substitutions are underlined)

[0233] or

[0234] (SEQ ID NO:94; "IgG1.3f", comprising substitutions L234A, L235E and G237A, wherein the substitutions are underlined).

[0235] For example, allotypes of IgG1 include K97R, D239E, and / or L241M (underlined and bolded) as numbered in SEQ ID NO:91. Within the full-length heavy chain constant region (e.g., MICA36 (SEQ ID NO:58)) and according to EU designations, these amino acid substitutions are designated K214R, D356E, and L358M. In some embodiments, the constant region of the anti-MICA / B antibody may further include one or more mutations or substitutions at amino acids L117, A118, G120, A213, and P214 (underlined) as numbered in SEQ ID NO:92, 93, and 94, or L234, A235, G237, A330, and P331 according to EU designations. In some embodiments, the constant region of the anti-MICA / B antibody includes one or more mutations or substitutions at amino acids L117A, A118E, G120A, A213S, and P214S in SEQ ID NO:91, or L234A, L235E, G237A, A330S, and P331S according to EU designations. The constant region of the anti-MICA / B antibody may also include one or more of the following mutations or substitutions: L117A, A118E, and G120A in SEQ ID NO:91, or L234A, L235E, and G237A according to EU designations.

[0236] Alternatively, the VH domain of the anti-MICA / B antibody may contain an amino acid sequence of any VH domain described herein fused with a human IgG4 constant region, such as the following human IgG4 amino acid sequence or a variant thereof:

[0237] (SEQ ID NO:95, containing S228P).

[0238] The VL domains described herein can be fused with constant domains of the human κ or λ light chain. For example, the VL domain of an anti-MICA / B antibody may comprise an amino acid sequence of any VL domain described herein fused with the following human IgG1κ light chain amino acid sequence:

[0239]

[0240] In some embodiments, the heavy chain constant region contains a lysine or another amino acid at its C-terminus, for example, it contains the following last amino acid in the heavy chain: LSPGK (SEQ ID NO:97). In some embodiments, the heavy chain constant region lacks one or more amino acids at its C-terminus and has, for example, the C-terminal sequence LSPG (SEQ ID NO:98) or LSP (SEQ ID NO:99).

[0241] The amino acid sequences of the illustrative heavy and light chains are described herein.

[0242] This article provides isolated anti-human MICA / B antibodies or their antigen-binding moieties, comprising:

[0243] (a1) Contains the heavy chain and light chain sequences of SEQ ID NO:58 and 60, respectively;

[0244] (a2) Contains the heavy chain and light chain sequences of SEQ ID NO:130 and 60, respectively;

[0245] (a3) Contains the heavy chain and light chain sequences of SEQ ID NO:62 and 64 respectively;

[0246] (a4) Contains the heavy chain and light chain sequences of SEQ ID NO:66 and 68, respectively;

[0247] (a5) Contains the heavy chain and light chain sequences of SEQ ID NO:70 and 72, respectively;

[0248] (a6) contains the heavy chain and light chain sequences of SEQ ID NO:74 and 76, respectively;

[0249] This antibody specifically binds to human MICA / B.

[0250] In some embodiments, the anti-MICA / B antibody comprises a combination of heavy and light chain sequences, such as those described herein in the preceding paragraphs, wherein the antibody comprises two heavy chains and two light chains, and may further comprise at least one disulfide bond linking the two heavy chains together. The antibody may also comprise a disulfide bond linking each light chain to each heavy chain.

[0251] Heavy and light chains (or their variable regions) containing at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% of the same amino acid sequence as any of the heavy or light chains described herein, such as SEQ ID NO: 58 and 60, can be used to form anti-human MICA / B antibodies with the desired characteristics, such as those further described herein. Exemplary variants are those containing allotropic variations (e.g., in constant domains) and / or mutations (such as those disclosed herein) in variable or constant regions. Heavy and light chains (or their variable regions) containing an amino acid sequence differing from any of the heavy or light chains described herein by at most 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 amino acid (by substitution, addition, or deletion) can be used to form anti-human MICA antibodies with the desired characteristics, such as those further described herein.

[0252] In some implementations, the anti-MICA / B antibody comprises a heavy chain variable region from a specific germline heavy chain immunoglobulin gene and / or a light chain variable region from a specific germline light chain immunoglobulin gene.

[0253] As demonstrated herein, human antibodies specific to MICA / B have been prepared, comprising either human germline products or variable regions derived from human germline heavy chains. Therefore, this document provides isolated monoclonal antibodies or their antigen-binding portions comprising either products derived from human VH germline genes or variable regions derived from human VH germline genes and any combination thereof, wherein the human VH germline gene is selected from the following: 3-09, 3-10, 3-33, 3-48, 5-51, 6-13, JH3b, JH4b, JH6b, and any combination thereof.

[0254] Human antibodies specific to MICA / B have been prepared, comprising either human germline products or light chain variable regions derived from human germline genes. Therefore, this document provides isolated monoclonal antibodies or their antigen-binding portions comprising either products derived from human VK germline genes or light chain variable regions derived from human VK germline genes, or any combination thereof, wherein the human VK germline gene is selected from the following: A27, L6, L18, JK1, JK2, JK3, JK5, or any combination thereof.

[0255] As shown in the figure, the anti-MICA / B antibodies described herein include those that contain both a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is itself a product of or derived from one of the human lineage VH genes listed above, and the light chain variable region is itself a product of or derived from one of the human lineage VK genes listed above.

[0256] As used herein, if the heavy and light chain variable regions of a human antibody are obtained from a system using human germline immunoglobulin genes, the antibody contains the variable regions that are themselves "products of a specific germline sequence" or "derived from a specific germline sequence." Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening human immunoglobulin gene libraries presented on phages with an antigen of interest. Human antibodies that are themselves "products of a human germline immunoglobulin sequence" or "derived from a human germline immunoglobulin sequence" can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is most similar to the human antibody sequence (i.e., the largest percentage of identity). Human antibodies that are themselves "products of a specific human germline immunoglobulin sequence" or "derived from a specific human germline immunoglobulin sequence" may contain amino acid differences compared to that germline sequence, due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, the selected human antibodies are typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and contain amino acid residues that identify the human antibody as human when compared with germline immunoglobulin amino acid sequences from other species (e.g., murine germline sequences). In some cases, the human antibody may be at least 95% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene, or even at least 96%, 97%, 98%, or 99%. Generally, human antibodies derived from a specific human germline sequence will show no more than 10 amino acid differences compared to the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, the human antibody may show no more than 5 amino acid differences compared to the amino acid sequence encoded by the germline immunoglobulin gene, or even no more than 4, 3, 2, or 1 amino acid differences.

[0257] Anti-MICA / B antibodies may include a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences and a light chain variable region containing CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences contain a specified amino acid sequence or a conserved modification thereof based on the anti-MICA / B antibody described herein, and wherein the antibody retains the desired functional properties of the anti-MICA / B antibody described herein.

[0258] Conserved amino acid substitutions may be performed in portions of the antibody, except in the CDR, or in portions of the antibody in addition to those in the CDR. For example, conserved amino acid modifications may be performed in the framework region or the Fc region. The variable region or heavy or light chain may contain 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 conserved amino acid substitutions relative to the anti-MICA / B antibody sequence provided herein. In some embodiments, the anti-MICA / B antibody comprises a combination of conserved and non-conserved amino acid modifications.

[0259] Engineered and modified antibodies are also provided, which can be prepared by engineering a modified antibody using an antibody having one or more of the VH and / or VL sequences disclosed herein as starting material. The modified antibody may have altered properties compared to the starting antibody. The antibody can be engineered by modifying one or more residues within one or two variable regions (i.e., VH and / or VL), such as one or more CDR regions and / or one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues within constant regions, for example, to alter the effector function of the antibody.

[0260] One type of variable region engineering that can be performed is CDR transplantation. Antibodies interact with target antigens primarily via amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequence within the CDR is more diverse among individual antibodies compared to the sequence outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the characteristics of naturally occurring specific antibodies by constructing expression vectors that graft CDR sequences from naturally occurring specific antibodies onto framework sequences from different antibodies with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; Winter's U.S. Patent No. 5,225,539; and Queen's U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370).

[0261] Therefore, some embodiments described herein relate to an isolated monoclonal antibody or its antigen-binding portion comprising a heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences described herein, and a light chain variable region containing the CDR1, CDR2, and CDR3 sequences described herein. Thus, such antibodies contain the VH and VL CDR sequences of the monoclonal antibodies described herein, and may also contain different framework sequences from these antibodies.

[0262] Such scaffold sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), and in Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M. et al. (1992), “The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups ofV H Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V H Segments Reveals a Strong Bias in their Usage, Eur. J. Immunol. 24: 827-836; the contents of each of these are expressly incorporated herein by reference.

[0263] In some embodiments, the framework sequence used in the anti-MICA / B antibody described herein is one that is structurally similar to the framework sequence used by the anti-MICA / B antibody described herein. The VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences may be transplanted to framework regions having sequences identical to those seen in germline immunoglobulin genes from which the framework sequence is derived, or the CDR sequences may be transplanted to framework regions containing one or more mutations compared to the germline sequence. For example, it has been found that, in certain circumstances, it is advantageous to mutate residues within the framework region to maintain or enhance the antigen-binding capacity of the antibody (see, for example, U.S. Patents Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0264] The engineered anti-MICA / B antibodies described herein include those in which scaffold residues within the VH and / or VL regions have been modified to, for example, improve antibody properties. Typically, such scaffold modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more scaffold residues to their corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain scaffold residues different from the germline sequence from which the antibody was derived. Such residues can be identified by comparing the antibody scaffold sequence with the germline sequence from which the antibody was derived. To restore the scaffold region sequence to its germline configuration, somatic mutations can be “backmutated” to germline sequences by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Antibodies exhibiting such “backmutations” are also intended to be covered. Another type of scaffold modification involves mutating one or more residues within the scaffold region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0265] Another type of variable region modification involves mutating amino acid residues in the VH and / or VL CDR1, CDR2, and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation, and the effect on antibody binding or other functions of interest can be assessed in in vitro or in vivo assays as described herein and provided in the examples. In some embodiments, conserved modifications are introduced (as discussed above). Mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically, no more than one, two, three, four, or five residues in the CDR regions are altered.

[0266] Methionine residues in the CDR of an antibody can be oxidized, causing potential chemical degradation and thus reducing antibody potency. Therefore, anti-MICA / B antibodies are also provided in which one or more methionine residues in the heavy chain and / or light chain CDRs are replaced with amino acid residues that do not undergo oxidative degradation. In some embodiments, the methionine residues in the CDRs of antibodies MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 are replaced with amino acid residues that do not undergo oxidative degradation.

[0267] Similarly, deamidation sites can be removed by anti-MICA / B antibodies, especially in CDRs.

[0268] The anti-MICA / B variable region described herein can be linked (e.g., covalently linked or fused) to Fc, such as IgG1, IgG2, IgG3, or IgG4 Fc, which can have any allotype or isoallotype, for example, for IgG1: G1m, G1ml(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3mll(b0), G3m5(b1), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Km1, Km2, Km3 (see, for example, Jefferies et al. (2009) mAbs 1:1).

[0269] Generally, the variable regions described herein can be linked to Fc regions containing one or more modifications to typically alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, antigen-dependent cytotoxicity, and / or antibody-dependent phagocytosis. Furthermore, the antibodies described herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation to change one or more functional properties of the antibody. Each of these embodiments is further described in detail below. The residues in the Fc region are numbered according to the Kabat EU index.

[0270] The Fc region encompasses domains derived from the constant region of immunoglobulins, including fragments, analogs, variants, mutants, or derivatives of that constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide homologous to the C-terminal region of an immunoglobulin, and may individually or in combination contain a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain.

[0271] Ig molecules interact with a variety of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγRs) that are specific to the IgG class of an antibody: FcγRI, FcγRII, and FcγRIII. It has been reported that sequences important for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is affected by its ability to bind to Fc receptors (FcRs).

[0272] In some embodiments, the Fc region is a variant Fc region, such as an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to the parental Fc sequence (e.g., an unmodified Fc polypeptide subsequently modified to produce a variant) to provide the desired structural features and / or biological activity.

[0273] Generally, variants of the constant region or a portion thereof (e.g., CH1, CL, hinge, CH2, or CH3 domains) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and / or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation or 1-10 or 1-5 mutations, or contain at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the corresponding wild-type region or domain (CH1, CL, hinge, CH2, or CH3 domains, respectively), subject to the constraint that the heavy chain constant region containing the specific variant retains the necessary biological activity.

