Detection of cell surface MICA and MICB using antibodies

Abstract

This disclosure relates to research and diagnostic tools for the specific detection of MICA and MICB polypeptides in paraffin-embedded tissue samples, as well as methods for using the tools to detect MICA and MICB polypeptides, particularly in tumor tissue.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 063,475, filed August 10, 2020, which is incorporated by reference herein in its entirety, including any drawings.

[0002] Sequence Listing Reference This application is filed with a Sequence Listing in electronic format. The Sequence Listing was created on August 4, 2021, and is provided as a 19 KB file entitled "MICA4_ST25." The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to a research and diagnostic tool for detecting proteins of interest in paraffin-embedded tissue samples. The present invention also relates to methods of using the tool to detect polypeptides, particularly in tumor tissue. [Background technology]

[0004] MICA (major histocompatibility complex class I-related chain A) and MICB (major histocompatibility complex class I-related chain B) are transmembrane proteins induced by stress signals on infected and tumor cells. MICA is a ligand for NKG2D (natural killer group 2, member D), an activating receptor expressed on natural killer (NK) cells and a subpopulation of gamma delta T cells. Interaction between MICA and NKG2D leads to the activation of effector cells that mediate target cell lysis. However, MICA protein is subject to shedding by metalloproteases in the tumor microenvironment, and soluble MICA is abundant and correlates with NKG2D downregulation on peripheral lymphocytes from patients with various cancers. In addition to soluble MICA, membrane-bound MICA is also highly effective at downregulating NKG2D. These mechanisms enable tumors to evade immune system control.

[0005] Many tumor types have been reported to express and / or shed MICA. Tumors have also been reported to express MICB. Antibodies for detection or diagnostic use have generally focused on detecting soluble (shed) MICA, rather than cell-bound MICA. However, assessment of cell-bound MICA and / or MICB by antibody-based methods may also be useful. This is because, due to the complex processes regulating these transcripts, quantification of MICA / B mRNA does not predict the amount of the corresponding protein expressed on the cell surface; therefore, assessment of cell-bound MICA and / or MICB is preferably performed by direct detection of the protein (Non-Patent Document 1).

[0006] New methods of cancer treatment are needed that can more specifically target cancer cells and exploit the immune system, for example, to avoid the typical side effects of conventional chemotherapy agents. To understand the tumor environment in more detail, it is often desirable to detect proteins of interest present in tumor tissue and / or tumor periphery or other nearby tissues. This can be done, for example, using frozen tissue samples. This is not only useful in research, but can also aid in decisions regarding what type of treatment should be used, for example, by detecting whether a tissue (e.g., tumor environment) is characterized by the presence of a protein that is the target of the treatment (e.g., immunotherapy). This information can be useful for selecting treatments that can modulate the activity of the protein and / or the cells that express it.

[0007] In addition to frozen tissues, markers can also be detected from tissue samples preserved as formaldehyde (e.g., formalin)-fixed, paraffin-embedded (FFPE) specimens. After deparaffinization, slides are ready for immunohistochemistry, for example, to detect the expression of specific proteins. This method is routinely used to detect tumor antigens in tumor tissue samples. Unfortunately, it is often impossible to find monoclonal antibodies that function effectively and specifically in FFPE sections. This is thought to be due to the effects of formalin fixation on protein structure. Epitopes bound by antibodies described as specific for recombinant proteins or cells are often present on other proteins when used in FFPE, rendering the antibodies nonspecific. In other cases, formalin fixation destroys many epitopes on native cellular proteins, rendering antibodies identified using recombinant proteins or cells ineffective for staining FFPE sections. As a result, many receptors are not suitable for generating ligands that bind specifically in paraffin-embedded sections (e.g., lacking specific epitopes that remain available after formalin fixation). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Raulet et al.2013 Annu.Rev.Immunol.2013;31:413-441 Summary of the Invention [Problem to be solved by the invention]

[0009] For example, there is a need for improved diagnostic methods and tools to identify the most appropriate treatment for a given patient. [Means for solving the problem]

[0010] The present invention relates, inter alia, to testing, detecting, and / or monitoring cell surface MICA and MICB proteins in tissue samples, particularly FFPE tissue samples. This disclosure stems from the development of methods for detecting MICA and MICB proteins in human tumor samples. This is through the development of highly specific test methods capable of detecting low levels of MICA*001, MICA*008, and MICB in FFPE tissue samples, particularly lower levels that may be present when considering only membrane or cell surface expression. Applicants provide staining of many tumors for MICA and MICB (e.g., a combination of MICA and MICB) in FFPE tissue samples. Staining, particularly membrane or cell surface staining, in FFPE samples, requiring the ability to detect low levels of protein may be particularly useful for identifying tumors suitable for treatment with depleting anti-MICA agents.

[0011] The present antibodies retain their specificity for MICA and MICB polypeptides in FFPE protocols; in particular, they bind to epitopes present on MICA and MICB polypeptides that remain present and specific after formalin fixation. Furthermore, the epitopes on MICA polypeptides remain present and specific for both highly prevalent MICA alleles in the human population, MICA*001 and *008, after formalin fixation. The present antibodies enable highly specific antigen detection in IHC protocols. Through the use of an antibody generation method using paraffin-embedded cell pellets prepared from cells bearing antibody-binding target antigens on their surfaces (cells expressing the target antigen pre-incubated with a therapeutic antibody against the target antigen), the present inventors obtained antibodies that can recognize multiple MICA alleles and even MICB, while recognizing antigen-specific epitopes in FFPE material. The resulting diagnostic antibodies can serve as universal single-antibody-based compositions, kits, systems, or methods for consistent detection of target antigens in FFPE samples from patients. The reagents can be used to detect MICA in tissue samples without the need for multiple additional allele-specific antibodies. The reagents can be used to detect MICA- and / or MICB-expressing tumors with relatively low levels of MICA and / or MICB. Because malignant cells in tumor tissue may express MICA and / or MICB, the reagents can provide increased sensitivity for detecting tumors that are positive for at least one of MICA and MICB, and can also improve the ability to identify subjects who may benefit from treatment with therapeutic agents that bind to both MICA and MICB agents (e.g., NKG2A protein or fragment, anti-MICA / B antibody). The reagents can be used to detect MICA and / or MICB on the cell surface or cell membrane of tumor cells (e.g., when levels of MICA and / or MICB are lower compared to when cytosolic proteins are also included in the assessment).The reagents can be used to select or identify individuals who can be treated with any suitable anti-MICA antibody, including MICA allele-specific therapeutic antibodies and MICA allele pan-specific therapeutic antibodies that can recognize multiple MICA alleles (e.g., two or more of the most common MICA alleles in the human population, such as MICA*001 and *008).

[0012] Formaldehyde-fixed (e.g., formalin-fixed, paraformaldehyde-fixed), paraffin-embedded (FFPE) tissue offers two major advantages over other immunological methods: (1) the tissue does not require special handling; and (2) its cytological and architectural characteristics are well-recognized, allowing for improved histopathological interpretation. In the examples herein, cells carrying different MICA alleles on their cell surface, cells carrying MICB on their cell surface, and cells carrying MICA or MICB polypeptides at different levels of combined expression were each separately prepared as formalin-fixed, paraffin-embedded (FFPE) samples. Use of these samples resulted in the discovery of an anti-MICA antibody for use in staining and testing MICA in human FFPE tissue samples that is useful as a single reagent across human populations and is even more suitable for detecting lower levels of combined MICA and MICB expression on the surface of cells in FFPE samples, such as tumor cells, or more generally, on the surface of cells in tumor tissue. The resulting antibodies were tested in various tumor tissues from human donors and were found to retain excellent performance in detecting the target antigen in FFPE tissue sections. As shown, when compared with a control antibody that was consistently unable to detect lower levels of combined MICA and MICB expression in cells in FFPE samples, the disclosed antibodies were able to identify tissue samples as MICA and / or MICB positive (membrane staining) at the cell surface for tumor types that tested negative for MICA or MICB at the cell surface using the control antibody. Thus, the present antibodies have the advantage of enabling the detection of a broader range of MICA- and / or MICB-positive tumors in FFPE samples from individuals, thereby enabling the treatment of individuals with MICA- and / or MICB-positive tumors with agents targeted to MICA and / or MICB (e.g., anti-MICA and / or anti-MICB depleting agents).

[0013] In one embodiment, the present disclosure provides a method for generating antibodies that specifically bind to MICA and MICB polypeptides in paraffin-embedded tissue, the method comprising: a) providing a plurality of candidate antibodies; and b) preparing or selecting antibodies from the plurality that specifically bind to MICA and MICB polypeptides expressed by cells prepared as a paraffin-embedded cell sample (e.g., compared to cells prepared as a paraffin-embedded cell sample that do not express MICA or MICB polypeptides).

[0014] In one embodiment, the present disclosure provides a method for producing antibodies that specifically bind to MICA and MICB polypeptides in paraffin-embedded tissue, the method comprising: a) providing cells that express a MICA polypeptide (e.g., do not express a MICB polypeptide) on their surface and preparing a paraffin-embedded cell sample from such cells; b) providing cells that express a MICB polypeptide (e.g., do not express a MICA polypeptide) on their surface and preparing a paraffin-embedded cell sample from such cells; c) providing a plurality of candidate antibodies, and preparing or selecting an antibody from said plurality that (i) binds to the MICA polypeptide in the paraffin-embedded cell sample of step a) and (ii) binds to the MICB polypeptide in the paraffin-embedded cell sample of step b), and optionally does not bind to a paraffin-embedded cell sample prepared from cells lacking expression of the MICA polypeptide and the MICB polypeptide; Includes.

[0015] In one embodiment, the present disclosure provides a method for producing antibodies that specifically bind to MICA and MICB polypeptides in paraffin-embedded tissue, the method comprising: a) providing cells that express a first allele of a MICA (e.g., MICA*001 or MICA*008) polypeptide on their surface (e.g., expressing the first allele as the only MICA polypeptide and not expressing a MICB polypeptide), and preparing a paraffin-embedded cell sample from such cells; b) providing cells that express a second allele of MICA on their surface that is not the same as the first allele (e.g., cells that express the second allele as the only MICA polypeptide and do not express a MICB polypeptide), and preparing a paraffin-embedded cell sample from such cells; c) providing cells that express a MICB polypeptide (e.g., do not express a MICA polypeptide) on their surface and preparing a paraffin-embedded cell sample from such cells; d) providing a plurality of candidate antibodies, and preparing or selecting an antibody from said plurality of antibodies that (i) binds to the first MICA allele polypeptide in the paraffin-embedded cell sample of step a), (ii) binds to the second MICA allele polypeptide in the paraffin-embedded cell sample of step b), and (iii) binds to the MICB polypeptide in the paraffin-embedded cell sample of step c), and optionally does not bind to the first and second MICA allele polypeptides and to a paraffin-embedded cell sample prepared from cells lacking expression of the MICB polypeptide; Optionally, step (d) includes providing a plurality of candidate antibodies, and optionally preparing or selecting an antibody from the plurality of antibodies that (i) binds to a MICA*001 polypeptide in the paraffin-embedded cell sample of step a), (ii) binds to a MICA*008 polypeptide in the paraffin-embedded cell sample of step b), and (iii) binds to a MICB polypeptide in the paraffin-embedded cell sample of step c), without binding to a paraffin-embedded cell sample prepared from cells lacking expression of a MICA*001 polypeptide, a MICA*001 polypeptide, and a MICB polypeptide.