[0274] For example, modifications can be made to the Fc region to produce Fc variants that, relative to the parental Fc, (a) increase or decrease antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP), (b) increase or decrease complement-mediated cytotoxicity (CDC), (c) increase or decrease affinity for C1q, and / or (d) increase or decrease affinity for the Fc receptor. Such Fc variants will generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications are considered particularly desirable. For example, the variant Fc region may include, for example, two, three, four, five, etc., substitutions at specific Fc region positions identified herein.

[0275] The variant Fc region may also contain sequence alterations involving the removal or substitution of amino acids involved in disulfide bond formation. Such removals prevent reactions with other cysteine-containing proteins present in host cells used to generate the anti-MICA / B antibody described herein. Even when cysteine ​​residues are removed, the single-stranded Fc domain can still form a non-covalently held dimer Fc domain. In some embodiments, the Fc region may be modified to make it more compatible with the selected host cell. For example, the PA sequence near the N-terminus of a typical native Fc region may be removed, which can be recognized by digestive enzymes in E. coli, such as proline iminopeptidase. In some embodiments, one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g., asparagine) confer a cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine). In some embodiments, sites involving interactions with complement, such as C1q binding sites, may be removed from the Fc region. For example, the EKK sequence of human IgG1 may be deleted or substituted. In some embodiments, sites affecting binding to the Fc receptor can be removed, preferably sites other than rescue receptor binding sites. In some embodiments, the Fc region can be modified to remove ADCC sites. ADCC sites are known in this art; for example, see Molec. Immunol. 29(5):633-9 (1992) regarding ADCC sites in IgG1. Specific examples of variant Fc domains are disclosed, for example, in WO 97 / 34631, WO 96 / 32478 and WO07 / 041635.

[0276] In some embodiments, the hinge region of the Fc is modified to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing them. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The alteration of the number of cysteine ​​residues in the hinge region of the Fc can, for example, promote the assembly of light and heavy chains, or increase or decrease antibody stability. In some embodiments, the Fc hinge region of the antibody is mutated to reduce the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain mediator of the Fc-hinge fragment to weaken the binding of the antibody to Staphylococcus aureus protein A (SpA) relative to the binding of the native Fc-hinge domain SpA. This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[0277] In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the antibody's effector function. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and / or 331 can be substituted with different amino acid residues to alter the antibody's affinity for the effector ligand while retaining the parent antibody's antigen-binding ability. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patents Nos. 5,624,821 and 5,648,260 to Winter et al.

[0278] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 may be substituted with different amino acid residues to give the antibody altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0279] In another example, one or more amino acid residues at positions 231 and 239 are altered to change the antibody's ability to fix complement. This method is further described in PCT Publication WO 94 / 29351 by Bodmer et al.

[0280] In another instance, the Fc region can be modified to enhance affinity for Fcγ and increase macrophage-mediated phagocytosis. See, for example, Richard et al., Mo. Cancer. Ther. 7(8):2517-27 (2008), which is incorporated herein by reference in its entirety. In some embodiments, the Fc region can be modified to increase affinity for FcγRIIa relative to repressive FcγRIIb. A specific point mutation, G236A (numbered according to the EU index), has been identified as increasing affinity for FcγRIIa relative to repressive FcγRIIb. This increased affinity for FcRIIa is associated with increased macrophage-mediated phagocytosis relative to native IgG1. In some embodiments, the Fc region of the anti-MICA / B antibody contains one or more mutations or combinations thereof selected from the following: G236A, I332E, S239 / I332E, I332E / G236A, and S239D / I332E / G236A. Other modifications to the Fc region can increase antibody-dependent cytotoxicity (ADCC), for example, by increasing affinity for activating receptors such as FcγRI and / or FcγRIIIa. For example, combinations of G236A substitution and G236A substitution with modifications that improve affinity for activating receptors such as FcγRI and / or FcγRIIIa (e.g., including, but not limited to, substitutions at 332 and 239) provide significantly improved ADCC relative to the parental WT antibody. See U.S. Patent No. 9,040,041, which is incorporated herein by reference in its entirety.

[0281] In another example, the Fc region can be modified to reduce antibody-dependent cytotoxicity (ADCC) and / or reduce affinity for the Fcγ receptor, by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 2 89, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications to enhance FcγR and complement interactions include (but are not limited to) substitutions of 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0282] Fc modifications that increase binding to the Fcγ receptor include amino acid modifications at any one or more of the following amino acid positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301 in the Fc region. 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, where the residues in the Fc region are numbered according to the EU index number in Kabat (WO00 / 42072).

[0283] As appropriate, the Fc region may contain non-naturally occurring amino acid residues at additional and / or alternative positions known to those skilled in the art (see, for example, U.S. Patents 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; 9,040,041; PCX Patent Publication WO 00 / 42072; WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; WO 04 / 074455; WO 04 / 099249; WO 04 / 063351; WO 05 / 070963; WO 05 / 040217; WO 05 / 092925; and WO 06 / 020114).

[0284] The affinity and binding properties of the Fc region to its ligands can be determined by a variety of in vitro analytical methods (based on biochemical or immunological assays) known in this technique. These methods include (but are not limited to) equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE assay) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize labeling on one or more components being tested and / or employ various detection methods, including (but not limited to) chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0285] In some embodiments, the antibody is modified to increase its biological half-life. Various methods are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn, for example, as described in U.S. Patent No. 6,277,375, by mutating one or more of the following residues: 252, 254, 256, 433, 435, 436. Specific illustrative substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al., to increase the biological half-life, the antibody can be modified in the CH1 or CL region to contain a rescue receptor-binding epitope acquired from two loops of the CH2 domain of the Fc region of IgG. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428 and 434, including, for example, 2591, 308F, 428L, 428M, 434S, 4341, 434F, 434Y and 434X1. Other variants that increase the binding of Fc to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al. 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Sciences). Chemistry,2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S(Dall Acqua et al. Journal of Immunology, 2002, 169: 5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry (281:23514-23524). Other modifications for regulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671.

[0286] In some embodiments, hybrid IgG isoforms with specific biological characteristics may be used. For example, an IgG1 / IgG3 hybrid variant may be constructed by substituting amino acids from IgG3 at different positions in the CH2 and / or CH3 regions of the IgG1 isoform. Thus, hybrid variant IgG antibodies may be constructed comprising one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In some embodiments described herein, an IgG1 / IgG2 hybrid variant may be constructed by substituting amino acids from IgG1 at different positions in the CH2 and / or CH3 regions of the IgG2 isoform. Therefore, hybrid variant IgG antibodies can be constructed, which contain one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (meaning the insertion of glycine at position 236) and 327A.

[0287] Furthermore, binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been located, and variants exhibiting modified binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 demonstrate modified binding to FcγRIII. Additionally, the following combined mutants, T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with the S239D / I332E and S239D / I332E / A330L mutations, which exhibit maximally increased affinity for FcγRIIIa, decreased binding to FcγRIIb, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introducing triple mutations into antibodies such as alemtuzumab (CD52 specific), trastuzumab (HER2 / neu specific), rituximab (CD20 specific), and cetuximab (EGFR specific) results in significantly enhanced in vitro ADCC activity, with the S239D / I332E variant showing enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgG1 mutants containing the L235V, F243L, R292P, Y300L, and P396L mutations have been identified in B-cell malignancy and breast cancer models. These mutants exhibit enhanced binding to FcγRIIIa and increased ADCC activity in transgenic mice expressing human FcγRIIIa (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other usable Fc mutants include: S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0288] Specific mutations at positions 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330, and 332 modify the binding of FcγRIIa and / or reduce the binding of FcγRIIb, resulting in enhanced ADCC and / or ADCP activity (Richards et al., Mol. Cancer Ther. 7(8): 2517-2527; U.S. Patent No. 9,040,041). Specifically, Fc variants that selectively modify the binding to one or more human activating receptors relative to FcγRIIb or selectively modify the binding to FcγRIIb relative to one or more activating receptors may include substitutions selected from the following: 234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D, 267D, 267E, 267Q, 268D, 268E, 293R, 295E, 324G, 324I, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, 332D, and 332E. Additional substitutions that can also be combined include other substitutions that modulate FcγR affinity and complement activity, including (but not limited to) 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333A, 333S, 334L, and 334A (US Patent No. 6,737,056; Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604; US Patent No. 6,528,624; Idusogie et al., 2001, J. Immunology 166:2571-2572). Preferred variants that are particularly suitable for combination with other Fc variants include those containing substitutions for 298A, 326A, 333A, and 334A. Additional substitutions that can be combined with selective variants of FcγR include 247L, 255L, 270E, 392T, 396L and 421K (US No. 10 / 754,922; US No. 10 / 902,588); and 280H, 280Q and 280Y (US No. 10 / 370,749).

[0289] When using the constant domain of IgG4, it may include a substitution for S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecule.

[0290] III. Non-fucosylation, low-fucosylation, and reduced fucosylation

[0291] In some embodiments, the glycosylation of the antibody can be modified. For example, glycosylated-free antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, which eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the antibody's affinity for the antigen. Such methods are further described in detail in U.S. Patents Nos. 5,714,350 and 6,350,861 to Co et al.

[0292] Glycosylation of the constant region on N297 can be prevented by mutating the N297 residue to another residue, such as N297A, and / or by mutating an adjacent amino acid, such as 298, thereby reducing glycosylation on N297.

[0293] The interaction between antibodies and FcγR can also be enhanced by modifying the glycan portion of each Fc fragment attached to the N297 residue. Specifically, the absence of the core fucose residue enhances ADCC by modifying the binding of IgG to the activating FcγRIIIA without altering antigen binding or CDC. Natsume et al. (2009) Drug Des. Devel. Ther. 3:7. There is compelling evidence that tumor-specific antibodies without fucosylation are converted to therapeutically enhanced antibodies in in vivo mouse models. Nimmerjahn and Ravetch (2005) Science 310:1510; Mossner et al. (2010) Blood 115:4393. Modification of antibody glycosylation can be achieved, for example, by expressing antibodies in host cells having a modified glycosylation mechanism. Cells having modified glycosylation mechanisms have been described in this technique and can be used as host cells for expressing the recombinant antibodies of the present invention to generate antibodies with modified glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α-(1,6)fucosyltransferase) (see U.S. Patent Application Publication No. 20040110704; Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87:614), so that antibodies expressed in these cell lines lack fucose on their carbohydrates. As another example, EP 1176195 also describes cell lines with a functionally disrupted FUT8 gene and cell lines with very little or no activity for adding fucose to N-acetylglucosamine (which binds to the Fc region of the antibody), such as the rat myeloma cell line YB2 / 0 (ATCCCRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line Lec13 with reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylated antibodies expressed in host cells. See also Shields et al. (2002) J. Biol. Chem. 277:26733. As described in PCT Publication WO 2006 / 089231, antibodies with modified glycosylation distributions can also be produced in eggs. Alternatively, antibodies with modified glycosylation distributions can be produced in plant cells such as Lemna. See, for example, U.S. Publication 2012 / 0276086. PCT Publication No. WO 99 / 54342 describes a cell line engineered to express a glycoprotein-modified glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminetransferase III (GnTIII)), such that the antibody expressed in the engineered cell line exhibits an increased bipartite GlcNac structure, resulting in increased ADCC activity of the antibody.See also: et al. (1999) Nat. Biotech. 17:176. Alternatively, the fucosylation residues of the antibody can be cleaved using a fucosidase. For example, the enzyme α-L-fucosidase removes fucosylation residues from the antibody. Tarentino et al. (1975) Biochem. 14:5516. As described in U.S. Patent No. 8,642,292, fucosylated degraded antibodies can also be produced in cells having a recombinant gene encoding an enzyme that uses GDP-6-deoxy-D-lythose-4-hexylose as a substrate, such as GDP-6-deoxy-D-lythose-4-hexylose reductase (RMD). Alternatively, cells can be grown in a culture medium containing a fucosylation analog that blocks the addition of fucosylation residues to N-linked glycans or glycoproteins, such as antibodies, produced by cells grown in that medium. U.S. Patent No. 8,163,551; WO 09 / 135181.