[0016] In one aspect, provided is an antibody or antibody fragment that specifically binds to MICA and MICB polypeptides in paraffin-embedded tissue, obtained by the antibody production method of the present disclosure. In one aspect, provided is a method for detecting MICA and / or MICB (e.g., MICA- and / or MICB-expressing cells) in formalin-treated and / or paraffin-embedded tissue samples using an antibody obtained by the antibody production method of the present disclosure.

[0017] In one aspect, a method for detecting MICA and / or MICB (e.g., MICA- and / or MICB-expressing cells) in a formalin-treated and / or paraffin-embedded tissue sample, optionally from a tumor or tumor-adjacent tissue sample, optionally from an individual pretreated with a therapeutic agent (e.g., a chemotherapeutic agent), is provided, comprising: a) contacting the tissue sample with an anti-MICA / B antibody (e.g., a diagnostic antibody of the present disclosure); and b) detecting the presence of bound antibody in the tissue sample. If MICA and / or MICB are detected, the sample can be determined to contain MICA / B (e.g., MICA-expressing cells, MICB-expressing tumor cells, or MICA- and MICB-expressing cells).

[0018] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells (e.g., cells that express MICA and / or MICB on their surface or cell membrane) in a sample from a human tumor, the method comprising contacting a paraffin-embedded tumor tissue sample from an individual with an antibody capable of specifically binding to human MICA and MICB polypeptides in the paraffin-embedded tumor tissue sample; detecting the presence of the bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody.

[0019] In any embodiment, an antibody capable of specifically binding to human MICA and MICB polypeptides in a paraffin-embedded tumor tissue sample may be characterized as being capable of binding to and / or staining BxPC-3 cells prepared as a paraffin-embedded cell pellet; optionally, the antibody or antibody fragment is capable of binding to and / or staining BxPC-3 cells prepared as a paraffin-embedded cell pellet when the antibody is provided at a low concentration (1 μg / mL), and optionally, further, the antibody or antibody fragment is capable of binding to and / or staining BxPC-3 cells prepared as a paraffin-embedded cell pellet at each of low (1 μg / mL), medium (5 μg / mL), and high (10 μg / mL) antibody concentrations. Optionally, the antibody or antibody fragment is capable of consistently binding to and / or staining paraffin-embedded BxPC-3 cells, e.g., binding and / or staining is observed each time the test is repeated multiple times (e.g., 10, 20, 100 or more times).

[0020] In one embodiment, the antibody capable of specifically binding to human MICA and MICB polypeptides in paraffin-embedded tumor tissue samples is antibody 12C9, an antibody having the heavy and light chain variable regions thereof, or a function-conservative variant of any of the foregoing.

[0021] In one embodiment, the antibody competes with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:7 and a light chain variable region having the amino acid sequence of SEQ ID NO:8 for binding to human MICA and / or MICB polypeptide in a paraffin-embedded cell sample (e.g., cells prepared as a paraffin-embedded cell sample expressing MICA and / or MICB).

[0022] In one aspect, an antibody or antibody fragment capable of specifically binding to human MICA and / or MICB polypeptide (e.g., MICA and / or MICB polypeptide in a sample of MICA-expressing cells prepared as a paraffin-embedded cell pellet) is provided, wherein the antibody or antibody fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 70%, 80%, or 90% identical to the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence that is at least 70%, 80%, or 90% identical to the amino acid sequence of SEQ ID NO:8.

[0023] In one embodiment, an antibody or antibody fragment, or a function-conservative variant thereof, is provided, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8.

[0024] In one aspect, an antibody or antibody fragment capable of specifically binding to human MICA and / or MICB polypeptide (e.g., MICA and / or MICB polypeptide in a sample of MICA-expressing cells prepared as a paraffin-embedded cell pellet) is provided, wherein the antibody or antibody fragment comprises three CDRs of the heavy chain variable region sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region sequence of SEQ ID NO: 8. In one embodiment, the antibody or antibody fragment is conjugated or covalently linked to a detectable moiety. In one embodiment, the antibody or antibody fragment binds to MICA polypeptide in a sample of MICA-expressing cells prepared as a paraffin-embedded cell pellet and binds to MICB polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA-negative and MICB-negative cells prepared as a paraffin-embedded cell pellet.

[0025] In any embodiment, the antibody or antibody fragment may be further characterized as being capable of binding to human MICA*004 polypeptide and / or human MICA*007 polypeptide, e.g., the antibody or antibody fragment binds to MICA*004 and / or MICA*007 polypeptide in a paraffin-embedded cell sample.

[0026] In any embodiment, the detection of MICA / B expressing cells comprises: obtaining a biological sample (e.g., as a biopsy, as a cell pellet) containing cells (e.g., tumor cells, MICA- and / or MICB-expressing cells); Fixing, embedding in paraffin, sectioning and deparaffinizing the sample, and optionally transferring the sample to a slide; contacting the section with an antibody that specifically binds to human MICA and MICB polypeptides; detecting the presence of bound antibodies in the section; Includes.

[0027] In other embodiments, methods for generating or producing antibodies are provided. In other embodiments, kits are provided that include a monoclonal antibody (diagnostic antibody) that binds to an antigen in a paraffin-embedded cell sample having the properties disclosed herein and a second antibody (e.g., a therapeutic antibody) capable of specifically binding to the same target antigen as the diagnostic antibody. In certain embodiments, the use of monoclonal antibodies having the properties disclosed herein in immunohistochemistry assays (including, but not limited to, antibodies and antibody fragments that bind to human MICA and MICB polypeptides), diagnostic methods (e.g., including, but not limited to, use in companion diagnostics to select individuals for treatment with therapeutic antibodies), prognostic methods, and patient monitoring methods are provided. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition As used herein, "a" or "an" may mean one or more. When used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0029] When "comprising" is used, this may optionally be replaced by "consisting essentially of" and more optionally by "consisting of."

[0030] As used herein, a "paraffin-embedded sample" (or paraffin-embedded "cells," "cell pellets," "slides," or "tissues") refers to cells or tissues taken from an organism or from an in vitro cell culture that have been fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide. It will be recognized that fixation and paraffin embedding is a general practice that can vary in many aspects, e.g., with respect to the fixation and embedding method used, with respect to the protocol followed, and that for the purposes of the present invention, all such modified methods are encompassed, so long as they involve fixing the tissue (such as by formalin treatment), embedding in paraffin or an equivalent substance, sectioning, and transfer to a slide.

[0031] The term "biological sample" or "sample" as used herein includes, but is not limited to, bodily fluids (e.g., serum, lymph, blood), cell samples, or tissue samples (e.g., bone marrow or tissue biopsies, e.g., skin, breast, lung, colon, ovary, stomach, etc., mucosal tissues, e.g., intestine, intestinal lamina propria, etc.).

[0032] The term "antibody" is used herein in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity. Techniques related to the production of antibodies are provided, for example, in Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). An "antibody fragment" comprises a portion of a full-length antibody, such as the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (generally the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (generally the VH and CH1 domains) and dAb (generally the VH domain) fragments; VH, VL, VhH and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies and kappabodies (see, e.g., Ill et al., Protein Eng 1997;10:949-57); camelid IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments and one or more isolated CDRs or functional paratopes, where isolated CDRs or antigen-binding residues or polypeptides are associated or linked together to form a functional antibody fragment. Various types of antibody fragments are described or reviewed, for example, in Holliger and Hudson, Nat Biotechnol 2005;23,1126-1136; WO2005040219 and US20050238646 and US20020161201.

[0033] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that contribute to antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., 1991) and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987;196:901-917). Generally, the numbering of amino acid residues in this region is performed according to the method described in Kabat et al., supra. Phrases such as "Kabat position," "variable domain residue numbering as in Kabat," and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a particular antibody by alignment of the regions of homology of the antibody's sequence with the "standard" Kabat numbered sequence. Application of either definition to refer to the CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by commonly used numbering schemes are shown below in Table 1 for comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of an antibody.

[0034] [Table 1]

[0035] "Framework" or "FR" residues, as used herein, refer to the regions of an antibody variable domain excluding the regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by the CDRs.

[0036] As defined herein, "constant region" refers to the constant region from an antibody encoded by one of the light or heavy chain immunoglobulin constant region genes. "Constant light chain" or "light chain constant region," as used herein, refers to the region of an antibody encoded by the kappa (Ckappa) or lambda (Clambda) light chain. The constant light chain generally contains a single domain and, as defined herein, refers to positions 108-214 of the Ckappa or Clambda, numbering according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). "Constant heavy chain" or "heavy chain constant region," as used herein, refers to the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon gene to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full-length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, and therefore includes positions 118 to 447, numbering according to the EU index.

[0037] "Fab" or "Fab region," as used herein, refers to a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region or polypeptide in isolation, a multispecific polypeptide, or this region in the context of an ABD or any other embodiment as outlined herein.

[0038] As used herein, "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a general review of methods for producing scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0039] By "Fv" or "Fv fragment" or "Fv region" as used herein is meant a polypeptide comprising the VL and VH domains of a single antibody.

[0040] As used herein, "Fc" or "Fc region" refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes immunoglobulin domains Cγ2 (CH2) and Cγ3 (CH3) and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined as including residues C226, P230, or A231 toward its carboxyl terminus, numbering according to the EU index. Fc may refer to this region in isolation or in the context of an Fc polypeptide, as described below. "Fc polypeptide" or "Fc-derived polypeptide," as used herein, refers to a polypeptide that comprises all or part of an Fc region. Fc polypeptides include, but are not limited to, antibodies, Fc fusions, and Fc fragments.

[0041] As used herein, "variable region" refers to the region of an antibody comprising one or more Ig domains substantially encoded by either the VL (including Vkappa (VK) and Vlambda) and / or VH genes that constitute the light (including kappa and lambda) and heavy chain immunoglobulin loci, respectively. A light or heavy chain variable region (VL or VH) consists of a "framework" or "FR" region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs has been precisely defined, for example, by Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., USDapartment of Health and Human Services, (1983)) and Chothia, et al. The framework region of an antibody, the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for antigen binding.

[0042] The term "deplete" with respect to MICA- and / or MICB-expressing cells refers to a process, method, or agent that can kill, remove, lyse, or induce such killing, removal, or lysis so as to negatively affect the number of MICA- and / or MICB-expressing cells present in a sample or subject. The agent can include, for example, an antibody that binds to MICA and / or MICB and directs ADCC (antibody-dependent cellular cytotoxicity) against MICA- and / or MICB-expressing cells, or the agent can be an antibody-drug conjugate that binds to MICA and directly causes death of MICA- and / or MICB-expressing cells by delivering its cytotoxic agent to the cells.

[0043] The terms "immunoconjugate" and "antibody conjugate" are used interchangeably and refer to an antigen-binding agent, such as an antibody-binding protein or antibody, conjugated to another moiety (e.g., a cytotoxic agent). Immunoconjugates comprising an antigen-binding agent conjugated to a cytotoxic agent may also be called "antibody drug conjugates" or "ADCs."

[0044] The term "drug" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials. The term "therapeutic agent" refers to an agent that has biological activity.