[0294] Because non-fucosylated antibodies exhibit significantly enhanced ADCC and / or ADCP compared to fucosylated antibodies, antibody formulations do not need to be completely free of fucosylated heavy chains to be suitable for use in this invention. Residual amounts of fucosylated heavy chains will not significantly interfere with the ADCC and / or ADCP activity of formulations that are substantially non-fucosylated heavy chains. Antibodies produced in conventional CHO cells fully capable of adding core fucose to N-glycans can still contain several percent to 15% non-fucosylated antibodies. Non-fucosylated antibodies can exhibit a tenfold higher affinity for CD16 and up to a 30- to 100-fold increase in ADCC and / or ADCP activity; therefore, even a small increase in the proportion of non-fucosylated antibodies can significantly increase the ADCC and / or ADCP activity of the formulation. Any formulation containing more non-fucosylated antibodies in the culture than produced in normal CHO cells will exhibit some level of ADCC and / or ADCP enhancement. Such antibody formulations are referred to herein as fucosylated-reduced formulations. Depending on the original level of non-fucosylation obtained from normal CHO cells, fucosylation-reduced formulations may contain 80%, 70%, 60%, 50%, 30%, 20%, 10%, and even 5% of non-fucosylated antibodies. Functionally, fucosylation reduction can be defined as a formulation exhibiting a 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, two-fold, three-fold, or greater enhancement of ADCC and / or ADCP compared to antibodies prepared in normal CHO cells, rather than referring to any fixed percentage of non-fucosylated species.

[0295] The level of non-fucosylation can be structurally defined. As used herein, “non-fucosylation” or “absence of fucosylation” (the terms are used synonymously) refers to a heavy chain deficiency of more than 95%, such as more than 96%, more than 97%, more than 98%, more than 99%, or 100%, in antibody formulations. The term “low fucosylation” refers to a heavy chain deficiency of more than 80% and less than or equal to 95% in antibody formulations; for example, a heavy chain deficiency of between 85% and 95%, between 80% and 85%, between 80% and 90%, between 85% and 90%, or between 90% and 95% in antibody formulations. The term “low fucosylation or non-fucosylation” refers to a heavy chain deficiency of 80% or more in antibody formulations. The term "reduced fucosylation" refers to a deficiency of fucose in the heavy chain of an antibody formulation, ranging from 10% to 80%, such as 20%-80%, 30%-80%, 40%-80%, 50%-80%, 60%-80%, 70%-80%, 20%-70%, 30%-70%, 40%-70%, 50%-70%, 60%-70%, 20%-60%, 30%-60%, 40%-60%, 50%-60%, 20%-50%, 30%-50%, 40%-50%, 20%-40%, 30%-40%, 10%-20%, 10%-30%, or 20%-30%.

[0296] In some embodiments, low-fucosylated or non-fucosylated anti-MICA / B antibodies may be generated in cells lacking enzymes essential for fucosylation (such as FUT8) (e.g., U.S. Patent No. 7,214,775), or in cells in which the exogenous enzyme is partially depleted in a pool of metabolic precursors for fucosylation (e.g., U.S. Patent No. 8,642,292), or in cells cultured in the presence of small molecule inhibitors of enzymes involved in fucosylation (e.g., WO09 / 135181).

[0297] The level of fucosylation in antibody formulations can be determined by any method known in this art, including (but not limited to) gel electrophoresis, capillary electrophoresis, liquid chromatography, and mass spectrometry. In some embodiments, the level of fucosylation in antibody formulations can be determined by hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC). In some embodiments, to determine the level of fucosylation in an antibody formulation, the sample is denatured and treated with PNG enzyme F to cleave the N-linked glycans, followed by analysis of the fucose content. LC / MS of the full-length antibody chain is an alternative method for detecting the level of fucosylation in antibody formulations, but the quantification of mass spectrometry is inherently less precise.

[0298] In some embodiments, the anti-MICA / B antibodies described herein may be de-fucosylated, or be hypo-fucosylated or non-fucosylated. In some embodiments, the anti-MICA / B antibodies described herein may be de-fucosylated. In some embodiments, the anti-MICA / B antibodies described herein may be hypo-fucosylated. In some embodiments, the anti-MICA / B antibodies described herein may be non-fucosylated.

[0299] In some embodiments, the anti-MICA / B antibody described herein may comprise i) one or more amino acid mutations in the Fc region to alter FcγR binding, and, where applicable ii) fucosylation reduction or elimination. In some embodiments, the anti-MICA / B antibody described herein may comprise one or more amino acid mutations in the Fc region to alter FcγR binding, and may be hypofucosylated or non-fucosylated. In some embodiments, the anti-MICA / B antibody described herein may comprise one or more amino acid mutations in the Fc region to alter FcγR binding, and may be non-fucosylated.

[0300] Another modification to the anti-MICA / B antibody described herein is PEGylation. Antibodies can be PEGylated to, for example, increase the antibody's biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. In some embodiments, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to cover any PEG form used to derive other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-cis-butenedimide. In some embodiments, the antibody to be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and are applicable to the anti-MICA / B antibody described herein. See, for example, Nishimura et al. EP 0 154 316 and Ishikawa et al. EP 0401 384.

[0301] IV. Antibody Physical Properties

[0302] Anti-MICA / B antibodies, such as those described herein, have some or all of the physical characteristics of the specific anti-MICA / B antibody described herein, such as those described in the examples.

[0303] The anti-MICA / B antibodies described in this article may contain one or more glycosylation sites in the variable regions of the light or heavy chains. Such glycosylation sites can lead to increased immunogenicity or altered pK of the antibody due to changes in antigen binding (Marshall et al., (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick et al., (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al., (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some cases, anti-MICA / B antibodies do not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0304] In some embodiments, the anti-MICA / B antibodies described herein do not contain an asparagine isomer site. Deamidation of asparagine can occur on the NG or DG sequence and result in the formation of isoaspartic residues, which introduce a kink into the polypeptide chain and reduce its stability (isoaspartic effect).

[0305] Each antibody will have a unique isoelectric point (pi), which is generally in the pH range between 6 and 9.5. The pi of IgG1 antibodies is typically in the pH range of 7 to 9.5, and the pi of IgG4 antibodies is typically in the pH range of 6 to 8. It is inferred that antibodies with pi outside the normal range may exhibit some degree of unfolding and instability under in vivo conditions. Therefore, anti-MICA / B antibodies can contain pi values ​​within the normal range. This can be achieved by selecting antibodies with pi within the normal range or by mutating charged surface residues.

[0306] Each antibody will have a characteristic melting temperature, with higher melting temperatures indicating greater overall in vivo stability (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). Generally, T MThe initial development temperature (i) can be greater than 60°C, greater than 65°C, or greater than 70°C. The melting point of the antibody can be measured using differential scanning calorimetry (Chen et al., (2003) Pharm Res 20:1952-60; Ghirlando et al., (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al., (2002) J. Chromatogr Sci 40:343-9).

[0307] In some implementations, antibodies that do not degrade rapidly are selected. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0308] In some embodiments, antibodies with minimal aggregation activity are selected, which could lead to triggering unwanted immune responses and / or altered or adverse pharmacokinetic properties. Generally, acceptable antibody aggregation is 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured using several techniques, including size exclusion column chromatography (SEC), high-performance liquid chromatography (HPLC), and light scattering. In some embodiments, the antibody exhibits a desired solubility, such as a commercially permissible solubility. In some embodiments, the antibodies described herein have a solubility of at least 10 mg / ml, at least 15 mg / ml, at least 20 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 60 mg / ml, or at least 70 mg / ml at neutral or weakly acidic pH.

[0309] In some embodiments, the antibody described herein has higher stability than a reference antibody. In some embodiments, the antibody described herein has a higher melting temperature than a reference antibody. In some embodiments, the antibody described herein has a lower tendency to aggregate than a reference antibody. In some embodiments, the antibody described herein has higher solubility than a reference antibody. In some embodiments, the antibody described herein has a higher absorption rate, lower toxicity, higher biological activity and / or target selectivity, better manufacturability, and / or lower immunogenicity compared to a reference antibody. The reference antibody may be another antibody or fragment thereof bound to MICA / B, or a conjugate thereof.

[0310] V. Methods for engineering antibodies

[0311] As discussed above, anti-MICA / B antibodies having the VH and VL sequences disclosed herein can be used to generate novel anti-MICA / B antibodies by modifying the VH and / or VL sequences or the constant regions attached thereto. Therefore, in another aspect described herein, the structural features of the anti-MICA / B antibodies described herein are used to generate structurally related anti-MICA / B antibodies that retain at least one functional property of the anti-MICA / B antibodies described herein, such as binding to human MICA / B and cynomolgus monkey MICA / B. For example, as discussed above, one or more CDR regions of MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 can be recombinantly combined with known framework regions and / or other CDRs to generate additional recombinantly engineered anti-MICA / B antibodies described herein. Other types of modifications include those described in the preceding sections. The starting material for the engineering methods is one or more of the VH and / or VL sequences provided herein or one or more of their CDR regions. To generate engineered antibodies, it is not necessary to actually prepare (i.e., express in protein form) an antibody having one or more of the VH and / or VL sequences or one or more of their CDR regions provided herein. Instead, the information contained in the sequence is used as starting material to generate a “second-generation” sequence derived from the initial sequence, and the “second-generation” sequence is subsequently prepared and expressed as a protein.

[0312] Therefore, this article provides a method for preparing the anti-MICA / B antibody described herein.

[0313] The modified antibody may exhibit at least one of the functional properties defined herein. The functional properties of the modified antibody may be assessed using standard analyses available in this art and / or described herein, such as those described in the examples (e.g., ELISA, FACS).

[0314] In some embodiments of the methods for engineering the anti-MICA / B antibodies described herein, mutations may be randomly or selectively introduced along all or part of the coding sequence of the anti-MICA / B antibody, and the resulting modified anti-MICA / B antibodies may be screened for binding activity and / or other functional properties as described herein. Mutation methods have been described in this art. For example, Short's PCT Publication WO 02 / 092780 describes a method for generating and screening antibody mutations using saturation mutagenesis, synthetic conjugation assembly, or a combination thereof. Alternatively, Lazar et al.'s PCT Publication WO 03 / 074679 describes a method for optimizing the physiochemical properties of antibodies using computational screening methods.

[0315] VI. Nucleic acid molecules

[0316] Another aspect described herein relates to nucleic acid molecules encoding the anti-MICA / B antibodies described herein. Nucleic acids may be present in whole cells, cell lysates, or in partially purified or substantially pure forms. A nucleic acid is “isolated” or “substantially pure” when purified to separate it from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, such as chromosomal DNA linked to isolated DNA in nature) or proteins, by means of standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in this field. See F. Ausubel et al., eds., (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule.

[0317] The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed from hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), the cDNA encoding the light and heavy chains of the antibody derived from the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage presentation technology), the nucleic acids encoding the antibody can be recovered from the library.

[0318] Some of the nucleic acid molecules described herein are those encoding the VH and VL sequences of antibodies MICA.36, MICA.52, MICA.54, MICA.2, and 71C2. Exemplary DNA sequences encoding the VH sequences of MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 are described in SEQ ID NO: 1, 11, 21, 31, and 41, respectively. Exemplary DNA sequences encoding the VL sequences of MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 are described in SEQ ID NO: 3, 13, 23, 33, and 43, respectively. Exemplary DNA sequences encoding the heavy chain sequences of MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 are described in SEQ ID NO: 59, 63, 67, 71, and 75, respectively. Exemplary DNA sequences encoding the light chain sequences MICA.36, MICA.52, MICA.54, MICA.2 and 71C2 are described in SEQ ID NO:61, 65, 69, 73 and 77.

[0319] Exemplary nucleic acids encoding the mature VH and VL domains of the MICA.36 antibody are described as SEQ ID NO:1 and 3, respectively. Exemplary nucleic acids encoding the mature heavy chain of the MICA.36 antibody are described as SEQ ID NO:59 and 131, and an exemplary nucleic acid encoding the mature light chain of the MICA.36 antibody is described as SEQ ID NO:61.

[0320] Exemplary nucleic acids encoding the mature VH and VL domains of the MICA.52 antibody are described as SEQ ID NO:11 and 13, respectively. Exemplary nucleic acids encoding the mature heavy chain of the MICA.52 antibody are described as SEQ ID NO:63, and exemplary nucleic acids encoding the mature light chain of the MICA.52 antibody are described as SEQ ID NO:65.

[0321] Exemplary nucleic acids encoding the mature VH and VL domains of the MICA.54 antibody are described as SEQ ID NO:21 and 23, respectively. Exemplary nucleic acids encoding the mature heavy chain of the MICA.54 antibody are described as SEQ ID NO:67, and exemplary nucleic acids encoding the mature light chain of the MICA.54 antibody are described as SEQ ID NO:69.

[0322] Exemplary nucleic acids encoding the mature VH and VL domains of the MICA.2 antibody are described as SEQ ID NO:31 and 33, respectively. Exemplary nucleic acids encoding the mature heavy chain of the MICA.2 antibody are described as SEQ ID NO:71, and exemplary nucleic acids encoding the mature light chain of the MICA.2 antibody are described as SEQ ID NO:73.