[0045] The term "specifically binds" means that an antibody or polypeptide can bind, preferably to a binding partner, e.g., MICA and MICB, in a competitive binding assay when assessed using either a recombinant form of the protein, an epitope therein, or the native protein present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0046] When an antibody or polypeptide is said to "compete with" a particular monoclonal antibody, this means that the antibody or polypeptide competes with the monoclonal antibody in a binding assay using an appropriate target molecule or a surface-expressed target molecule, such as MICA, expressed by cells in paraffin-embedded cell pellets. For example, if a test antibody reduces binding of 12C9 to a MICA polypeptide or MICA-expressing cells in a binding assay, the antibody is said to compete with 12C9.

[0047] "Function-conservative variants" are variants in which certain amino acid residues in a protein or enzyme have been modified without altering the overall conformation and function of the polypeptide, including, but not limited to, replacing amino acids with amino acids having similar properties (e.g., polarity, hydrogen-bonding ability, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those shown to be conserved may vary in the protein such that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary, e.g., 70%-99% as determined according to an alignment scheme such as by the Cluster method, where similarity is based on the MEGALIGN algorithm. "Function-conservative variants" also include polypeptides having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95%, as determined by the BLAST or FASTA algorithm, and have the same or substantially similar properties or functions as the native or parent protein (e.g., heavy or light chain or variable region thereof) to which they are being compared.

[0048] The term "affinity," as used herein, refers to the strength of binding of an antibody or polypeptide to an epitope. The affinity of an antibody is given by the dissociation constant, KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant, KA, is defined by 1 / KD. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are incorporated herein by reference in their entireties. One preferred standard method known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (e.g., by analysis on a BIAcore™ SPR analyzer).

[0049] The term "epitope" refers to an antigenic determinant, an area or region on an antigen to which an antibody or polypeptide binds. A protein epitope can include amino acid residues directly involved in binding as well as those effectively blocked by a specific antigen-binding antibody or peptide, i.e., within the "footprint" of an antibody. This is the simplest form or smallest structural region on a complex antigen molecule that can be combined with, for example, an antibody or receptor. Epitopes can be linear or conformational. The term "linear epitope" is defined as an epitope composed of amino acid residues that are adjacent in a linear sequence of amino acids (primary structure). The term "conformational or conformational epitope" is defined as an epitope composed of amino acid residues that are not all adjacent and therefore represent individual portions of the linear sequence of amino acids, but are brought into proximity with each other by molecular folding (secondary, tertiary, and / or quaternary structure). Conformational epitopes depend on the three-dimensional structure. Thus, the term "conformational" is often used interchangeably with "structure."

[0050] The term "amino acid modification" as used herein refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. An example of an amino acid modification herein is a substitution. An "amino acid modification" as used herein refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. An "amino acid substitution" or "substitution" as used herein refers to the replacement of an amino acid at a position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent polypeptide in which the tyrosine at position 50 is replaced with a tryptophan. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, generally a native or "parent" polypeptide. A polypeptide variant may possess one or more amino acid substitutions, deletions, and / or insertions at a position within the native amino acid sequence.

[0051] "Conservative" amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues with similar side chains are known in the art and 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0052] The terms "identity" or "identical," when used in the context of two or more polypeptide sequences, refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between two or more amino acid residue strings. "Identity" measures the percent of identical matches between the smaller of two or more sequences, with gap alignment (if necessary) accommodated by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0053] Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)), including those in the GCG program package. The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith-Waterman algorithm may also be used to determine identity.

[0054] An "isolated" molecule is one that is the predominant species in the composition in which it is found with respect to the class of molecules to which it belongs (i.e., it constitutes at least about 50% of the molecular type in the composition, and generally constitutes at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more of the molecular species, e.g., peptides, in the composition). Generally, a composition of polypeptides will exhibit 98%, 98%, or 99% homogeneity with respect to the polypeptide with respect to all of the peptide species in the composition, or peptide species that are at least substantially active in connection with the proposed use.

[0055] In the context of this specification, "treatment" or "treating" refers, unless contradictory by context, to preventing, alleviating, managing, curing, or ameliorating one or more symptoms or clinically relevant findings of a disease or disorder. For example, "treatment" of a patient in whom symptoms or clinically relevant findings of a disease or disorder have not been identified is preventative or prophylactic treatment, whereas "treatment" of a patient in whom symptoms or clinically relevant findings of a disease or disorder have been identified generally does not constitute preventative or prophylactic treatment.

[0056] Throughout this specification, whenever reference is made to "treatment of cancer" or the like in relation to an anti-MICA binding agent (e.g., an anti-MICA / MICB antibody), it means: (a) a method of treating cancer comprising administering (for at least one treatment) an anti-MICA binding agent (preferably in a pharmaceutically acceptable carrier substance) to an individual, mammal, particularly a human, in need of such treatment, preferably in a dose (amount) as specified herein, in a dose (therapeutically effective amount) that allows for treatment of cancer; (b) the use of an anti-MICA binding agent for the treatment of cancer or for use in said treatment (particularly in a human); (c) the use of an anti-MICA binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, a method of using an anti-MICA binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, comprising combining the anti-MICA binding agent with a pharmaceutically acceptable carrier or pharmaceutical preparation comprising an effective dose of the anti-MICA binding agent that is suitable for the treatment of cancer; or (d) any combination of a), b), and c), in accordance with subject matter that is permissible for patenting in the country in which this application is filed.

[0057] Antibodies for detecting MICA and MICB MICA (PERB11.1) refers to MHC class I polypeptide-related sequence A (see, e.g., UniProtKB / Swiss-Prot Q29983), its gene and cDNA, and its gene product or naturally occurring variants thereof. The nomenclature of the MICA gene and protein, together with reference to the sequence accession numbers for different alleles, is described in Frigoul A. and Lefranc, MP. Recent Res. Devel. Human Genet., 3 (2005): 95-145 ISBN: 81-7736-244-5, the disclosure of which is incorporated herein by reference. The MICA gene and protein sequence, including polymorphisms at the protein and DNA levels, is also available from http: / / www.ebi.ac.uk / ipd / imgt / hla / align.html, maintained by Cancer Research UK and the European Bioinformatics Institute (EBI).

[0058] The amino acid sequence of MICA was first described in Bahram et al. (1994) Proc. Nat. Acad. Sci. 91:6259-6263 and Bahram et al. (1996) Immunogenetics 44:80-81, the disclosures of which are incorporated herein by reference. The MICA gene is polymorphic, exhibiting a unique distribution of multiple variant amino acids in the extracellular α1, α2, and α3 domains. To further define the MICA polymorphism, Petersdorf et al. (1999) tested its alleles in 275 individuals with common and rare HLA genotypes. The amino acid sequences of the extracellular α1, α2, and α3 domains of human MICA are shown in SEQ ID NOs: 1-5. The complete MICA sequence further includes a 23-amino acid leader sequence and transmembrane and cytoplasmic domains. The amino acid sequence of MICA*001 is shown in SEQ ID NO: 1, which corresponds to GenBank accession number AAB41060. The amino acid sequence of human MICA allele MICA*004 is set forth in SEQ ID NO:2, which corresponds to Genbank accession number AAB41063. The amino acid sequence of human MICA allele MICA*007 is set forth in SEQ ID NO:3, which corresponds to Genbank accession number AAB41066. The amino acid sequence of human MICA allele MICA*008 is set forth in SEQ ID NO:4, which corresponds to Genbank accession number AAB41067. The amino acid sequence of human MICA allele MICA*019 is set forth in SEQ ID NO:5, which corresponds to Genbank accession number AAD27008.

[0059] MICB (also known as PERB11.2) refers to MHC class I polypeptide-related sequence B (see, e.g., UniProtKB / Swiss-Prot Q29980). The amino acid sequence of a representative human MICB polypeptide is set forth in Genbank accession number CAI18747 (SEQ ID NO: 6).

[0060] [Table 2]

[0061] [Table 3]

[0062] While MICA is constitutively expressed in some cells, low levels of MICA expression do not normally result in host immune cell attack. However, MICA is upregulated on rapidly proliferating cells, such as tumor cells. Of all NKG2D ligands, MICA is the most highly expressed and has been found across a wide range of tumor types (e.g., common carcinomas, bladder cancer, melanoma, lung cancer, hepatocellular carcinoma, glioblastoma, prostate cancer, common hematologic malignancies, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia). Recently, Tsuboi et al. (2011) (EMBO J:1-13) reported that the O-glycan branching enzyme, core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT), is active in MICA-expressing tumor cells and that MICA from tumor cells contains core 2 O-glycans (O-glycans containing an N-acetylglucosamine branch linked to an N-acetylgalactosamine).

[0063] Bauer et al., Science 285:727-729, 1999, provided a role for MICA as a stress-inducible ligand for NKG2D. As used herein, "MICA" refers to any MICA polypeptide, including any variant, derivative, or isoform of the MICA gene or the encoded protein to which they refer. The MICA gene is polymorphic and displays a unique distribution of multiple variant amino acids in the extracellular alpha-1, alpha-2, and alpha-3 domains. Various allelic variants have been reported for MICA polypeptides (e.g., MICA), each of which is referred to by a respective term, e.g., the human MICA polypeptides MICA*001, MICA*002, MICA*004, MICA*005, MICA*006, MICA*007, MICA*008, MICA*009, MICA*010, MICA*011, MICA*012, MICA*013, MICA*014, MICA*015, MICA*016, MICA*017, MICA*018, MICA*019, MICA*020, MICA*022, MICA*023, MICA*024, MICA*025, MICA*026, MICA*027, MICA*028, MICA*029, MICA*030, MICA*031, MICA*032, MICA*033, MICA*034, MICA*035, MICA*036, MICA*037, MICA*038, MICA*039, MICA*040, MICA*041, MICA*042, MICA*043, MICA*044, MICA*045, MICA*046, MICA*047, MICA*048, MICA*049, MICA*100, MICA*101, MICA*102, MICA*103, MICA*104, MICA*105, MICA*106, MICA*107, MICA*108, MICA*109, MICA*110, MICA*111, MICA*112, MICA*113, MICA*114, MICA*115, MICA*116, MICA*117, MICA*118, MICA*1 ICA*027, MICA*028, MICA*029, MICA*030, MICA*031, MICA*032, MICA*033, MICA*034, MICA *035, MICA*036, MICA*037, MICA*038, MICA*039, MICA*040, MICA*041, MICA*042, MICA*043 , MICA*044, MICA*045, MICA*046, MICA*047, MICA*048, MICA*049, MICA*050, MICA*051, MICA*052, MICA*053, MICA*054, MICA*055, MICA*056 and further encompassed by alleles MICA*057 to MICA*087.

[0064] As used herein, "NKG2D," and unless otherwise indicated or contradicted by context, the terms "hNKG2D," "NKG2-D," "CD314," "D12S2489E," "KLRK1," "killer cell lectin-like receptor subfamily K, member 1," or "KLRK1" refer to the human killer cell-activating receptor gene, its cDNA (e.g., GenBank Accession No. NM_007360) and its gene product (GenBank Accession No. NP_031386), or naturally occurring variants thereof. In NK and T cells, hNKG2D can form heterodimers or higher order complexes with proteins such as DAP10 (GenBank Accession Nos. AAG29425, AAD50293). Any activity attributed to hNKG2D herein, e.g., cell activation, antibody recognition, etc., may also be attributed to hNKG2D in the form of a heterodimer, e.g., hNKG2D-DAP10, or a higher order complex with these two (and / or other) components.