[0323] Exemplary nucleic acids encoding the mature VH and VL domains of the 71C2 antibody are described as SEQ ID NO:41 and 43, respectively. Exemplary nucleic acids encoding the mature heavy chain of the 71C2 antibody are described as SEQ ID NO:75, and exemplary nucleic acids encoding the mature light chain of the 71C2 antibody are described as SEQ ID NO:77.

[0324] The exemplary nucleic acids described above may further include the signal peptide described in SEQ ID NO:100-105. The nucleotide sequence encoding the signal peptide is set forth in SEQ ID NO:106-107. In some embodiments, the signal peptide comprises MRAWIFFLLCLAGRALA (SEQ ID NO:105).

[0325] The nucleic acid molecules described in this article can be modified to remove specific sequences (e.g., restriction enzyme recognition sequences) or to optimize codons.

[0326] A method for obtaining MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 may include expressing the heavy and light chains in a cell line containing nucleotide sequences encoding a heavy chain and a light chain and a signal peptide (e.g., SEQ ID NO:59 and 61, or SEQ ID NO:131 and 61, respectively, for MICA.36). A method for obtaining MICA.36, MICA.52, MICA.54, MICA.2, and 71C2 may include expressing the heavy and light chains in a cell line containing nucleotide sequences encoding a heavy chain and a light chain and a signal peptide. Host cells containing these nucleotide sequences are included herein.

[0327] Once the DNA fragments encoding the VH and VL regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques to, for example, convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. In this context, the term "operatively linked" is intended to mean that the two DNA fragments are linked in such a way that the amino acid sequences encoded by the two DNA fragments remain within the same reading frame.

[0328] Separated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably ligating the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in this technique (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, such as the IgG1 region. For Fab fragment heavy chain genes, the VH-encoding DNA is operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0329] Separated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in this technique (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be either a κ or λ constant region.

[0330] To generate the scFv gene, DNA fragments encoding VH and VL are operatively ligated to a flexible linker, such as another fragment encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as adjacent single-stranded proteins, wherein the VL and VH regions are linked by the flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0331] This article also provides nucleic acid molecules encoding sequences homologous to the VH and VL sequences of antibodies MICA.36, MICA.52, MICA.54, MICA.2, and 71C2. Exemplary nucleic acid molecules encode VH and VL sequences that are at least 70% identical to those encoding the VH and VL sequences of antibodies MICA.36, MICA.52, MICA.54, MICA.2, and 71C2, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. This article also provides nucleic acid molecules with conserved substitutions (i.e., substitutions that do not alter the resulting amino acid sequence during nucleic acid molecule translation), for example, for codon optimization.

[0332] Also provided are nucleic acids encoding the VH and / or VL regions of anti-MICA / B antibodies, such as those described herein, said nucleic acid containing at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same nucleotide sequence as any nucleotide sequence encoding the VH and / or VL regions of the anti-MICA / B antibodies described herein.

[0333] Also provided are nucleic acids encoding anti-MICA / B antibodies, such as the heavy and / or light chains of the anti-MICA / B antibodies described herein, said nucleic acid containing at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same nucleotide sequence as any of the heavy and / or light chains encoding the anti-MICA / B antibodies described herein.

[0334] VII. Antibody production

[0335] The monoclonal anti-MICA / B antibodies described herein can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization is preferred in principle, other techniques for producing monoclonal antibodies may also be used, such as viral or oncogenic transformation of B lymphocytes, and phage presentation using human antibody gene libraries.

[0336] The preferred animal system for preparing hybridomas is the mouse system. The production of hybridomas in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in this art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0337] The chimeric or humanized anti-MICA / B antibodies described herein can be prepared based on the sequences of murine monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest using standard molecular biology techniques and engineered to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to generate chimeric antibodies, murine variable regions can be linked to human constant regions using methods known in this art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To generate humanized antibodies, murine CDR regions can be inserted into human scaffolds using methods known in this art (see, for example, U.S. Patent No. 5,225,539 to Winter; and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0338] In some implementations, the anti-MICA / B antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies against MICA / B can be produced using transgenic or transchromosomal mice carrying a portion of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “human Ig mice.”

[0339] (Medarex, Inc.) contains a human immunoglobulin gene microlobe encoding sequences of unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulins; and target mutations that inactivate endogenous μ and κ chain loci (see, for example, Lonberg et al., (1994) Nature 368(6474):856-859). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations to produce high-affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), ibid.; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. NYAcad. Sci. 764:536-546). The preparation and uses of HuMab mice and the genomic modifications carried by these mice are further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851.In addition, see all of Lonberg and Kay’s U.S. Patents Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299 and 5,770,429; Surani et al.’s U.S. Patent No. 5,545,807; and Lonberg and Kay’s PCT Publications Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884 and WO Case No. 99 / 45962; and PCT Publication No. WO 01 / 14424 by Korman et al.

[0340] In some embodiments, the anti-MICA / B antibodies described herein are bred using mice carrying human immunoglobulin sequences on transgenic and transchromosomal translocations, such as mice carrying human heavy chain transgenic and human light chain transchromosomal translocations. Such mice (referred to herein as "KM mice") are described in detail in PCT Publication WO 02 / 43478 by Ishida et al.

[0341] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in this technology and can be used to breed the anti-MICA / B antibodies described herein. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described, for example, in U.S. Patents Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 to Kucherlapati et al.

[0342] Furthermore, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in this technology and can be used to breed the anti-MICA / B antibodies described herein. For example, mice carrying both human heavy chain transchromosomal and human light chain transchromosomal chromosomes, known as "TC mice," can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Additionally, cattle carrying both human heavy chain and light chain transchromosomal chromosomes have been described in this technology (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to breed the anti-MICA / B antibodies described herein.

[0343] The additional mouse system described in this technology for cultivating human antibodies, such as human anti-MICA / B antibodies, includes (i) (ii) Mice (Regeneron Pharmaceuticals, Inc.), wherein endogenous mouse heavy and light chain variable regions have been replaced by homologous recombination with human heavy and light chain variable regions, the variable regions being operatively linked to endogenous mouse constant regions, such that a chimeric antibody (human V / mouse C) is cultured in mice and subsequently transformed into a fully human antibody using standard recombinant DNA technology; and (ii) The mice (Merus Biopharmaceuticals, Inc.) contain unrearranged human heavy chain variable regions but a single rearranged human common light chain variable region. The use of such mice and antibodies derived from them is described in, for example, WO 2009 / 15777, US2010 / 0069614, WO 2011 / 072204, WO 2011 / 097603, WO 2011 / 163311, WO 2011 / 163314, WO 2012 / 148873, US 2012 / 0070861, and US 2012 / 0073004.

[0344] The human monoclonal anti-MICA / B antibodies described herein can also be prepared using phage presentation methods for screening human immunoglobulin gene libraries. Such phage presentation methods for isolating human antibodies are established in this art. See, for example: U.S. Patents 5,223,409, 5,403,484, and 5,571,698 to Ladner et al.; U.S. Patents 5,427,908 and 5,580,717 to Dower et al.; U.S. Patents 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patents 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 to Griffiths et al.

[0345] The human monoclonal anti-MICA / B antibody described herein can also be prepared using SCID mice in which human immune cells have been reconstituted to produce a human antibody response upon immunization. Such mice are described, for example, in U.S. Patents 5,476,996 and 5,698,767 to Wilson et al.

[0346] VII.A. Immunity

[0347] To generate fully human antibodies against MICA / B, transgenic or transchromatic mice (e.g., HCo2, HCo7, or KM mice) containing human immunoglobulin genes can be immunized with purified or enriched formulations of human MICA / B antigens (e.g., MICA*002, MICA*004, MICA*008, MICA*009, MICB*005, or any combination thereof) and / or cells expressing MICA or fragments thereof, as described, for example, by Lonberg et al., (1994) Nature 368(6474):856-859; Fishwild et al., (1996) Nature Biotechnology 14:845-851; and WO 98 / 24884 for other antigens. Alternatively, mice can be immunized with DNA encoding human MICA / B or fragments thereof. In some embodiments, mice may be 6-16 weeks old at the time of the first infusion. For example, purified or enriched formulations of recombinant MICA / B antigen (5-50 μg) can be used for intraperitoneal immunization of HuMAb mice. If immunization with purified or enriched formulations of MICA / B antigen does not produce antibodies, mice can be immunized with MICA / B-expressing cells, such as cell lines, to promote an immune response. Exemplary cell lines include stable CHO and Raji cell lines that overexpress MICA / B.

[0348] Cumulative experience with various antigens has shown that HuMAb transgenic mice respond best when initially immunized with an antigen-containing Ribi adjuvant via intraperitoneal (IP) or subcutaneous (SC) immunization, followed by weekly IP / SC immunizations with the antigen-containing Ribi adjuvant (up to a total of 10 times). Immune responses can be monitored throughout the immunization protocol, with plasma samples obtained via retroorbital blood collection. Plasma can be screened by ELISA and FACS (described below), and mice with sufficient titers of anti-MICA / B human immunoglobulin can be used for fusion. Mice are boosted with the antigen intravenously for 3 days, followed by sacrifice and removal of the spleen and lymph nodes. Two to three fusions are expected for each immunization. Typically, 6 to 24 mice are immunized for each antigen. HCo7, HCl2, and KM strains are commonly used. Alternatively, both HCo7 and HCl2 transgenes can be co-bred into a single mouse carrying two different human heavy chain transgenes (HCo7 / HCol2).

[0349] VII.B. Hybridomas that produce monoclonal antibodies against MICA / B.

[0350] To generate hybridomas for producing the human monoclonal anti-MICA / B antibodies described herein, spleen cells and / or lymph node cells from immunized mice can be isolated and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of spleen lymphocytes from immunized mice can be fused with Sp2 / 0 non-secreting mouse myeloma cells (ATCC, CRL 1581) in the presence of PEG. Cells can be seeded in a flat-bottomed microtiter dish and then cultured in selective medium. After several weeks, cells can be cultured in medium. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once significant hybridoma growth has occurred, the culture medium can typically be monitored after 10–14 days. Antibody-secreting hybridomas can be re-inoculated for further screening, and if still positive for human IgG, the monoclonal antibody can be subcloned at least twice using a limiting dilution method. Stable subclones can then be cultured in vitro to produce small amounts of antibodies in tissue culture medium, which are then used for characterization.

[0351] To purify human monoclonal antibodies, the selected hybridomas were grown in two-liter rotary flasks for purification. The supernatant was filtered and concentrated, followed by affinity chromatography using protein A-agarose (Pharmacia, Piscataway, NJ). The eluted IgG was examined for purity by gel electrophoresis and high-performance liquid chromatography. The buffer solution could be replaced with PBS, and the concentration could be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies could be aliquoted and stored.

[0352] VII.C. Transfected tumors that produce monoclonal antibodies against MICA / B

[0353] Antibodies can be generated in host cell transfection tumors using, for example, a combination of recombinant DNA technology and gene transfection methods well known in this technique (Morrison, S. (1985) Science 229:1202).

[0354] For example, to express an antibody or an antibody fragment thereof, DNA encoding a portion or full length of the light and heavy chains can be obtained using standard molecular biology techniques (e.g., PCR amplification using a hybridoma expressing the antibody of interest or cDNA cloning), and said DNA can be inserted into an expression vector such that the gene is operatively linked to transcriptional and translational control sequences. In this case, the term "operatively linked" means that the antibody gene is attached to the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the host cell used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector using standard methods (e.g., attachment of the antibody gene fragment to complementary restriction sites on the vector, or blunt-end ligation where no restriction sites are present). The light and heavy chain variable regions of the anti-MICA / B antibody described herein can be used to generate full-length antibody genes of any antibody isotype by inserting the variable regions into an expression vector that encodes the heavy and light chain constant regions of the desired isotype, thereby enabling V H The C segment is operably connected within the carrier. H Section and V L The C segment is operably connected within the carrier. L It is done in sections.

[0355] Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector so that the signal peptide is co-located with the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0356] In some embodiments, the following signal peptides derived from the heavy and light chains of human antibodies may be used: MDWTWRVFCLLAVAPGAHS (SEQ ID NO: 100); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 101); MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 102); MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 103); MDMRVPAQLLGLLLWLPGARC (SEQ ID NO: 104) or MRAWIFFLLCLAGRALA (SEQ ID NO: 105). In some embodiments, the signal sequence used to express any of the anti-MICA / B antibodies described herein is SEQ ID NO: 105. The heavy and light chains of the anti-MICA / B antibody may be expressed together with individual signal sequences linked to the chains in a hybridoma in which the heavy and light chains are cloned. The following are signal sequences for various anti-MICA / B antibodies, as present in hybridomas from which the antibodies are cloned. These signal sequences can be used to express the same antibody or another antibody:

[0357] In some embodiments, the heavy and light chains of the anti-MICA / B antibody (e.g., MICA.36) may be engineered to have signal sequences different from those present in hybridomas from which the heavy and light chains are cloned. Examples of such sequences include (but are not limited to) the following:

[0358] (i) Nucleic acid sequence used as the signal sequence for the heavy chain:

[0359]

[0360] (ii) Nucleic acid sequences used for signal sequences in the light chain:

[0361]

[0362] (iii) The amino acid sequence used for the signal sequence of the heavy chain and the light chain: MRAWIFFLLCLAGRALA (SEQ ID NO: 105).