[0065] The three-dimensional structure of MICA in complex with NKG2D has been determined (see, e.g., Li et al., Nat. Immunol. 2001;2:443-451; code 1hyr and IMGT / 3Dstructure-DB (Kaas et al., Nucl. Acids Res. 2004;32:D208-D210)). When MICA is in complex with NKG2D homodimers, residues 63-73 of MICAα2 (IGMT numbering) are aligned, conferring approximately two helical turns. The two monomers of NKG2D contribute equally to the interaction with MICA, with seven positions in each NKG2D monomer interacting with one of the MICAα1 or α2 helix domains.

[0066] The antibodies (e.g., diagnostic antibodies) of the present disclosure specifically bind to human MICA (MICA*001 and MICA*008, optionally further including MICA*004, MICA*007, and / or MICA*019) and MICB, particularly in fixed samples such as paraffin-embedded tissue sections. The antibodies can specifically bind to their target antigens in biological samples containing MICA- and MICB-expressing cells that have been prepared as paraffin-embedded cell pellets (e.g., FFPE sections).

[0067] The antibody may optionally be characterized as an antibody or antibody fragment that binds to human MICA*001 polypeptide in a sample of MICA*001-expressing cells prepared as a paraffin-embedded cell pellet, binds to human MICA*008 polypeptide in a sample of MICA*008-expressing cells prepared as a paraffin-embedded cell pellet, and binds to human MICB polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICB-negative cells (e.g., Raji cells) prepared as a paraffin-embedded cell pellet. In either embodiment, the antibody or antibody fragment may optionally be further characterized as an antibody or antibody fragment that binds to human MICA*002 polypeptide in a sample of MICA*002-expressing cells prepared as a paraffin-embedded cell pellet. In either embodiment, the antibody or antibody fragment may optionally be further characterized as an antibody or antibody fragment that binds to human MICA*007 polypeptide in a sample of MICA*007-expressing cells prepared as a paraffin-embedded cell pellet. In either embodiment, the antibody or antibody fragment may optionally be further characterized as an antibody or antibody fragment that binds to a human MICA*004 polypeptide in a sample of MICA*004-expressing cells that has been prepared as a paraffin-embedded cell pellet.

[0068] As described herein, the ability of the antibodies to specifically bind to MICA and MICB in paraffin-embedded tissue sections makes them useful for many applications, particularly for detecting MICA- and / or MICB-expressing cells (e.g., tumor cells, cells contributing to tumor progression, cells contributing to tumor evasion from control or lysis by the host immune system) and the levels or distribution of MICA- and / or MICB-expressing cells for diagnostic or therapeutic purposes. In certain embodiments, the antibodies are used to determine the presence or levels of MICA- and / or MICB-expressing cells in or near tumor tissue in a sample (e.g., biopsy) taken from an individual, e.g., an individual with cancer or tumor. Optionally, in one embodiment, the presence of MICA and / or MICB at cell (e.g., tumor cell) membranes is assessed and / or detected in the tissue sample. Optionally, in one embodiment, MICA- and / or MICB-expressing cells are determined to be present if MICA and / or MICB are detected in the tissue sample. Optionally, in another embodiment, if MICA and / or MICB are detected in the tissue sample (optionally, if a predetermined level of MICA and / or MICB staining is detected), the individual is determined to be suitable for treatment with a therapeutic antibody that binds MICA and / or MICB, e.g., a depleting anti-MICA and / or MICB antibody. Optionally, in one embodiment, the individual has been treated (e.g., during an ongoing or previous course of treatment) with a therapeutic antibody that binds to the target antigen.

[0069] Detection of antibody binding to MICA and / or MICB can be carried out in any of a number of ways. For example, the antibody can be directly labeled with a detectable moiety, such as a luminescent compound, e.g., a fluorescent moiety or a radioactive compound, gold, biotin (allowing subsequent amplified binding to avidin, e.g., avidin-AP), or an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). Alternatively, binding of the antibody to the target antigen in the sample can be assessed indirectly, e.g., by using a secondary antibody that binds to the primary anti-target antigen antibody and is itself preferably labeled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); however, it will be recognized that the secondary antibody can be labeled or detected using any suitable method. In a preferred embodiment, an amplification system is used to enhance the signal provided by the secondary antibody, such as the EnVision system, in which the secondary antibody is conjugated to a polymer (e.g., dextran) to which many copies of a detectable compound or enzyme, such as HRP or AP, are attached (see, e.g., Wiedorn et al., (2001) The Journal of Histochemistry & Cytochemistry, Volume 49(9):1067-1071; Kaemmerer et al., (2001) Journal of Histochemistry and Cytochemistry, Vol. 49, 623-630, the entire disclosures of which are incorporated herein by reference).

[0070] In one aspect, the present disclosure provides a method for producing an antibody capable of detecting MICA and / or MICB in an FFPE sample. MICA and / or MICB or one or more immunogenic fragments thereof can be used as an immunogen to generate an antibody, which can recognize an epitope within a target antigen polypeptide in a paraffin-embedded sample as described herein. Preferably, the recognized epitope is present on the cell surface, i.e., it is accessible to the antibody on the outside of the cell. In one aspect, the epitope is an epitope specifically recognized by antibody 12C9 in a paraffin-embedded cell pellet sample. In one aspect, the antibody competes with antibody 12C9 for binding to the epitope specifically recognized by antibody 12C9 in a paraffin-embedded cell pellet sample. Furthermore, to bind MICA and / or MICB with maximal efficacy and breadth in a population of human individuals who may have undergone or have undergone different prior therapies, particularly those who may have been treated with therapeutic antibodies against target antigen polypeptides (e.g., MICA and / or MICB, MICA*001 and MICA*008 and further MICB), antibodies that recognize the same epitope within MICA as that recognized by antibody 12C9 or that compete for binding with 12C9 can be used.

[0071] The antibodies can be produced by a variety of techniques known in the art, as demonstrated, for example, in Example 2 herein. Generally, they are produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a MICA and / or MICB polypeptide (e.g., MICA*001, MICA*008, and MICB polypeptides), preferably a human polypeptide. The polypeptide may comprise the full-length sequence of the human polypeptide or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of the polypeptide comprising an epitope exposed on the surface of cells expressing the MICA polypeptide, preferably an epitope recognized by the 12C9 antibody. Such fragments typically contain at least about 7 contiguous amino acids of the mature polypeptide sequence, and even more preferably at least about 10 contiguous amino acids thereof. Fragments typically are derived essentially from the extracellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human MICA polypeptide or a fragment thereof. In a specific embodiment, the immunogen comprises an intact cell, particularly an intact human cell, that is optionally treated or lysed.

[0072] The step of immunizing a non-human mammal with an antigen can be carried out in any manner known in the art for stimulating antibody production in mice (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)). The immunogen is optionally suspended or dissolved in a buffer solution with an adjuvant, such as complete or incomplete Freund's adjuvant. Methods for determining the amount of immunogen, the type of buffer, and the amount of adjuvant are well known to those skilled in the art and are not intended to limit the present invention in any way. These parameters may vary for different immunogens but are easily ascertained.

[0073] Similarly, the location and frequency of immunization sufficient to stimulate antibody production are well known in the art. In a typical immunization protocol, non-human animals are intraperitoneally injected with an antigen on day 1 and again about one week later. This is followed by a recall injection of the antigen, optionally with an adjuvant, such as incomplete Freund's adjuvant, on about day 20. Recall injections are performed intravenously and can be repeated over several consecutive days. This is followed by a booster injection, typically without an adjuvant, either intravenously or intraperitoneally on day 40. This protocol results in the production of B cells that produce antigen-specific antibodies after about 40 days. Other protocols can also be used, so long as they result in the production of B cells that express antibodies directed against the antigen used in immunization.

[0074] In an alternative embodiment, lymphocytes from a non-immunized non-human mammal are isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out.

[0075] Splenocytes can be isolated from immunized non-human mammals, followed by fusing the splenocytes with immortalized cells to form antibody-producing hybridomas. Isolation of splenocytes from non-human mammals is well known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces, and squeezing the splenocytes from the splenic membrane through a nylon mesh of a cell strainer into an appropriate buffer to produce a single cell suspension. The cells are washed, centrifuged, and resuspended in a buffer that lyses any red blood cells. The solution is centrifuged again, and finally, the remaining lymphocytes in the pellet are resuspended in a new buffer.

[0076] Once isolated and in single-cell suspension, lymphocytes can be fused with an immortal cell line. This is typically a mouse myeloma cell line, although numerous other immortal cell lines useful for generating hybridomas are known in the art. Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, USA, and X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Fusion is accomplished using polyethylene glycol, or the like. The resulting hybridomas are then grown in a selective medium containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0077] Hybridomas are typically grown on a feeder layer of macrophages. The macrophages are preferably from the littermate of the non-human mammal used to isolate the splenocytes, and are typically primed with incomplete Freund's adjuvant or similar several days before plating the hybridomas. Fusion methods are described in Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59-103 (Academic Press, 1986), the disclosure of which is incorporated herein by reference.

[0078] The cells are allowed to grow in the selective medium for a time sufficient for colony formation and antibody production, which is usually from about 7 to about 14 days.

[0079] Hybridoma colonies can be assayed for the production of antibodies that specifically bind to MICA and / or MICB on fixed, paraffin-embedded MICA- and / or MICB-expressing cell pellets; optionally, hybridomas are assayed for competition with antibody 12C9 for binding to fixed, paraffin-embedded MICA- and / or MICB-expressing cell pellets. Wells positive for the production of the desired antibody are tested to determine whether one or more distinct colonies are present. If multiple colonies are present, the cells can be recloned and expanded to confirm that only a single cell gave rise to the colony producing the desired antibody. Cells that do not naturally express the target antigen (e.g., MICA) can be made to express the target antigen (e.g., by transfection with a nucleic acid expressing the target antigen), prepared as a cell pellet, formalin-fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to slides. Control cells that do not express the target antigen (e.g., the same cells as above but not transfected with the target antigen) can be used as a negative control.

[0080] Hybridomas that are confirmed to produce the desired monoclonal antibody can be grown in larger quantities in an appropriate medium, such as DMEM or RPMI-1640. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in an animal. The growth medium containing the monoclonal antibody (or ascites fluid) is allowed to grow sufficiently to produce the desired monoclonal antibody, after which it is separated from the cells and the monoclonal antibody present therein is purified. Purification is typically accomplished by gel electrophoresis, dialysis, chromatography using Protein A or Protein G-Sepharose, or anti-mouse Ig bound to solid supports, such as agarose or Sepharose beads (all as described, for example, in Antibody Purification Handbook, Biosciences, publication No. 18-1037-46, Edition AC, the disclosures of which are incorporated herein by reference). Bound antibodies are typically eluted from the Protein A / Protein G column using low pH buffers (glycine or acetate buffers at pH 3.0 or below) with immediate neutralization of the antibody-containing fractions, which are then pooled, dialyzed, and concentrated as necessary.

[0081] Positive wells, with an apparent single colony, are typically recloned and reassayed to ensure that only one monoclonal antibody is detected and produced.

[0082] Antibodies can also be produced by selecting combinatorial libraries of immunoglobulins, for example, as disclosed in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is incorporated herein by reference). For example, phage display technology can be used to generate libraries.