[0363] In addition to the antibody chain gene, recombinant expression vectors can also carry regulatory sequences that control the expression of the antibody chain gene in host cells. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control components (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those familiar with this technique will understand that the design of expression vectors (including the selection of regulatory sequences) depends on factors such as the choice of host cells to be transformed and the desired protein expression level. Preferred regulatory sequences for mammalian host cell expression include viral components that guide high protein expression levels in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-hemoglobin promoters, can be used. Furthermore, the regulatory components consist of sequences from different sources, such as the SRa promoter system, which contains sequences from the early SV40 promoter and long terminal repeat sequences from human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466-472).

[0364] In addition to the antibody chain gene and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and optional marker genes. Optional marker genes facilitate selection of host cells already incorporating the vector (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017, all granted to Axel et al.). For instance, in host cells already incorporating the vector, optional marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate. Preferred optional marker genes include the dihydrofolate reductase (DHFR) gene (for dhfr-host cell selection / amplification with methotrexate) and the neo gene (for G418 selection).

[0365] For the expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, and similar techniques.

[0366] It is possible to express the anti-MICA / B antibodies described herein in prokaryotic or eukaryotic host cells, such as mammalian cells.

[0367] Certain mammalian host cells used to express the recombinant anti-MICA / B antibody described herein include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with optional DHFR markers, such as those described in RJ Kaufman and P.A. Sharp (1982) Mol. Biol. 759:601-621), NSO myeloma cells, COS cells, and SP2 cells. Specifically, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient to allow expression of the antibody in the host cell or, more preferably, by secreting the antibody into the culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0368] VIII. Immunoconjugates, Antibody Derivatives, and Diagnostics

[0369] The anti-MICA / B antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose, the antibody (or its binding fragment) can be conjugated with a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents for whole-body imaging are detectable labels including radioisotopes, while for sample testing, suitable agents are radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags.

[0370] The detectable label that can be attached to any anti-MICA / B antibody described herein can be any of the various types currently used in the field of in vitro diagnostics, including microparticle labels, including metal sols such as colloidal gold; isotopes, such as I... 125 or Tc 99This includes, for example, peptide chelators of the N2S2, N3S, or N4 type; chromophores, including fluorescent markers, luminescent markers, phosphorescent markers, and analogs; and enzyme labels that convert a given substrate into a detectable label, and polynucleotide tags that are revealed after amplification (such as by polymerase chain reaction). Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and analogs. For example, the label can be alkaline phosphatase, which is presented by reacting 1,2-dioxane substrates (such as adamantyl methoxyphosphoryloxyphenyl dioxane dioxane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decyl}-4-yl)phenyl phosphate disodium salt (CSPD)) and CDP and Alternatively, the presence or formation of chemiluminescence can be measured after conversion of other luminescent substrates familiar to those skilled in this technique (e.g., suitable lanthanide elements, such as chelates of terbium(III) and europium(III)). The detection method is determined by the chosen label. The presence of the label or its reaction product can be observed with the naked eye if it is labeled as a particulate and accumulates in an appropriate amount, or using instruments such as spectrophotometers, luminometers, fluorometers, and the like, all according to standard practice.

[0371] In some implementations, the conjugation method produces substantially (or nearly) non-immunogenic bonds, such as peptide bonds (i.e., amide bonds), sulfur bonds (sterically hindered), disulfide bonds, hydrazone bonds, and ether bonds. These bonds are nearly non-immunogenic and exhibit reasonable stability in serum (see, for example, Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009 / 059278; WO 95 / 17886).

[0372] Depending on the biochemical properties of the moiety and the antibody, different conjugation strategies may be employed. In the case of a naturally occurring or recombinant moiety having 50 to 500 amino acids, standard procedures describing the chemistry for synthesizing protein conjugates exist in textbooks and can be readily followed by a skilled technician (see, for example, Hackenberger, CPR and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, the maleic imide moiety is used to react with cysteine ​​residues within the antibody or moiety. This is a particularly suitable conjugation chemistry when using, for example, a Fab or Fab' fragment of the antibody. Alternatively, in some embodiments, conjugation with the C-terminus of the antibody or moiety is performed. C-terminal modification of proteins (e.g., Fab fragments) can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0373] Generally, site-specific reactions and covalent coupling are based on converting native amino acids into amino acids with reactivity orthogonal to that of other functional groups present. For example, specific cysteine ​​residues in rare sequence cases can be enzymatically converted into aldehydes (see Frese, MA and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain desired amino acid modifications by utilizing the specific enzymatic reactivity of certain enzymes with native amino acids in given sequence cases (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al., Chem. Biol. 15 (2008) 128-136; and the protease-catalyzed formation of CN bonds is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reactions and covalent coupling can also be achieved through selective reactions of terminal amino acids with appropriate modifying agents.

[0374] The reactivity of the N-terminal cysteine ​​with benzonitrile (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling.

[0375] Natural chemical linkages can also depend on C-terminal cysteine ​​residues (Taylor, E. Vogel; Imperiali, B., Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0376] US6437095B1 describes a conjugation method based on the fact that cysteine ​​in a negatively charged amino acid chain reacts faster with cysteine ​​in a positively charged amino acid chain.

[0377] Some may also be synthetic peptides or peptide mimics. In cases where peptides are chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf, AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295). Because a variety of orthogonal functional groups are essential and can be introduced into synthetic peptides, the conjugation of such peptides with linkers is standard chemistry.

[0378] To obtain monolabeled peptides, stoichiometric 1:1 conjugates can be separated from other conjugation by chromatography. This procedure can be facilitated by using dye-labeled binding pair members and charged linkers. By using such labeled and highly negatively charged binding pair members, monolabeled peptides are readily separated from unlabeled peptides and peptides carrying more than one linker, because differences in charge and molecular weight can be used for separation. Fluorescent dyes can be used to purify and separate the complex from unbound components (such as labeled monovalent binders).

[0379] In some implementations, the portion attached to the anti-MICA / B antibody is selected from the following: binding portion, labeling portion, and biologically active portion.

[0380] The anti-MICA / B antibodies described herein can also be conjugated with therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binding agents, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably bound via cleavable linkers, such as peptide, disulfide, or hydrazone linkers. In some embodiments, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 108), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600, 6,989,452 and 7,129,261; PCT Publications WO 02 / 096910, WO 07 / 038658, WO 07 / 051081, WO 07 / 059404, WO 08 / 083312 and WO 08 / 103693; U.S. Patent Publications 20060024317, 20060004081 and 20060247295.

[0381] In some implementations, the therapeutic agent is selected from the following: cytotoxic agents, non-cytotoxic drugs, radioactive agents, secondary antibodies, enzymes, anti-hypertrophic agents, and any combination thereof.

[0382] In some embodiments, the immunoconjugate comprises an anti-MICA / B antibody and a cytotoxin. The cytotoxin may be selected from any cytotoxin known in the art. In some embodiments, the cytotoxin is selected from: saurustoxin, monomethylolpropionate E (MMAE), maytansine, buprofen, chalcogenide, pyrrolobenzodiazepine, buprofen, sentamycin, SN38, doxorubicin, its derivatives, synthetic analogs, and any combination thereof. In some embodiments, the immunoconjugate comprises an anti-MICA / B antibody and cytotoxin A. In other embodiments, the immunoconjugate comprises an anti-MICA / B antibody and a non-cytotoxic drug.

[0383] In some embodiments, the immunoconjugate comprises an anti-MICA / B antibody and a radioactive agent. In some embodiments, the radioactive agent is a radioactive nucleotide. In some embodiments, the radioactive agent comprises radioactive iodine. In a particular embodiment, the radioactive agent comprises 131-iodine. In other embodiments, the radioactive agent comprises the radioactive isotope yttrium-90.

[0384] In some embodiments, the immunoconjugate comprises an anti-MICA / B antibody and a secondary antibody. The secondary antibody may be any antibody described in this invention, including (but not limited to) antibodies that specifically bind to proteins selected from: PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-2, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, VISTA, CD96, CD27, GITR, and any combination thereof. In one embodiment, the immunoconjugate comprises an anti-MICA / B antibody and an anti-PD-1 antibody. In another embodiment, the immunoconjugate comprises an anti-MICA / B antibody and nivolumab.

[0385] In one embodiment, the immunoconjugate comprises an anti-MICA / B antibody and PEGylated IL-2 or PEGylated IL-10.

[0386] In some embodiments, the immunoconjugate comprises an anti-MICA / B antibody and an enzyme. In some embodiments, the enzyme comprises glucose oxidase. In some embodiments, the enzyme comprises a peroxidase. In some embodiments, the enzyme comprises myeloperoxidase. In some embodiments, the enzyme comprises glucose oxidase. In some embodiments, the enzyme comprises horseradish peroxidase.

[0387] In some embodiments, the immunoconjugate comprises an anti-MICA / B antibody and an anti-hypertrophic agent. The anti-hypertrophic agent may be any such agent known in the art. In some embodiments, the anti-hypertrophic agent is epirubicin. In some embodiments, the anti-hypertrophic agent is a superantigen. In some embodiments, the superantigen is staphylococcal enterotoxin A (SEA / E-120; estafenatox).

[0388] Anti-MICA / B antibodies (such as those described herein) can also be used to detect MICA / B, such as human MICA / B, for example, human MICA / B on the cell surface or soluble MICA / B in serum. Antibodies can be used, for example, in assays or flow cytometry. In some embodiments, the anti-MICA / B antibody is contacted with cells or serum for a duration suitable for specific binding to occur, and then a reagent for detecting the anti-MICA / B antibody, such as an antibody, is added. Illustrative analyses are provided in the examples. An exemplary method for detecting MICA / B (e.g., surface-expressed MICA / B or soluble MICA / B (sMICA / B) in a sample (serum)) includes (i) contacting the sample with the anti-MICA / B antibody for a duration sufficient to allow the anti-MICA / B antibody to specifically bind to MICA / B in the sample, and (2) contacting the sample with a detection reagent (e.g., an antibody) specifically bound to the anti-MICA / B antibody (such as the Fc region of the anti-MICA / B antibody), thereby detecting MICA / B bound to the anti-MICA / B antibody. The washing step may be included after incubation with the antibody and / or detection reagent. Since a separate detection reagent can be used, the anti-MICA / B antibody used in these methods does not necessarily need to be linked to a label or detection reagent.

[0389] Other uses of anti-MICA / B antibodies, such as as monotherapy or combination therapy, are provided elsewhere in this article, such as in the section on combination therapy.

[0390] IX. Bispecific molecules

[0391] The anti-MICA / B antibodies described herein can be used to form bispecific molecules. Anti-MICA / B antibodies or their antigen-binding moieties can be derived from or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to produce bispecific molecules that bind to at least two different binding sites or target molecules. For example, anti-MICA / B antibodies can be linked to an antibody or scFv that specifically binds to any protein that can be used as a potential target for combination therapy, such as the proteins described herein (e.g., antibodies against PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-2, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, CD96, VISTA or GITR, or pegylated IL-2 or pegylated IL-10). The antibodies described herein can actually be derived from or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be covered by the term "bispecific molecule" as used herein. In order to produce the bispecific molecules described herein, the antibodies described herein can be functionally linked (e.g., by chemical conjugation, gene fusion, non-covalent association, or otherwise) to one or more other binding molecules (such as another antibody, antibody fragment, peptide, or binding mimic) to obtain bispecific molecules.

[0392] Therefore, this document provides a bispecific molecule comprising at least one first binding specificity for MICA / B and a second binding specificity for a second target epitope. In some embodiments described herein, wherein the bispecific molecule has multiple specificities, the molecule may further comprise a third binding specificity.

[0393] In some embodiments, the bispecific molecules described herein comprise at least one antibody or an antibody fragment thereof as a binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv). As described in U.S. Patent No. 4,946,778 to Ladner et al., the antibody may also be a light chain or heavy chain dimer or any of its smallest fragments, such as Fv or a single-chain construct.

[0394] While human monoclonal antibodies are preferred, other antibodies that may be used in the bispecific molecules described herein are murine, chimeric, and humanized monoclonal antibodies.

[0395] The bispecific molecules described herein can be prepared by constitutively conjugating specific binders using methods known in this art. For example, each binder of the bispecific molecule can be generated separately and then conjugated together. When the binders are proteins or peptides, various coupling agents or cross-linking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, S-acetyl-thioacetic acid N-succinimide (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenyl dicis-butenedimide (oPDM), 3-(2-pyridyldithio)propionate N-succinimide (SPDP), and 4-(N-cis-butenedimide-methyl)cyclohexane-1-carboxylic acid sulfosuccinimide (sulfon-SMCC) (see, for example, Karpovsky et al. (1984) J.Exp.Med. 160:1686; Liu, MA et al. (1985) Proc.Natl.Acad.Sci.USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83; and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Some conjugates are SATA and sulfonyl-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0396] When the binding specificity is an antibody, it can be conjugated via thiohide (HH) bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge regions are modified to contain an odd number, preferably one HH residue, prior to binding.

[0397] Alternatively, the two binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly suitable for bispecific molecules that are mAb×mAb, mAb×Fab, mAb×(scFv)2, Fab×F(ab')2, or ligand×Fab fusion proteins. Bispecific antibodies may be antibodies containing scFv at the C-terminus of each heavy chain. The bispecific molecules described herein may be single-chain molecules containing one single-chain antibody and one binding determinant, or single-chain bispecific molecules containing two binding determinants. Bispecific molecules may contain at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498, and 5,482,858.

[0398] The binding of a bispecific molecule to its specific target can be confirmed using methods recognized in this technique, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS, bioassays (e.g., growth inhibition), or Western blotting. Each of these analyses typically detects the presence of the complex of interest by employing a labeling reagent (e.g., an antibody) that is specific to the protein-antibody complex of particular interest.

[0399] X. Composition

[0400] Further, compositions, such as pharmaceutical compositions, are provided comprising an anti-MICA / B antibody or a combination thereof as described herein, or a combination thereof with antibodies against other targets, or the antigen-binding portion thereof, formulated together with a pharmaceutically acceptable carrier. Such compositions may comprise an antibody or immunoconjugate or bispecific molecule as described herein, or (e.g., a combination of two or more different) antibodies or immunoconjugates or bispecific molecules as described herein. For example, the pharmaceutical compositions described herein may comprise a combination of antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on a target antigen or have complementary activities.

[0401] In some embodiments, the composition comprises an anti-MICA / B antibody at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1-300 mg / ml, or 100-300 mg / ml.

[0402] The pharmaceutical compositions described herein can also be administered in combination therapies, i.e., in combination with other agents. For example, a combination therapy may include the anti-MICA / B antibody described herein combined with at least one other anticancer agent and / or immunomodulator, such as a T-cell stimulant (e.g., an activator). Examples of therapeutic agents that can be used in combination therapies are described in more detail in the section below regarding the use of the antibodies described herein.

[0403] In some embodiments, the anti-MICA / B antibody may be combined with at least one other agent selected from chemotherapy drugs, small molecule drugs, and antibodies that stimulate an immune response against a given cancer. In some cases, the anti-MICA / B antibody may be combined with one or more of the following: anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody (e.g., BMS986178 or MDX-1803), anti-CD137 antibody, anti-LAG-3 antibody, anti-GITR antibody, anti-KIR antibody, anti-TGFβ antibody, anti-IL-10 antibody, long-acting IL-10 molecule (e.g., IL-10-Fc fusion or PEGylated IL-10, such as ARMO). BioSciences' AM0010), long-acting IL-2 (e.g., PEGylated IL-2 molecules, such as Nektar's NKTR-214; see US8,252,275, WO12 / 065086 and WO15 / 125159), anti-VISTA antibody, anti-CD96 antibody, anti-IL-8 antibody, anti-B7-H4, anti-Fas ligand antibody, anti-CXCR4 antibody, anti-mesothelin antibody, anti-CD27 antibody, or any combination thereof.

[0404] In other embodiments, the anti-MICA / B antibody may be formulated together with a secondary antibody. In some embodiments, the secondary antibody specifically binds to proteins selected from the following: PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-2, VISTA, CD96, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, and any combination thereof.

[0405] In some embodiments, the second antibody may be an anti-PD-1 antibody. The anti-PD-1 antibody may be any antibody that binds to PD-1 and inhibits the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-1 antibody is any anti-PD-1 antibody disclosed herein. In some embodiments, the second antibody may be nivolumab. In some embodiments, the second antibody may be pembrolizumab.

[0406] In some embodiments, the second antibody may be an anti-PD-L1 antibody. The anti-PD-L1 antibody may be any antibody that binds to PD-L1 and inhibits the interaction between PD-1 and PD-L1. In some embodiments, the anti-PD-L1 antibody is any anti-PD-L1 antibody disclosed herein. In some embodiments, the second antibody may be atezolizumab. In some embodiments, the second antibody may be durvalumab. In some embodiments, the second antibody may be avelumab.

[0407] In some embodiments, the second antibody may be an anti-CTLA-4 antibody. The anti-CTLA-4 antibody may be any antibody that binds to CTLA-4 and inhibits its activity. In some embodiments, the anti-CTLA-4 antibody is any anti-CTLA-4 antibody disclosed herein. In some embodiments, the second antibody may be tremelimumab. In some embodiments, the second antibody may be ipilimumab.

[0408] In some embodiments, the second antibody may be an anti-LAG3 antibody. The anti-LAG3 antibody may be any antibody that binds to LAG-3 and inhibits its activity. In some embodiments, the anti-LAG3 antibody is any anti-LAG3 antibody disclosed herein. In some embodiments, the second antibody may be 25F7.

[0409] In some embodiments, the second antibody may be an anti-CD137 antibody. The anti-CD137 antibody may be any antibody that binds to CD137 and inhibits its activity. In some embodiments, the anti-CD137 antibody may be any anti-CD137 antibody disclosed herein. In some embodiments, the second antibody may be urelumab.

[0410] In some embodiments, the second antibody may be an anti-KIR antibody. An anti-KIR antibody may be any antibody that binds to KIR and inhibits its activity. In some embodiments, the anti-KIR antibody is any anti-KIR antibody disclosed herein. In some embodiments, the second antibody may be lirilumab.

[0411] In some embodiments, the second antibody may be an anti-GITR antibody. An anti-GITR antibody may be any antibody that binds to GITR and inhibits its activity. In some embodiments, the anti-GITR antibody is any anti-GITR antibody disclosed herein. In some embodiments, the second antibody may be MK4166. In some embodiments, the second antibody may be TRX518.

[0412] In some implementations, the second antibody may be an anti-CD96 antibody.

[0413] In some implementations, the second antibody may be an anti-TIM3 antibody.

[0414] In some implementations, the second antibody may be an anti-VISTA antibody.

[0415] In some implementations, the second antibody may be an anti-NKG2a antibody.

[0416] In some implementations, the second antibody may be an anti-ICOS antibody.

[0417] In some implementations, the second antibody may be an anti-OX40 antibody.

[0418] In some implementations, the second antibody may be an anti-IL8 antibody, such as... -IL8(BMS-986253).

[0419] In some embodiments, the anti-MICA / B antibody may be formulated together with a long-acting IL-10 molecule. In some embodiments, the anti-MICA / B antibody may be formulated together with an IL-10-Fc fusion molecule. In some embodiments, the anti-MICA / B antibody may be formulated together with pegylated IL-10, such as ARMO BioSciences' AM0010.

[0420] In some embodiments, the anti-MICA / B antibody may be formulated together with a long-acting IL-2. In some embodiments, the anti-MICA / B antibody may be formulated together with a pegylated IL-2 molecule, such as Nektar's NKTR-214; see US8,252,275, WO12 / 065086 and WO15 / 125159.

[0421] In some embodiments, the compositions of the present invention further comprise a filler. The filler may be selected from the following: NaCl, mannitol, glycine, alanine, and any combination thereof. In other embodiments, the compositions of the present invention comprise a stabilizer. The stabilizer may be selected from the following: sucrose, fucose, raffinose, arginine, or any combination thereof. In other embodiments, the compositions of the present invention comprise a surfactant. The surfactant may be selected from the following: polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In some embodiments, the compositions further comprise a chelating agent. The chelating agent may be selected from the following: diethyltriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, hypochlorous acid, and any combination thereof.

[0422] In other embodiments, the composition comprises a third antibody. In some embodiments, the third antibody is any antibody disclosed herein.

[0423] In one embodiment, the composition comprises NaCl, mannitol, pentetic acid (DTPA), sucrose, PS80, and any combination thereof.

[0424] As used herein, “pharmaceutically acceptable carriers” include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, absorption delayers, and the like. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or transdermal administration (e.g., by injection or infusion). The option for subcutaneous injection is based on the description in Halozyme Therapeutics. Drug delivery technology involves a co-formulation of an antibody (Ab) and recombinant human hyaluronidase (rHuPH20) that can be delivered subcutaneously via the extracellular matrix, removing traditional volume limitations on biologics and drugs (US Patent No. 7,767,429). Depending on the route of administration, the active compound (i.e., antibody, immunoconjugate, or bispecific molecule) can be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate it.

[0425] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound without conferring any unwanted toxicological effects (see, for example, Berge, SM et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like; and those derived from non-toxic organic acids such as aliphatic monocarboxylic acids and aliphatic dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Alkali addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like; and those derived from non-toxic organic amines such as N,N'-diphenylmethylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0426] The pharmaceutical compositions described herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and analogues thereof; (2) oil-soluble antioxidants, such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and analogues thereof; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and analogues thereof.

[0427] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate flowability can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0428] These compositions may also contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial presence can be ensured by sterilization procedures (as previously described) and by the use of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenolic sorbic acid, and their analogues. It may also be necessary to include isotonic agents in the composition, such as sugars, sodium chloride, and their analogues. Furthermore, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0429] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in this art. Unless any known media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is permissible. Pharmaceutical compositions may contain preservatives or may not contain preservatives. Additional active compounds may be incorporated into the composition.

[0430] Therapeutic compositions must generally be sterile and stable under manufacturing and storage conditions. Compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, compositions may include isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Prolonged absorption of injectable compositions may be achieved by including delayed absorption agents (e.g., monostearate and gelatin) in the composition.

[0431] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound, along with one or more of the ingredients listed above, into a suitable solvent, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile medium containing an alkaline dispersion medium and any other desired ingredients from those listed above. In cases where sterile powders are used to prepare sterile injectable solutions, some methods involve vacuum drying and freeze-drying (lyophilization), yielding a powder containing the active ingredient plus any additional desired ingredients from a pre-sterile filtered solution.

[0432] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the individual being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces the therapeutic effect. Generally, on a 100% basis, this amount will be in the range of about 0.01% to about 99% of the active ingredient, about 0.1% to about 70% of the active ingredient, or about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0433] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several fractions can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions into unit dosage forms for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable for use as a single dose to the individual to be treated; each unit contains a predetermined amount of active compound, calculated to produce the desired therapeutic effect, in combination with the desired pharmaceutical carrier. The specifications of the unit dosage forms described herein are specified and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the techniques for compounding such active compounds for treating an individual's allergy.

[0434] For administration of anti-MICA / B antibodies, such as those described herein, the dose ranges from about 0.0001 to 100 mg / kg. Anti-MICA / B antibodies can be administered at a flat dose (flat-dose regimen). In some embodiments, anti-MICA / B antibodies can be administered at a fixed dose along with another antibody. In some embodiments, anti-MICA / B antibodies are administered at a weight-based dose.

[0435] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the indicated range. Antibodies are typically administered multiple times. The time interval between single administrations can be, for example, weekly, monthly, every three months, or annually. The interval can also be irregular, as indicated by measuring the blood antibody levels against the target antigen in the patient. In some methods, the dose is adjusted to achieve plasma antibody concentrations of approximately 1–1000 μg / ml, and in other methods, approximately 25–300 μg / ml.

[0436] Anti-MICA / B antibodies can be administered together with another antibody in the same dosing regimen. For example, anti-MICA / B antibodies can be administered with anti-PD-1 antibodies, such as nivolumab. Administer together every two weeks via intravenous infusion over 60 minutes until disease progression or unacceptable toxicity occurs. Anti-MICA / B antibody can be used in conjunction with pembrolizumab. Administer every 3 weeks via intravenous infusion over 30 minutes until disease progression or unacceptable toxicity occurs. Anti-MICA / B antibody can be used in conjunction with atezolizumab (TECENTRIQ). TM ) Administer together every 3 weeks via intravenous infusion over 60 or 30 minutes until the disease worsens or unacceptable toxicity occurs.