[0083] According to an alternative embodiment, hybridomas may first be assayed for the production of antibodies that specifically bind to target antigen polypeptides (e.g., MICA and / or MICB), and DNA encoding the antibodies may be isolated from the hybridomas and placed into a suitable expression vector for gene transfer into suitable host cells. The host cells may then be used for recombinant production of the antibodies or variants thereof, such as functional fragments of the antibodies, chimeric antibodies comprising the antigen-recognition portion of the antibodies, or versions comprising a detectable moiety. The recombinantly produced antibodies may then be assayed for the production of antibodies that specifically bind to MICA and / or MICB on fixed, paraffin-embedded MICA- and / or MICB-expressing cell pellets, and optionally, the hybridomas may be assayed for competition with antibody 12C9 for binding to fixed, paraffin-embedded MICA- and / or MICB-expressing cell pellets.

[0084] Identification of one or more antibodies that bind to MICA and / or MICB, and in particular compete with monoclonal antibody 12C9 for binding to MICA and / or MICB (or epitopes thereon to which 12C9 binds), can be readily determined using any one of a variety of immunological screening assays in which antibody competition can be assessed, according to the methods described herein or any other suitable method.

[0085] In certain embodiments, varying amounts of test antibody and control antibody (e.g., 12C9) are premixed (e.g., about 1:10 or about 1:100) for a period of time before application to a MICA and / or MICB antigen sample (e.g., a paraffin-embedded MICA and / or MICB-expressing cell pellet sample). In other embodiments, the control and varying amounts of test antibody can simply be mixed during exposure to the MICA and / or MICB antigen sample. As long as it is possible to distinguish bound antibody from free antibody (e.g., by using separation or washing techniques to remove unbound antibody) and 12C9 from test antibody (e.g., by using a species- or isotype-specific secondary antibody or by specifically labeling 12C9 with a detectable label), it can be determined whether the test antibody reduces binding of 12C9 to the antigen, indicating that the test antibody competes with 12C9 for binding to the antigen. Binding of a (labeled) control antibody in the absence of a completely unrelated antibody can serve as a high-value control. A low control can be obtained by incubating labeled (12C9) antibody with an unlabeled antibody of the exact same type (12C9), which will compete and reduce binding of the labeled antibody. A significant reduction in the reactivity of the labeled antibody in the presence of the test antibody in the test assay indicates a test antibody that "cross-reacts" with the labeled (12C9) antibody. Any test antibody that reduces binding of 12C9 to MICA and / or MICB by at least about 50%, e.g., at least about 60%, or more preferably at least about 70% (e.g., about 65-100%) is considered to be an antibody that competes with 12C9 at any ratio of 12C9:test antibody from about 1:10 to about 1:100. Preferably, such a test antibody reduces binding of 12C9 to MICA and / or MICB antigens by at least about 90% (e.g., about 95%).

[0086] Upon immunization and antibody production in a vertebrate animal or cell (or, for example, generation of a library of candidate antibodies, optionally a library of nucleic acid or amino acid sequences for antibodies), certain selection steps may be performed to isolate the claimed antibody. In this regard, in certain embodiments, the present invention also relates to methods for producing such antibodies, comprising: (a) providing a library of antibodies and / or immunizing a non-human mammal with an immunogen comprising MICA and / or MICB polypeptides and preparing antibodies from said immunized animal; and (b) selecting antibodies from step (a) capable of specifically binding to said MICA and / or MICB polypeptides in paraffin-embedded cell pellets, e.g., FFPE cell pellets.

[0087] DNA encoding a monoclonal antibody of the invention, e.g., antibody 12C9, can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the murine antibody). Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells that do not otherwise produce immunoglobulin protein, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. As described elsewhere herein, such DNA sequences can be modified for any of a number of purposes, e.g., to humanize the antibody, to produce fragments or derivatives, or to modify the sequence of the antibody, e.g., in the antigen-binding site, to optimize the binding specificity of the antibody.

[0088] The present disclosure further provides screening methods based on formaldehyde-treated, paraffin-embedded cell pellets (FFPE cell pellets) that reveal MICA and / or MICB epitopes present after formalin treatment, including when the cells are incubated with other anti-MICA antibodies (e.g., neutralizing antibodies) prior to fixation. Accordingly, in one aspect, the present disclosure provides monoclonal antibodies that specifically bind to MICA and / or MICB polypeptide-expressing cells (e.g., cells that have been made to express MICA and / or MICB) in samples stored as paraffin-embedded cell pellets (and deparaffinized prior to analysis). Optionally, the antibodies are further characterized by their lack of binding to MICA- and MICB-negative cells (cells that do not express either MICA or MICB) in paraffin-embedded cell pellets. Optionally, the antibodies are further characterized by their binding to MICA and / or MICB polypeptide-expressing cells in paraffin-embedded tissue sections.

[0089] In one aspect, the present disclosure provides a monoclonal antibody (e.g., a first antibody) that specifically binds to human MICA and MICB polypeptides, wherein the antibody (e.g., the first antibody) specifically binds to the MICA and MICB polypeptides in a biological sample that has been treated (or fixed) with formaldehyde (e.g., formalin, paraformaldehyde). Formaldehyde fixation can be used, inter alia, in the preparation of paraffin-embedded tissue sections that can then be deparaffinized and analyzed for the presence of a marker of interest, such as MICA.

[0090] In one aspect, a monoclonal antibody is provided that specifically binds to a human MICA polypeptide expressed by cells preserved in paraffin, e.g., cells preserved as a paraffin-embedded cell pellet. Optionally, the cells are pelleted, formaldehyde-treated (e.g., formaldehyde, formalin, paraformaldehyde), and then paraffin-embedded. Optionally, the cells expressing the human MICA polypeptide are in a biological sample that is deparaffinized prior to antibody binding and analysis.

[0091] In one aspect, the antibody binds to an antigenic determinant present on MICA and MICB, optionally MICA*001, MICA*008, and MICB, in an FFPE cell pellet sample. In one aspect, the antibody binds to substantially the same epitope or determinant as antibody 12C9 or competes with 12C9 for binding to such an epitope on MICA and / or MICB in an FFPE cell pellet sample. In one embodiment, the antibody binds to an epitope of MICA and / or MICB that at least partially overlaps with or includes at least one residue in the epitope bound by antibody 12C9. The residue to which the antibody binds can be identified as being present on the surface of MICA and MICB polypeptides, optionally further expressed on the surface of cells, or optionally further expressed on the surface of cells preserved as paraffin-embedded cell pellets.

[0092] The amino acid sequence of the heavy chain variable region of antibody 12C9 is set forth as SEQ ID NO:7, and the amino acid sequence of the light chain variable region is set forth as SEQ ID NO:8. In certain embodiments, the antibody essentially binds to the same epitope or determinant as monoclonal antibody 12C9; optionally, the antibody comprises the hypervariable region of antibody 12C9. In any of the embodiments herein, antibody 12C9 may be characterized by its amino acid sequence and / or its encoding nucleic acid sequence. In one embodiment, the monoclonal antibody comprises the Fab or F(ab')2 portion of 12C9. Also provided are monoclonal antibodies comprising the heavy chain variable region of 12C9 or function-conservative variants thereof. According to one embodiment, the monoclonal antibody comprises the three CDRs of the heavy chain variable region of 12C9. Also provided are monoclonal antibodies further comprising the variable light chain variable region of 12C9 or function-conservative variants thereof. According to one embodiment, the monoclonal antibody comprises the three CDRs of the light chain variable region of 12C9. Optionally, any one or more of the light or heavy chain CDRs may contain one, two, three, four, five, or more amino acid modifications (e.g., substitutions, insertions, or deletions). Optionally, antibodies are provided in which any of the light and / or heavy chain variable regions, comprising part or all of the antigen-binding region of antibody 12C9, optionally further comprises amino acid substitutions to, for example, modulate (e.g., reduce) effector function (binding to human Fcγ receptors) or provide for conjugation of a moiety of interest (e.g., a detectable moiety), in which the light and / or heavy chain variable regions are fused to an IgG-type immunoglobulin constant region, optionally a human constant region, optionally of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0093] In one embodiment, the anti-MICA antibody comprises a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99% or more identity) to a heavy chain variable region having the amino acid sequence of SEQ ID NO:7.

[0094] In one embodiment, the anti-MICA antibody comprises a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99% or more identity) to a light chain variable region having the amino acid sequence of SEQ ID NO:8.

[0095] In one embodiment, the antibody comprises an HCDR1 comprising the amino acid sequence: GYYMN (SEQ ID NO: 9), or a sequence of at least 4 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; an HCDR2 comprising the amino acid sequence: TINPYYGSSTYNQKFKG (SEQ ID NO: 10), or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; an HCDR3 comprising the amino acid sequence: VDGDHGYFDY (SEQ ID NO: 11), or a sequence of at least 4, 5, or 6 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and an amino acid sequence: RSSQ. an LCDR1 region comprising the amino acid sequence: SLVHSNGNTYLH (SEQ ID NO: 12) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (optionally, one or more of these amino acids may be substituted with a different amino acid); an LCDR2 region comprising the amino acid sequence: KVSTRFS (SEQ ID NO: 13) or a sequence of at least 4, 5 or 6 consecutive amino acids thereof (optionally, one or more of these amino acids may be substituted with a different amino acid); and / or an LCDR3 region comprising the amino acid sequence: SQSTHVPFT (SEQ ID NO: 14) or a sequence of at least 4, 5, 6, 7 or 8 consecutive amino acids thereof (optionally, one or more of these amino acids may be deleted or substituted with a different amino acid).

[0096] Particular heavy chain, light chain, variable region, framework and / or CDR sequences may contain sequence modifications, e.g., substitutions (1, 2, 3, 4, 5, 6, 7, 8, or more sequence modifications). In one embodiment, the amino acid sequence contains one, two, three, or more amino acid substitutions, and the substituted residues are residues present in the sequence of human origin. In one embodiment, the substitutions are conservative modifications. Conservative sequence modifications refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions typically replace an amino acid residue with an amino acid residue having a side chain with similar physicochemical properties. A defined amino acid sequence may contain one, two, three, four, or more amino acid insertions, deletions, or substitutions. When substitutions are made, conservative modifications are preferred. Families of amino acid residues having similar side chains have been defined in the art. 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention can be replaced with another amino acid residue from the same side chain family, or the altered antibody can be tested for retained function (i.e., the properties described herein) using the assays described herein.

[0097] In one embodiment, the antibodies of the present invention are antibody fragments that retain their binding and / or functional properties. Fragments and derivatives of the antibodies of the present invention (which are encompassed by the terms "antibody" or "antibodies" as used in this application unless otherwise specified or clearly contradicted by the context), preferably 12C9-like antibodies, can be produced by techniques known in the art. A "fragment" comprises a portion of an intact antibody, generally the antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, (1) a single-chain Fv molecule, (2) a single-chain polypeptide containing only one light-chain variable domain, without any associated heavy-chain portion, or a fragment thereof containing the three CDRs of the light-chain variable domain, and (3) a single-chain polypeptide containing only one heavy-chain variable region, without any associated light-chain portion, or a fragment thereof containing the three CDRs of the heavy-chain variable domain; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody or antibody fragment is derivatized by conjugating or covalently linking the antibody or antibody fragment to a detectable moiety.

[0098] FFPE sample preparation and staining The present antibodies have the particular property of being able to efficiently and specifically bind to polypeptides (e.g., MICA and MICB polypeptides) present in fixed tissue or cell samples. Various methods for preparing and using such tissue preparations are well known in the art, and any suitable method or type of preparation may be used. The present antibodies are further capable of binding to their target antigens in samples in which therapeutic (e.g., function-neutralizing) antibodies were present at or before fixation.

[0099] The FFPE material in a biological sample from an individual is generally tissue. FFPE tissue is a piece of tissue that is first isolated from a specimen animal (e.g., a human individual) by dissection or biopsy. The tissue is then fixed to prevent its decay or degradation and to allow it to be clearly examined under a microscope for histological, pathological, or cytological studies. Fixation is a process that immobilizes, kills, and preserves tissue for the purpose of staining and examining it under a microscope. Post-fixation treatments allow the tissue to be permeated with staining reagents and cross-link its macromolecules so that they are stabilized and locked in place. The fixed tissue is then embedded in wax and allowed to be sectioned and stained with hematoxylin and eosin stain. It is then microtomed by sectioning for examination of antibody staining under a microscope.

[0100] For example, it will be appreciated that the present antibodies can be used with different suitable fixed cell or tissue specimens, and different specific fixation or embedding methods will be used. For example, while the most common formaldehyde-based fixation procedure involves formalin (e.g., 10%), there are alternative methods such as paraformaldehyde (PFA), Bouin's solution (formalin / picric acid), alcohol, zinc-based solutions (for one example, see, e.g., Lykidis et al., (2007) Nucleic Acids Research, 2007, pp. 1-10, the disclosure of which is incorporated herein by reference in its entirety), and others (see, e.g., the HOPE method, Pathology Research and Practice, Volume 197, Number 12, December 2001, pp. 823-826(4), the disclosure of which is incorporated herein by reference in its entirety). Similarly, while paraffin is preferred, other materials can also be used for embedding, such as polyester wax, polyethylene glycol-based formulations, glycol methacrylate, JB-4 plastic, and the like. For reviews of methods for preparing and using tissue preparations, see, e.g., Gillespie et al., (2002) Am J Pathol. 2002 February; 160(2):449-457; Fischer et al. CSH Protocols; 2008; Renshaw (2007), Immunohistochemistry: Methods Express Series; Bancroft (2007) Theory and Practice of Histological Techniques; and WO 06074392, the entire disclosures of which are incorporated herein by reference.

[0101] In one embodiment of the present invention, the FFPE tissue is tumor tissue or tumor-adjacent tissue, such as human tumor tissue, which may be, for example, head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, or melanoma.

[0102] The antibody (e.g., anti-MICA and / or MICB antibody) is incubated with FFPE material for the detection of MICA and / or MICB polypeptides. The term incubation step involves contacting the FFPE material with the antibody of the present invention for a distinct period of time depending on the type of material, antibody, and / or antigen. The incubation process also depends on various other parameters, such as detection sensitivity, and optimization follows routine procedures known to those skilled in the art. The addition of chemical solutions and / or application of physical procedures, such as the influence of heat, can improve the accessibility of target structures in the sample. Specific incubation products are formed as a result of incubation.

[0103] Suitable tests for the detection of antibody / antigen complexes formed are known to those skilled in the art or can be routinely easily designed. Many different types of assays are known, examples of which are given below.

[0104] For example, the sample (tissue or cells) to be examined can be obtained by biopsy and sectioned (e.g., 3 mm or less thick) from body fluids, tumor tissue, or healthy tissue, and fixed using formalin or an equivalent fixation method (see above). The time of fixation depends on the application, but can range from a few hours to 24 hours or more. After fixation, the tissue is embedded in paraffin (or an equivalent material), cut into very thin sections (e.g., 5 microns) with a microtome, and then mounted on a preferably coated slide. The slide is then dried, e.g., air-dried.

[0105] The fixed and embedded tissue sections on the slides can be dried and stored indefinitely. For immunohistochemistry, the slides are deparaffinized and then rehydrated. For example, they are subjected to a series of washes, first with xylene, then with xylene with ethanol, and then with decreasing percentages of ethanol in water.

[0106] Prior to antibody staining, tissues can be subjected to an antigen retrieval step, e.g., enzymatic or heat-based, to disrupt methane bridges that form during fixation and can mask epitopes. In a preferred embodiment, treatment in boiling 10 mM citrate buffer, pH 6, is used.

[0107] Once the slides are rehydrated and ideally antigen retrieval has been performed, they can be incubated with the primary antibody. First, the slides can be washed, e.g., with TBS, and then, after a blocking step, e.g., with serum / BSA, the antibody can be applied. The concentration of the antibody depends on its form (e.g., purified), its affinity, and the tissue sample used, but a suitable concentration is, e.g., 1-10 μg / ml. In one embodiment, the concentration used is 10 μg / ml. The incubation time can also vary, but overnight incubation is generally appropriate. After a post-antibody wash step, e.g., in TBS, the slides are then processed for detection of antibody binding.

[0108] The detection method used will depend on the antibody, tissue, etc. used, and may involve, for example, detection of luminescence or otherwise visible or detectable moieties through the use of a secondary antibody conjugated to or detectable by the primary antibody. Methods of antibody detection are well known in the art and are taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1st edition (December 1, 1988); Fischer et al. CSH Protocols; 2008; Renshaw (2007), Immunohistochemistry: Methods Express Series; Bancroft (2007) Theory and Practice of Histological Techniques; WO 06074392; the entire disclosures of each of which are incorporated herein in their entirety.

[0109] Many direct or indirect detection methods are known and can be adapted for use. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, etc., linked to antibodies. Antibodies labeled with iodine-125 (125I) can be used. Chemiluminescent assays using protein-specific chemiluminescent antibodies are suitable for sensitive, non-radioactive detection of protein levels. Antibodies labeled with fluorescent dyes are also suitable. Examples of fluorescent dyes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas Red, and Lissamine.

[0110] Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, and urease. Covalent conjugation of anti-MICA antibodies to enzymes can be achieved by different methods, such as coupling with glutaraldehyde. Both the enzyme and antibody are linked to glutaraldehyde via their free amino groups, followed by removal of the networked enzyme by-products and antibody. Alternatively, enzymes, such as peroxidase, can be coupled to antibodies via sugar residues if they are glycoproteins. The enzyme is oxidized with sodium periodate and directly linked to the amino groups of the antibody. Other carbohydrate-containing enzymes can also be coupled to antibodies in this manner. Enzyme coupling can also be achieved by linking the free thiol group of an enzyme, such as β-galactosidase, to the amino group of an antibody using a heterobifunctional linker, such as succinimidyl 6-(N-maleimido)hexanoate. For example, a horseradish-peroxidase detection system can be used with the chromogenic substrate tetramethylbenzidine (TMB), which produces a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, which produces a soluble product that is easily detectable at, for example, 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoxide (ONPG), which produces a soluble product that is detectable at 410 nm. A urease detection system can be used with a substrate such as urea-bromocresol purple.

[0111] In one embodiment, binding of the primary antibody is detected by binding to a labeled secondary antibody, preferably a secondary antibody covalently linked to an enzyme such as HRP or AP. In a particularly preferred embodiment, any of a number of methods for amplifying antibody detection is used to amplify the signal generated by binding of the secondary antibody. For example, the EnVision method can be used (see, e.g., U.S. Pat. No. 5,543,332 and European Patent No. 594,772; Kaemmerer et al. (2001) Journal of Histochemistry and Cytochemistry, Vol. 49, 623-630; Wiedorn et al. (2001) The Journal of Histochemistry & Cytochemistry, Volume 49(9):1067-1071; the entire disclosures of which are incorporated herein by reference), in which the secondary antibody is linked to a polymer (e.g., dextran) that is itself linked to multiple copies of AP or HRP.

[0112] In one example, formalin-fixed, paraffin-embedded blocks were sliced ​​into 5-μm-thick sections and immunostained on a Discovery Ultra or Benchmark Ultra automaton (Ventana). After pretreatment with Cell Conditioning 1, sections were incubated with 2 μg / mL (for staining on the Discovery Ultra) or 6.6 μg / mL (for staining on the Benchmark Ultra) anti-MICA / B primary antibody or mouse IgG1 isotype control for 1 hour at 37°C. Signal amplification was then performed using a discovery Amp HQ™ kit or UltraView™ kit. After development with 3,3-diaminobenzidine, counterstaining was performed with hematoxylin and bluing agent, and the sections were washed, dehydrated, cleared, and cover-slipped. Finally, the stained sections were scanned on a slide scanner (S60 Nanozoomer™ Hamamatsu or Pannoramic scan II, 3DHistech™). Staining is interpreted and scored by trained pathologists who determine MICA / B expression on tumor cells. Samples with more than a specified percentage of MICA / B-positive tumor cells (e.g., 1%, 5%, 10%, etc.) are considered MICA / B-positive.

[0113] Compositions and Uses in Diagnostics, Prognosis and Therapy The antibodies of the present disclosure are particularly effective in detecting MICA and / or MICB (e.g., containing multiple of the most prevalent alleles of MICA in the human population, including at least the MICA*001 and MICA*008 alleles) in biological samples prepared as FFPE, without nonspecific staining on tissues or cells that do not express MICA or MICB polypeptides. Thus, the antibodies have advantages for use in the testing, evaluation, diagnosis, prognosis, and / or monitoring of diseases in which detection and / or localization of MICA and / or MICB polypeptides and / or MICA and / or MICB-expressing cells is of interest. For example, patients whose tumors or tumor-adjacent tissues are characterized by MICA and / or MICB-expressing cells (e.g., MICA and / or MICB-expressing tumor cells) may have an unfavorable prognosis for tumor progression. Such patients may benefit from treatment with therapeutic agents and regimens appropriate for their prognosis and / or MICA and / or MICB expression profile, including, for example, immunotherapy, chemotherapy, and certain combination treatments.

[0114] Thus, methods for detecting, diagnosing, or monitoring cancer in a subject are provided, comprising contacting tumor cells (e.g., in vitro) with an anti-MICA / MICB antibody or antibody fragment of the present disclosure and detecting tumor-associated MICA and / or MICB polypeptides. In related embodiments, the diagnostic method comprises immunohistochemistry (IHC). In certain embodiments, tumor samples are chemically fixed and / or paraffin-embedded. Those skilled in the art will further recognize that such MICA / MICB detection agents can be labeled or associated with effectors, markers, or reporters and can be detected using any one of a number of standard imaging techniques. In other embodiments, the anti-MICA / MICB antibody is not directly labeled but is detected using a detectable secondary agent (e.g., a labeled anti-mouse antibody). In certain embodiments, the present disclosure provides methods for diagnosing individuals using any of the anti-MICA / MICB compositions and the detection methods of the present disclosure, and identifying or selecting individuals for administration of a therapeutic agent (e.g., chemotherapy, immunotherapy), including individualizing a course of treatment based on the outcome.

[0115] In one embodiment, the present disclosure provides a method for detecting MICA / MICB-expressing cells in a sample from an individual having head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, or melanoma, the method comprising contacting a paraffin-embedded tumor tissue sample from the individual with an antibody capable of specifically binding to human MICA and MICB polypeptides in the paraffin-embedded tumor tissue sample; detecting the presence of bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody. Upon detection of bound antibody in the section, and optionally upon detection of cell membrane (cell surface) staining by the antibody in the section, the individual can be determined or considered to have a tumor that is MICA and / or MICB-positive.