[0437] Antibodies can be administered in the form of sustained-release formulations, in which case the frequency of administration is less. The dosage and frequency vary depending on the antibody's half-life in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low doses are administered at relatively infrequent intervals over a long period. Some patients continue treatment for the rest of their lives. In therapeutic use, relatively high doses are sometimes administered at relatively short intervals until disease progression decreases or ceases, and until the patient shows partial or complete improvement in symptoms. Afterward, a prophylactic regimen can be administered to the patient.

[0438] The actual dose level of the active ingredient in the pharmaceutical compositions described herein may vary to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and administration mode. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition described herein or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, disease, general health condition, and medical history of the patient being treated, and similar factors well known in medical technology.

[0439] The "therapeuticly effective dose" of the anti-MICA / B antibody described herein can cause a reduction in the severity of disease symptoms, an increase in the frequency or duration of asymptomatic periods, or prevention of damage or disability resulting from the disease. In the case of cancer, a therapeutically effective dose can cause a prolongation of survival (e.g., overall survival) and / or prevention of further deterioration of cancer-related physical symptoms. The symptoms of cancer are well known in this technique and include, for example, abnormal mole features; changes in the appearance of moles, including asymmetry, borders, color and / or diameter; new areas of colored skin; abnormal moles; darkening of the area under the nails; breast lumps; nipple changes; breast cysts; breast pain; death; weight loss; weakness; excessive fatigue; difficulty eating; loss of appetite; chronic cough; worsening dyspnea; hemoptysis, hematuria, hematochezia, nausea, vomiting, liver cancer metastasis, lung cancer metastasis, bone cancer metastasis, abdominal pain and heaviness, abdominal distension, peritoneal fluid, vaginal bleeding, constipation, abdominal bloating, colonic perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon cancer metastasis, lung cancer metastasis, bladder cancer metastasis, liver cancer metastasis, bone cancer metastasis, kidney cancer metastasis and pancreatic cancer metastasis, dysphagia and similar symptoms.

[0440] Effective therapeutic doses can prevent or delay the onset of cancer, such as when early or initial symptoms of the disease are present. Laboratory tests used in cancer diagnosis include chemical (including measurements of MICA / B levels), hematological, serological, and radiological tests. Therefore, any clinical or biochemical analysis monitoring any of the foregoing can be used to determine whether a particular treatment is an effective dose for cancer. A person skilled in the art will be able to determine such doses based on factors such as individual body size, the severity of individual symptoms, and the specific composition or route of administration chosen.

[0441] The compositions described herein can be administered via one or more routes of administration, using one or more of the various methods known in the art. As those skilled in the art will understand, the route of administration and / or mode of administration will vary depending on the desired outcome. Routes of administration of the anti-MICA / B antibodies described herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” means a mode of administration other than enteral and local administration, which is normally administered by injection, and includes (but is not limited to) intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intrabursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0442] Alternatively, the antibodies described herein may be administered via non-parenteral routes, such as local, transdermal, or transmucosal administration, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0443] The active compound can be prepared together with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or are generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

[0444] The therapeutic composition can be administered using medical devices known in the art. For example, in some embodiments, the therapeutic composition described herein can be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules used with the anti-MICA / B antibodies described herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs via the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system with multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses a permeable drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0445] In some embodiments, the anti-MICA / B antibodies described herein can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes a variety of highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if needed, e.g., for brain cancer), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811, 5,374,548, and 5,399,331. Liposomes may contain one or more portions selectively transported to specific cells or organs, thereby enhancing the delivery of targeted drugs (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting components include folic acid or biotin (see, for example, U.S. Patent 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PGBloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al., (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0446] XI. Applications and Methods

[0447] Some aspects of the present invention relate to methods of treating an individual, including administering to the subject an anti-MICA / B antibody disclosed herein, a polynucleotide encoding the anti-MICA / B antibody, a vector containing the polynucleotide, a host cell containing the polynucleotide, an immunoconjugate containing the anti-MICA / B antibody, or any combination thereof.

[0448] Some aspects of this invention relate to a method of treating cancer in an individual in need, comprising administering to the subject an effective dose of a composition disclosed herein (e.g., an antibody, polynucleotide, carrier, host cell, immunoconjugate, or pharmaceutical composition). In other aspects, the invention relates to a method of inhibiting the shedding of MICA from tumor cells in an individual in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the invention relates to a method of reducing shed MICA / B in serum and / or retaining MICA / B on cell surfaces in an individual in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the invention relates to a method of killing tumor cells in an individual in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the invention relates to a method of reducing tumor size in an individual in need, comprising administering to the subject an effective dose of a composition disclosed herein. In other embodiments, the invention relates to inhibiting cancer metastasis of a tumor in an individual in need, comprising administering an effective dose of the composition disclosed herein to the subject. In some embodiments, the individual is a human.

[0449] The compositions of the present invention can be administered via any pharmaceutically acceptable route. In some embodiments, the compositions (e.g., antibodies, polynucleotides, carriers, host cells, immunoconjugates, or pharmaceutical compositions) are administered intravenously, intraperitoneally, intramuscularly, intraarterially, intrasheathically, intralymphaticly, intralesionally, intracapsularly, intracardiacly, intradermally, subcutaneously, subepidermally, intra-articularly, subcapsularly, subarachnoidly, intravertebrally, epidurally, intrasternally, locally, transepidermally, transmucosally, or any combination thereof. In some embodiments, the compositions are administered intravenously. In some embodiments, the compositions are administered subcutaneously.

[0450] In some embodiments, the method reduces the size of cancer, such as tumor size, in an individual. In some embodiments, the cancer size is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0451] In some embodiments, the method increases an individual's overall survival. In some embodiments, overall survival is increased relative to the average overall survival of individuals with the same cancer but treated with different therapies. In some embodiments, overall survival is increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 3 times, or at least about 5 times. In some embodiments, overall survival is increased by at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about one month, at least about twelve months, at least about fifteen months, at least about eighteen months, at least about twenty-one months, at least about two years, at least about three years, at least about four years, at least about five years, or at least about ten years.

[0452] In some embodiments, the method increases an individual's progression-free survival. In some embodiments, progression-free survival is increased relative to the average progression-free survival of individuals with the same cancer but treated with different therapies. In some embodiments, progression-free survival is increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 3 times, or at least about 5 times. In some embodiments, progression-free survival is increased by at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about one month, at least about twelve months, at least about fifteen months, at least about eighteen months, at least about twenty-one months, at least about two years, at least about three years, at least about four years, at least about five years, or at least about ten years.

[0453] In some implementations, the method increases the objective response rate of individuals. In some implementations, the method induces a complete response in individuals. In some implementations, the method induces a partial response in individuals.

[0454] In some embodiments, the method includes administering the anti-MICA / B antibody (or polynucleotide, vector, host cell, or immune conjugate) disclosed herein along with a second therapy. In some embodiments, the second therapy is administered prior to the anti-MICA / B antibody. In some embodiments, the second therapy is administered after the anti-MICA / B antibody. In some embodiments, the second therapy is administered simultaneously with the anti-MICA / B antibody. In some embodiments, the anti-MICA / B antibody and the second therapy are administered separately. In other embodiments, the anti-MICA / B antibody and the second therapy are administered in a single formulation.

[0455] The second therapy can be any other therapy known in this technology. In some embodiments, the second therapy comprises immunotherapy. In some embodiments, the second therapy comprises chemotherapy. In some embodiments, the second therapy comprises radiation therapy. In some embodiments, the second therapy comprises surgery. In some embodiments, the second therapy comprises administration of a second therapeutic agent.

[0456] In some embodiments, the second therapeutic agent comprises a second antibody. In some embodiments, the second therapeutic agent comprises an effective amount of an antibody that specifically binds to a protein selected from: inducible T cell costimulator (ICOS), CD137 (4-1BB), CD134 (OX40), NKG2A, CD27, glucocorticoid-induced TNFR-related protein (GITR) and herpesvirus entry mediator (HVEM), programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1), CTLA-4, B and T lymphocyte attenuators (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), and lymphocytes. Lymphocyte activation gene-3 (LAG-3), adenosine A2A receptor (A2aR), cytotoxic lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immune receptor with Ig and ITIM domains (TIGIT) and receptors for V domain Ig repressor (VISTA) for T cell activation, NKG2a, KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CEACAM-1, CD96, CD52, HER2 and any combination thereof.

[0457] XI.A. Anti-PD-1 antibody

[0458] In some embodiments, the second antibody may be an anti-PD-1 antibody. Anti-PD-1 antibodies known in this art can be used in the compositions and methods described in this invention. Various human monoclonal antibodies that bind specifically to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449. It has been shown that the anti-PD-1 human antibody disclosed in U.S. Patent No. 8,008,449 exhibits one or more of the following characteristics: (a) as measured by surface plasmon resonance using a Biacore biosensor system, at 1 × 10⁻⁶. -7 M or smaller K D(a) binds to human PD-1; (b) does not significantly bind to human CD28, CTLA-4, or ICOS; (c) increases T cell proliferation in mixed lymphocyte reaction (MLR) assays; (d) increases interferon-γ production in MLR assays; (e) increases IL-2 secretion in MLR assays; (f) binds to both human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses; (i) stimulates antibody responses; and (j) inhibits in vivo tumor cell growth. Anti-PD-1 antibodies that can be used in this invention comprise monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments, at least five of the aforementioned characteristics.

[0459] Other anti-PD-1 monoclonal antibodies have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509; U.S. Publication No. 2016 / 0272708; and PCT Publications Nos. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, and WO No. 2017 / 020858, No. WO 2016 / 197367, No. WO 2017 / 024515, No. WO 2017 / 025051, No. WO 2017 / 123557, No. WO 2016 / 106159, No. WO 2014 / 194302, No. WO No. 2017 / 040790, No. WO 2017 / 133540, No. WO 2017 / 132827, No. WO 2017 / 024465, No. WO 2017 / 025016, No. WO 2017 / 106061, No. WO 2017 / 19846, No. WO No. 2017 / 024465, WO The following documents are incorporated herein by reference in their entirety: WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540.

[0460] In some implementations, the anti-PD-1 antibody is selected from the following: nivolumab (also known as...) 5C4, BMS-936558, MDX-1106 and ONO-4538), larizumab (Merck; also known as Lambrolizumab and MK-3475; see WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), Regeneron (also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136(2017)), BGB-A317 (Beigene; see WO 2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2015 / 085847; ...). 2017 / 19846) and IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540).

[0461] In one embodiment, the anti-PD-1 antibody is nivoluma...

Claims

1. An antibody that specifically binds to human MHC class I polypeptide-related sequence A (MICA) and / or human MHC class I polypeptide-related sequence B (MICB), comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1, a VH-CDR2 and a VH-CDR3, and the VL comprises a VL-CDR1, a VL-CDR2 and a VL-CDR3; wherein the amino acid sequence of VH-CDR1 is as shown in SEQ ID NO:5; the amino acid sequence of VH-CDR2 is as shown in SEQ ID NO:6; the amino acid sequence of VH-CDR3 is as shown in SEQ ID NO:7; the amino acid sequence of VL-CDR1 is as shown in SEQ ID NO:8; the amino acid sequence of VL-CDR2 is as shown in SEQ ID NO:9; and the amino acid sequence of VL-CDR3 is as shown in SEQ ID NO:

10.

2. The antibody according to claim 1, wherein the antibody has one or more properties selected from the group consisting of: (a) The antibody inhibits MICA shedding by tumor cells; (b) The antibody increases membrane-bound MICA on tumor cells; (c) the antibody reduces the level of soluble MICA in the patient's serum; (d) the antibody mediates enhanced ADCC and / or ADCP; (e) the antibody mediates enhanced antigen processing and / or cross-presentation by cells; (f) the antibody inhibits tumor growth and / or cancer metastasis; (g) the antibody reduces tumor volume; (h) the antibody increases progression-free survival; (i) the antibody increases overall survival; and (j) any combination thereof.

3. The antibody of claim 1, wherein the VH comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:

2. The antibody according to claim 1 , wherein the VH comprises the amino acid sequence of SEQ ID NO:

2.

5. The antibody of claim 1, wherein the VL comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:

4. The antibody according to claim 1 , wherein the VL comprises the amino acid sequence of SEQ ID NO:

4.

7. The antibody of claim 1, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:2, and the VL comprises the amino acid sequence set forth in SEQ ID NO:

4.

8. The antibody according to claim 1, wherein (a) the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:58 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:60; or (b) The antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 130 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

60.