[0116] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells in a sample from an individual with urothelial carcinoma, pancreatic cancer, hepatocellular carcinoma (HCC), or endometrial cancer, the method comprising contacting a paraffin-embedded tumor tissue sample from the individual with an antibody capable of specifically binding to human MICA and MICB polypeptides in the paraffin-embedded tumor tissue sample; detecting the presence of the bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody. Upon detection of the bound antibody in the section, and optionally upon detection of cell membrane (cell surface) staining by the antibody in the section, the individual may be determined or considered to have a tumor that is MICA and / or MICB positive.

[0117] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells (e.g., cells that express MICA and / or MICB on their surface or cell membrane) in a sample from a human tumor, the method comprising contacting a paraffin-embedded tumor tissue sample from an individual with an antibody capable of specifically binding to human MICA and MICB polypeptides in the paraffin-embedded tumor tissue sample; detecting the presence of the bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody.

[0118] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells in a sample from an individual having head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, or melanoma, the method comprising contacting a paraffin-embedded tumor tissue sample from the individual with an antibody capable of specifically binding to human MICA and MICB polypeptides in the paraffin-embedded tumor tissue sample; detecting the presence of the bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody.

[0119] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells in a sample from a human tumor, the method comprising contacting a paraffin-embedded tumor tissue sample from an individual with an antibody capable of specifically binding to human MICA and MICB polypeptides on the surface of cells that have been prepared as a paraffin-embedded pellet; detecting the presence of the bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody.

[0120] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells in a sample from a human tumor, the method comprising contacting a paraffin-embedded tumor tissue sample from an individual with an antibody that has been assessed for its ability to specifically bind (or determined to specifically bind) human MICA and MICB polypeptides on the surface of cells that have been prepared as a paraffin-embedded cell pellet; detecting the presence of the bound antibody in the section, and optionally further detecting cell membrane (cell surface) staining by the antibody.

[0121] In one embodiment, the present disclosure provides a method for detecting MICA / B-expressing cells in a tumor tissue sample from an individual having a tumor, the method comprising: (a) providing a paraffin-embedded tumor tissue sample from an individual and contacting the sample with a monoclonal antibody that does not bind to MICA / MICB negative cells in the paraffin-embedded cell pellet and is capable of binding to MICA and / or MICB expressing cells in the paraffin-embedded cell pellet in vitro; (b) assessing whether this antibody binds to the surface of tumor cells; and a determination that the antibody binds to the surface of tumor cells indicates that the tumor is positive for MICA / B-expressing cells and / or is suitable for treatment with a depleting anti-MICA agent.

[0122] In either embodiment, the FFPE tissue may be tumor or tumor-adjacent tissue obtained from an individual pretreated with an anti-cancer treatment (e.g., a chemotherapeutic agent), optionally an anti-cancer treatment known to be capable of upregulating MICA and / or MICB-expressing cancer cells. Certain chemotherapeutic agents or other treatments have been shown to be capable of inducing and / or increasing MICA and / or MICB expression (and optionally additional NKG2D ligands) on tumor cells. This includes well-known chemotherapeutics, including ionizing and UV radiation, inhibitors of DNA replication, inhibitors of DNA polymerase, chromatin-modulating treatments, antimetabolites (e.g., halogenated analogs of pyruvate, such as 3-bromopyruvate), and apoptosis inducers, such as the HDAC inhibitors trichostatin A and valproic acid. Exemplary agents are those that activate DNA damage response pathways, such as those that activate ATM (ataxia telangiectasia mutated) or ATR (ATM- and Rad3-related) protein kinases, or CHK1 or even CHK2, or p53. Examples of the latter include ionizing radiation, inhibitors of DNA replication, DNA polymerase inhibitors, and chromatin-altering agents or treatments, such as HDAC inhibitors. Compositions that upregulate NKG2D ligands are further described in Gasser et al (2005) Nature 436(7054):1186-90. Additional chemotherapeutic agents include alkylating agents, cytotoxic antibiotics, such as topoisomerase I inhibitors, topoisomerase II inhibitors, plant derivatives, RNA / DNA antimetabolites, and antimitotic agents. Preferred examples include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, taxol, gemcitabine, navelbine, transplatin, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or any analog or derivative variant thereof.Individuals who have been pretreated with anti-cancer treatment and who have MICA and / or MICB expression on their tumor cells may be deemed suitable for treatment with anti-MICA and / or MICB antibodies.

[0123] In one embodiment, if MICA and / or MICB are detected, the FFPE tissue can be tumor or tumor-adjacent tissue obtained from an individual who is a candidate for treatment with anti-MICA and / or MICB antibodies.

[0124] In one embodiment, where MICA and / or MICB are detected, the FFPE tissue may be tumor or tumor-adjacent tissue obtained from an individual that has been treated with an anti-MICA and / or MICB antibody, e.g., has undergone or is undergoing a course of treatment with such an antibody.

[0125] The present disclosure further provides methods for selecting individuals with MICA- and / or MICB-expressing tumors for therapeutic intervention. Examples of therapeutic intervention include antibodies that bind to MICA and / or MICB, optionally causing depletion of MICA- and / or MICB-expressing cells. In one example, the therapeutic intervention is an agent that alleviates or reduces the immunosuppressive effects of MICA and / or MICB (e.g., an antibody that binds to MICA), such as an agent that alleviates or reduces the downregulation of NKG2D on the surface of NK and / or CD8 T cells induced by a soluble MICA polypeptide. If an individual is determined or deemed to have a tumor that is MICA- and / or MICB-positive, the methods of the present disclosure may optionally be further specified as including treating the individual with the therapeutic intervention, e.g., if the individual is determined to have a MICA- and / or MICB-expressing tumor, as determined by staining, optionally cell membrane (cell surface) staining, on the FFPE sample using an antibody capable of specifically binding to human MICA and / or MICB in the FFPE sample.

[0126] In either embodiment, the antibody can be used without the further or prior step of determining or assessing which alleles of MICA are expressed by an individual, hi either embodiment, the antibody can be used across a human population.

[0127] The antibodies described herein can be used to detect the presence of MICA- and / or MICB-expressing cells, e.g., tumor cells, preferably in vitro. Such methods generally involve contacting an antibody according to the present disclosure with a biological sample from an individual (e.g., an FFPE, deparaffinized sample) and detecting the formation of an immunological complex resulting from an immunological reaction between the antibody and the biological sample. The complex can be detected directly by labeling the antibody according to the present disclosure or indirectly by adding a molecule that reveals the presence of the antibody (e.g., a secondary antibody, a streptavidin / biotin tag, etc.). For example, labeling can be accomplished by coupling the antibody to a radioactive or fluorescent tag. These methods are well known to those of skill in the art. Accordingly, the present invention also relates to the use of antibodies according to the present disclosure to prepare diagnostic compositions that can be used to detect the presence of MICA- and / or MICB-expressing cells (e.g., tumor cells), optionally to detect the presence of a pathology in which MICA- and / or MICB-expressing cells are present, and optionally to characterize cancer or other pathologies in vivo or in vitro.

[0128] In some embodiments, the antibodies of the present disclosure are useful for predicting cancer progression. Cancer prognosis, prognosis for cancer or cancer progression, includes providing a forecast or prediction (prognosis for) any one or more of the following: survival time of a subject prone to or diagnosed with cancer, recurrence-free survival time, progression-free survival time of a subject prone to or diagnosed with cancer, response rate and / or duration of response to treatment in a subject or group of subjects prone to or diagnosed with cancer, degree of response, or survival rate of a subject after treatment. Exemplary survival endpoints include, for example, TTP (time to progression), PFS (progression-free survival), DOR (duration of response), and OS (overall survival). Generally, disease progression and response may be assessed according to standard tumor response criteria conventions, e.g., according to "Response Evaluation Criteria in Solid Tumors" (RECIST) v1.1 as detailed by Eisenhauer, EA, et al., New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1), Eur J Cancer 2009:45:228-247, the disclosure of which is incorporated herein by reference.

[0129] Diagnosing MICA / MICB-positive tumors, predicting cancer progression, and / or individualizing treatment regimens for individuals with cancer can be based, for example, on measuring the percentage of positively staining MICA cells in tumor or tumor-adjacent tissue samples. In this regard, patients who exhibit a certain percentage of positively staining cells in fixed IHC samples when examined with anti-MICA / MICB antibodies are considered MICA / MICB+ and selected for treatment according to the teachings herein. In such embodiments, tumor samples that exhibit greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50% positive cell staining, as measured as percent positive cells, can be classified as MICA / MICB+. In certain aspects, MICA / MICB+ tumors express MICA and / or MICB in >50% of their constituent cells, as measured as percent positive.

[0130] In other embodiments, patient diagnosis and / or selection may be predicted by the percentage of MICA and / or MICB positive cells staining at a particular intensity. By way of example, tumors with >10%, optionally >20%, of cells exhibiting 2+ intensity or higher may be considered suitable for chemotherapy or immunotherapy treatment. In other embodiments, a patient is a candidate for chemotherapy or immunotherapy treatment if >10%, >20%, >30%, >40%, or >50% of tumor cells exhibit 1+ intensity or higher when stained with anti-MICA / MICB antibodies and examined according to standard IHC protocols, e.g., as disclosed herein. In other specific embodiments, an individual is suitable for chemotherapy or immunotherapy treatment if >10%, >20%, >30%, >40%, or >50% of tumor cells exhibit 2+ intensity or higher when stained with anti-MICA / MICB antibodies and examined according to standard IHC protocols, e.g., as disclosed herein.

[0131] In some embodiments, tissue samples (e.g., tumor or tumor-adjacent tissue samples) from individuals with cancer may be characterized or evaluated using the antibodies disclosed herein to assess MICA and / or MICB polypeptides and / or MICA and / or MICB-expressing cells in or around the tumor.

[0132] In one embodiment, a cancer or tumor (or an individual having such a cancer or tumor) characterized by MICA and / or MICB-expressing cells may be identified as being suitable for (e.g., would benefit from) treatment with a chemotherapeutic or immunotherapeutic agent (e.g., a depleting anti-MICA and / or -MICB antibody).

[0133] In one embodiment, the present disclosure provides an in vitro method for diagnosing, prognosing, monitoring, and / or characterizing cancer in an individual in need thereof, the method comprising providing a paraffin-embedded tumor or tumor-adjacent sample from the individual and detecting MICA and / or MICB polypeptides (e.g., MICA and / or MICB-expressing cells) in the sample, optionally the paraffin-embedded tissue sample, using a monoclonal antibody that specifically binds to human MICA and MICB polypeptides in the fixed tissue sample, wherein detection of MICA and / or MICB polypeptides indicates that the individual is suitable for (e.g., will benefit from) treatment with a chemotherapeutic agent or an anti-MICA and / or MICB therapeutic agent. The anti-MICA and / or MICB therapeutic agent can be, for example, an agent that binds to human MICA and MICB polypeptides, and optionally, the agent is a depleting agent, such as an anti-MICA antibody (or a function-conservative variant thereof) having the heavy and light chain CDRs or variable regions of any of the known antibodies disclosed in WO 2013 / 117647, WO 2013 / 049527, WO 2014 / 040903, WO 2015 / 085210, WO 2018 / 217688 (e.g., the 7C6 antibody), WO 2018 / 081648, and WO 2019 / 183551 (e.g., the 1D5 antibody), the disclosures of which are incorporated herein by reference. Such agents may be useful for treating individuals with tumors or tumor tissue characterized by detectable and / or high levels of MICA and / or MICB expression.