9. The antibody according to any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA as determined by yeast surface display, which comprises one or more amino acid residues in human MICA selected from the group consisting of G254, D255, L257, Y264, W267 corresponding to SEQ ID NO: 51, and any combination thereof.

10. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising G254, D255, L257, Y264 and W267 corresponding to SEQ ID NO: 51 as determined by yeast surface display.

11. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising amino acid residues W150-M163 corresponding to SEQ ID NO:51 as determined by hydrogen / deuterium exchange mass spectrometry.

12. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising amino acid residues Y231-T238 corresponding to SEQ ID NO: 51 as determined by hydrogen / deuterium exchange mass spectrometry.

13. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising amino acid residues D255-Q265 corresponding to SEQ ID NO: 51 as determined by hydrogen / deuterium exchange mass spectrometry.

14. The antibody of claim 13, wherein the epitope comprises amino acid residues W253-W267 corresponding to SEQ ID NO:51 as determined by hydrogen / deuterium exchange mass spectrometry.

15. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising L201-N220 corresponding to SEQ ID NO:51 as determined by hydrogen / deuterium exchange mass spectrometry.

16. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising T238-Q252 corresponding to SEQ ID NO: 51 as determined by hydrogen / deuterium exchange mass spectrometry.

17. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising L201-N220 and T238-Q252 corresponding to SEQ ID NO:51 as determined by hydrogen / deuterium exchange mass spectrometry.

18. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising R240, Q241, D242, V244 and R279 corresponding to SEQ ID NO:51 as determined by yeast surface display.

19. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising P258, G260, G262 and Y264 corresponding to SEQ ID NO:51 as determined by yeast surface display.

20. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising at least one residue selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269 as determined by X-ray co-crystallography.

21. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising at least two residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267 and A269 as determined by X-ray co-crystallography.

22. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising at least three residues selected from T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269 as determined by X-ray co-crystallography.

23. The antibody of any one of claims 1 to 8, wherein the antibody binds to an epitope on human MICA comprising T222, T224, R226, W233, N234, H248, D249, Q251, Q252, W253, G254, D255, V256, L257, P258, D259, G260, N261, Y264, Q265, W267, and A269 as determined by X-ray co-crystallography.

24. The antibody according to any one of claims 1 to 8, wherein the antibody is expressed at 1×10 -4 MM or smaller K D Specific binding to human MICA, with K measured by surface plasmon resonance analysis D .

25. The antibody according to any one of claims 1 to 8, wherein the antibody is expressed at 1×10 -4 The on-rates of M 1 / Ms or greater specifically bind to human MICA, where the on-rates are measured by surface plasmon resonance analysis.

26. The antibody according to any one of claims 1 to 8, wherein the antibody is expressed at 1×10 -4 The off-rates were M 1 / s or less and specifically bound to human MICA, where the off-rates were measured by surface plasmon resonance analysis.

27. The antibody according to any one of claims 1 to 8, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 or variants thereof.

28. The antibody according to any one of claims 1 to 8, wherein the antibody is an IgG1 antibody.

29. The antibody of any one of claims 1 to 8, wherein the antibody is afucosylated.

30. The antibody of any one of claims 1 to 8, wherein the antibody comprises a constant region in the heavy chain, and wherein the constant region comprises a G to A mutation at the position corresponding to residue 234 in SEQ ID NO:

58.

31. The antibody of any one of claims 1 to 8, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

32. An antibody that specifically binds to human MHC class I polypeptide-related sequence A (MICA) and / or human MHC class I polypeptide-related sequence B (MICB), comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a VH complementarity determining region (CDR) 1, a VH-CDR2 and a VH-CDR3, and the VL comprises a VL-CDR1, a VL-CDR2 and a VL-CDR3; wherein the amino acid sequence of VH-CDR1 is as shown in SEQ ID NO:5; the amino acid sequence of VH-CDR2 is as shown in SEQ ID NO:6; the amino acid sequence of VH-CDR3 is as shown in SEQ ID NO:7; the amino acid sequence of VL-CDR1 is as shown in SEQ ID NO:8; the amino acid sequence of VL-CDR2 is as shown in SEQ ID NO:9; and the amino acid sequence of VL-CDR3 is as shown in SEQ ID NO:10; and Wherein the antibody is non-fucosylated.

33. The antibody of claim 32, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:2, and the VL comprises the amino acid sequence set forth in SEQ ID NO:

4.

34. The antibody of claim 33, wherein the antibody comprises a constant region in the heavy chain comprising a G to A mutation at the position corresponding to residue 234 in SEQ ID NO:

58.

35. The antibody according to any one of claims 32 to 34, wherein the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO:58 and a light chain comprising the amino acid sequence described in SEQ ID NO:

60.

36. The antibody according to claim 32 or 33, wherein the antibody comprises a heavy chain comprising the amino acid sequence described in SEQ ID NO: 130 and a light chain comprising the amino acid sequence described in SEQ ID NO:

60.

37. An antibody that specifically binds to human MHC class I polypeptide-related sequence A (MICA) and / or human MHC class I polypeptide-related sequence B (MICB), comprising a heavy chain and a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO:58, and the amino acid sequence of the light chain being as shown in SEQ ID NO:60, wherein the antibody is non-fucosylated.

38. The antibody of claim 37, which is produced by a cell line in which the expression of fucosyltransferase has been reduced or eliminated.

39. The antibody of claim 37, which is produced by a cell line lacking the FUT8 gene or having a functionally disrupted FUT8 gene.

40. A polynucleotide or set of polynucleotides encoding the antibody of any one of claims 1 to 39.

41. A vector or a vector set comprising the polynucleotide or a polynucleotide set according to claim 40.

42. A host cell comprising the antibody of any one of claims 1 to 39, the polynucleotide or polynucleotide set of claim 40, or the vector or vector set of claim 41.

43. An immunoconjugate comprising the antibody of any one of claims 1 to 39 and a therapeutic agent.

44. The immunoconjugate of claim 43, wherein the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-neoplastic agent, and any combination thereof.

45. The immunoconjugate of claim 44, wherein the cytotoxin is selected from the group consisting of dolastatin, monomethyl auristatin E (MMAE), maytansine, duocarmycin, calicheamicin, pyrrolobenzodiazepine, duocarmycin, sentanycin, SN38, doxorubicin, derivatives thereof, synthetic analogs thereof, and any combination thereof.

46. ​​The immunoconjugate of claim 45, wherein the cytotoxin comprises cytotoxin A.

47. A pharmaceutical composition comprising the antibody of any one of claims 1 to 39, the polynucleotide or polynucleotide group of claim 40, the vector or vector group of claim 41, or the immunoconjugate of any one of claims 43 to 46, and a pharmaceutically acceptable excipient.

48. A pharmaceutical composition comprising the antibody of any one of claims 1 to 39, and a second antibody.

49. The pharmaceutical composition of claim 48, wherein the second antibody specifically binds to a protein selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, and any combination thereof.

50. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-PD-1 antibody.

51. The pharmaceutical composition of claim 48, wherein the second antibody comprises nivolumab or pembrolizumab.

52. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-PD-L1 antibody.

53. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-CTLA-4 antibody.

54. The pharmaceutical composition of claim 53, wherein the anti-CTLA-4 antibody comprises Tremelimumab or Ipilimumab.

55. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-LAG3 antibody.

56. The pharmaceutical composition of claim 55, wherein the anti-LAG3 antibody comprises 25F7.

57. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-CD137 antibody.

58. The pharmaceutical composition of claim 57, wherein the anti-CD137 antibody comprises usurelumab.

59. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-KIR antibody.

60. The pharmaceutical composition of claim 59, wherein the anti-KIR antibody comprises lirelumab.

61. The pharmaceutical composition of claim 48, wherein the second antibody comprises an anti-GITR antibody.

62. The pharmaceutical composition of claim 61, wherein the anti-GITR antibody comprises MK4166 or TRX518.

63. according to the pharmaceutical composition described in claim 48, its preparation is for intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, local, through epidermal or through mucosal administration.

64. Use of the antibody of any one of claims 1 to 39, the polynucleotide or polynucleotide group of claim 40, the vector or vector group of claim 41, the host cell of claim 42, the immunoconjugate of any one of claims 43 to 46, or the pharmaceutical composition of any one of claims 47 to 63 for the preparation of a medicament for treating lung cancer in a subject in need thereof.

65. The use according to claim 64, wherein the lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, and a combination of said small cell lung cancer and non-small cell lung cancer.

66. The use according to claim 65, wherein the non-small cell lung cancer is squamous non-small cell lung cancer or non-squamous non-small cell lung cancer.

67. The method of claim 64, wherein the antibody, polynucleotide or polynucleotide group, vector or vector group, host cell, immunoconjugate or pharmaceutical composition is administered intravenously, intraperitoneally, intramuscularly, intraarterially, intrathecally, intralymphatically, intralesionally, intracapsularly, intraorbitally, intracardially, intradermally, transtracheally, subcutaneously, subcutaneously, intraarticularly, subcapsularly, subarachnoidally, intraspinally, epidurally, intrasternally, topically, transdermally or transmucosally.

68. The use according to claim 64, wherein the subject is a human.

69. The use of claim 64, further comprising administering a second therapy to the subject.

70. The use of claim 69, wherein the second therapy comprises a second therapeutic agent, and wherein the second therapeutic agent comprises an effective amount of an antibody that specifically binds to a protein selected from the group consisting of: inducible T cell co-stimulator (ICOS), CD137 (4-1BB), CD134 (OX40), NKG2A, CD27, CD96, glucocorticoid-induced TNFR-related protein (GITR) and herpes virus entry mediator (HVEM), programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), CTLA-4, B and T lymphocyte attenuator (BTLA), T cell immune immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), adenosine A2a receptor (A2aR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immunoreceptor with Ig and ITIM domains (TIGIT) and receptor for V-domain Ig inhibitor of T cell activation (VISTA), KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CEACAM-1, CD52, HER2, and any combination thereof.

71. The use according to claim 70, wherein the second therapeutic agent comprises an anti-PD-1 antibody.

72. The use of claim 70, wherein the second therapeutic agent comprises nivolumab or pembrolizumab.

73. The use according to claim 70, wherein the second therapeutic agent comprises an anti-PD-L1 antibody.

74. The use according to claim 70, wherein the second therapeutic agent comprises an anti-PD-L1 antibody selected from atezolizumab, durvalumab and ivelumab.

75. The use of claim 70, wherein the second therapeutic agent comprises an anti-CTLA-4 antibody.

76. The use of claim 70, wherein the second therapeutic agent comprises Tremelimumab or Ipilimumab.

77. The use of claim 70, wherein the second therapeutic agent comprises an anti-LAG3 antibody.

78. The use of claim 77, wherein the anti-LAG3 antibody comprises 25F7.

79. The use of claim 70, wherein the second therapeutic agent comprises an anti-CD137 antibody.

80. The use according to claim 79, wherein the anti-CD137 antibody comprises usurelumab.

81. The use of claim 70, wherein the second therapeutic agent comprises an anti-KIR antibody.

82. The use according to claim 81, wherein the anti-KIR antibody comprises lirelumab.

83. The use of claim 70, wherein the second therapeutic agent comprises an anti-GITR antibody.

84. The use of claim 83, wherein the anti-GITR antibody comprises MK4166 or TRX518.

85. The use according to claim 69, wherein the second therapy comprises a chemotherapeutic agent for cancer, radiation, an agent that activates innate immune cells and / or an agent that enhances the survival of NK and / or CD8+ T cells.

86. The use according to claim 85, wherein the second therapy comprises a chemotherapeutic agent that induces MICA / B expression on tumor cells.

87. The use according to claim 85, wherein the second therapy comprises a chemotherapeutic agent selected from a proteasome inhibitor, an IMiD and a Bet inhibitor.

88. The use according to claim 87, wherein the proteasome inhibitor is bortezomib.

89. The use according to claim 85, wherein the second therapy comprises PEGylated IL-10 and IL-10-Fc fusion.

90. The use according to claim 85, wherein the second therapy comprises an agent that enhances the survival of NK and / or CD8+ T cells, the agent being selected from pegylated IL-2.

91. The use of claim 69, wherein the second therapy comprises an agent selected from the group consisting of doxorubicin, cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide, lenalidomide, bortezomib, dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine, rituximab, ifosfamide, vincristine, fludarabine, thalidomide, alemtuzumab, ofavumab, everolimus, and carfilzomib.

92. The use of claim 69, wherein the second therapy comprises an IDO antagonist.

93. A method for preparing an antibody, comprising expressing the polynucleotide or polynucleotide set according to claim 40, or the vector or vector set according to claim 41 in a host cell under suitable conditions.

94. The method of claim 93, further comprising collecting the antibody.

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