[0134] In one embodiment, the present disclosure provides a method of treating or preventing head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, melanoma, urothelial cancer, pancreatic cancer, hepatocellular carcinoma (HCC), or endometrial cancer in an individual in need thereof, the method comprising: a) detecting MICA and / or MICB polypeptides in a formalin-treated and / or paraffin-embedded tumor tissue sample (or tumor cell sample) from an individual, optionally at the surface of cells (e.g., membrane MICA and / or MICB staining); b) upon determining that the tumor sample (or tumor cells) contain a MICA polypeptide (e.g., MICA-expressing cells), optionally at a level that is elevated compared to a reference level, administering to the individual an anti-cancer agent, optionally an antibody or chemotherapeutic agent that binds to human MICA and / or MICB polypeptide; In one embodiment, the individual has been pre-treated with a chemotherapeutic agent (prior to step (a)). Detection of MICA and / or MICB polypeptides can be performed using the antibodies of the present disclosure.

[0135] In either aspect, detecting MICA and / or MICB polypeptides in a sample using an antibody can include contacting a biological sample from an individual (e.g., a deparaffinized, FFPE sample) with the antibody and detecting the formation of an immunological complex resulting from an immunological reaction between the antibody and the biological sample.

[0136] Also provided are diagnostic or prognostic kits for detecting MICA and / or MICB, e.g., for cancer, comprising antibodies according to the present disclosure. Optionally, the kits include an antibody of the invention and antibodies (e.g., 1, 2, 3, 4, 5, 10, or more antibodies) that bind to non-MICA / MICB polypeptides for use as a diagnostic or prognostic. The kits may further include means for detecting immunological complexes resulting from an immunological reaction between a biological (e.g., tumor tissue) sample and the antibodies, particularly reagents that allow for the detection of the antibodies.

[0137] The methods may be useful in the testing, evaluation, diagnosis, prognosis and / or monitoring of a range of cancers, such as head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, melanoma, urothelial cancer, pancreatic cancer, hepatocellular carcinoma (HCC) or endometrial cancer.

[0138] Embodiments: 1. An antibody or antibody fragment capable of specifically binding to a human MICA polypeptide and a human MICB polypeptide, comprising three CDRs of the heavy chain variable region sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region sequence of SEQ ID NO: 8, wherein the CDRs are determined according to Kabat numbering.

[0139] 2. An antibody or antibody fragment capable of specifically binding to human MICA and MICB polypeptides, wherein such binding is in a sample of cells expressing such MICA and / or MICB polypeptides and prepared as a paraffin-embedded cell pellet, the antibody or antibody fragment comprising a heavy chain variable domain comprising an amino acid sequence at least 80%, optionally at least 90%, identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable domain comprising an amino acid sequence at least 80%, optionally at least 90% identical to the amino acid sequence of SEQ ID NO:8.

[0140] 3. The antibody or antibody fragment of embodiment 1 or 2, which is conjugated or covalently attached to a detectable moiety.

[0141] 4. The antibody or antibody fragment of any one of the preceding embodiments, which binds to a MICA polypeptide in a sample of MICA-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA- and MICB-negative cells, optionally Raji cells, prepared as a paraffin-embedded cell pellet.

[0142] 5. An antibody or antibody fragment according to any one of the preceding embodiments, which binds to a MICA*001 polypeptide in a sample of MICA*001-expressing cells prepared as a paraffin-embedded cell pellet, and further binds to a MICA*008 polypeptide in a sample of MICA*008-expressing cells prepared as a paraffin-embedded cell pellet.

[0143] 6. The antibody or antibody fragment of any one of the preceding embodiments, which binds to a MICB polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA- and MICB-negative cells, optionally Raji cells, prepared as a paraffin-embedded cell pellet.

[0144] 7. The antibody or antibody fragment of any one of the preceding embodiments, which binds to BxPC-3 cells prepared as a paraffin-embedded cell pellet; optionally, when the antibody is provided at a low concentration (1 μg / mL), it is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet, and optionally, further, when the antibody is provided at low (1 μg / mL), medium (5 μg / mL), and high (10 μg / mL) concentrations, it is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet.

[0145] 8. The antibody or antibody fragment of any one of embodiments 2 to 7, comprising three CDRs of the heavy chain variable region sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region sequence of SEQ ID NO: 8, wherein the CDRs are determined according to Kabat numbering.

[0146] 9. The antibody or antibody fragment of any one of the preceding embodiments, which competes with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:7 and a light chain variable region having the amino acid sequence of SEQ ID NO:8 for binding to human MICA polypeptide expressed by cells (e.g., MICA-expressing cells) prepared as a paraffin-embedded cell sample.

[0147] 10. An in vitro method for detecting MICA and / or MICB polypeptides in a sample from a human individual, comprising providing a paraffin-embedded sample from said individual and detecting MICA polypeptides in said sample using the antibody or antibody fragment of any one of embodiments 1 to 9.

[0148] 11. An in vitro method for detecting MICA and / or MICB polypeptides in a sample from a human individual, comprising providing a paraffin-embedded sample from the individual; and detecting MICA polypeptides in the sample using an antibody or antibody fragment that binds to human MICA*001 polypeptide in a sample of MICA*001-expressing cells prepared as a paraffin-embedded cell pellet, binds to human MICA*008 polypeptide in a sample of MICA*008-expressing cells prepared as a paraffin-embedded cell pellet, and binds to human MICB polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA- and MICB-negative cells, optionally Raji cells, prepared as a paraffin-embedded cell pellet.

[0149] 12. The method of embodiment 11, wherein the antibody or antibody fragment binds to BxPC-3 cells prepared as a paraffin-embedded cell pellet; optionally, the antibody or antibody fragment is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet when the antibody is provided at a low concentration (1 μg / mL), and optionally, further, the antibody or antibody fragment is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet when the antibody is provided at a low concentration (1 μg / mL), a medium concentration (5 μg / mL), and a high concentration (10 μg / mL).

[0150] 13. The method according to embodiment 11 or 12, wherein the antibody or antibody fragment is an antibody or antibody fragment according to embodiments 1 to 9.

[0151] 14. The method of embodiment 11 or 12, wherein the antibody or antibody fragment is an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8; an antibody or antibody fragment that competes with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8 for binding to human MICA polypeptide expressed by cells prepared as a paraffin-embedded cell sample; or a function-conservative variant of the antibody or antibody fragment comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8.

[0152] 15. The method of any one of embodiments 10 to 14, wherein the step of detecting MICA and / or MICB polypeptides comprises contacting the sample with the antibody or antibody fragment and detecting the formation of an immune complex resulting from an immune reaction between the antibody or antibody fragment and the sample.

[0153] 16. The method of any one of embodiments 10 to 14, wherein the step of detecting MICA and / or MICB polypeptides comprises contacting the sample with the antibody or antibody fragment and detecting the formation of an immune complex at the cell membrane resulting from an immune reaction between the antibody or antibody fragment and the sample.

[0154] 17. The method of any one of embodiments 10 to 16, wherein the sample is tumor tissue.

[0155] 18. The method of any one of embodiments 10-17, wherein the individual has head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, melanoma, urothelial cancer, pancreatic cancer, hepatocellular carcinoma (HCC), or endometrial cancer.

[0156] 19. The method of any one of embodiments 10-18, wherein the individual is an individual who has undergone, is undergoing, or is a candidate for treatment with an anti-MICA antibody or antibody fragment.

[0157] 20. The method of any one of embodiments 10-19, wherein the individual has been pretreated with a chemotherapeutic agent.

[0158] 21. A method or composition according to any one of the above embodiments, wherein the paraffin-embedded tissue sample has been fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide.

[0159] 22. A method or composition according to any one of the previous embodiments, wherein the MICA and / or MICB polypeptide is detected using a secondary antibody that specifically binds to the antibody that binds the MICA and / or MICB polypeptide.

[0160] 23. An in vitro method for assessing MICA and MICB expression in an individual who has been pretreated with a chemotherapeutic agent, comprising: providing a paraffin-embedded tumor or tumor-adjacent tissue sample from the individual; and detecting MICA and / or MICB polypeptides in the sample using an antibody or antibody fragment that binds to human MICA*001 polypeptide in a sample of MICA*001-expressing cells prepared as a paraffin-embedded cell pellet, binds to human MICA*008 polypeptide in a sample of MICA*008-expressing cells prepared as a paraffin-embedded cell pellet, and binds to human MICB polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA and MICB-negative cells prepared as a paraffin-embedded cell pellet, wherein detection of MICA and / or MICB polypeptide indicates that the individual is suitable for treatment with a therapeutic agent, and optionally, the therapeutic agent is an antibody that binds to human MICA and / or MICB polypeptide.

[0161] 24. An in vitro method for assessing the suitability of an individual having a tumor for treatment with a therapeutic agent, comprising providing a paraffin-embedded tumor or tumor-adjacent tissue sample from the individual and detecting MICA and / or MICB polypeptides in the sample using the antibody or antibody fragment of embodiments 1-9 or the method of any one of embodiments 10-22, wherein detection of MICA and / or MICB polypeptides indicates that the individual is suitable for treatment with a therapeutic agent, and optionally, the therapeutic agent is an antibody that binds to human MICA and / or MICB polypeptides.

[0162] 25. The method of embodiments 23-24, further comprising administering to said individual an antibody that binds to human MICA and / or MICB polypeptides.

[0163] 26. The method of embodiments 10-25, wherein the individual has been pretreated with a radioactive or chemotherapeutic agent known to be capable of causing upregulation of MICA and / or MICB expression by tumor cells.

[0164] 27. A method for predicting cancer progression in an individual having cancer, comprising providing a paraffin-embedded tumor tissue sample from said individual, and detecting a MICA polypeptide in said sample according to the method of any one of embodiments 9 to 21 or using the antibody or antibody fragment of any one of embodiments 1 to 9.

[0165] 28. The method of embodiment 27, wherein detection of a MICA polypeptide in the sample (or detection of a higher number of such MICA-expressing cells compared to a reference value) indicates that the subject has an unfavorable prognosis for cancer progression.

[0166] 29. Detecting cells in the sample using antibodies comprises: Obtaining a biological sample containing cells (e.g., as a biopsy); Fixing, embedding in paraffin, sectioning and deparaffinizing the sample, and optionally transferring the sample to a slide; contacting the section with the antibody; detecting the presence of bound antibody within said section; 29. The method of any one of embodiments 10 to 28, comprising:

[0167] 30. A kit comprising an antibody or antibody fragment according to any one of embodiments 1 to 9, optionally further comprising a labeled secondary antibody that specifically recognizes the antibody according to any one of embodiments 1 to 9.

[0168] 31. A kit comprising the antibody or antibody fragment of any one of embodiments 1 to 9 and a therapeutic agent, optionally a depleting and / or neutralizing anti-MICA antibody.

[0169] 32. A nucleic acid or set of nucleic acids encoding the antibody or antibody fragment according to any one of embodiments 1 to 9.

[0170] 33. A hybridoma or recombinant host cell producing an antibody according to embodiments 1 to 9 or comprising a nucleic acid according to embodiment 32.

[0171] 34. A method for producing antibodies that specifically bind to human MICA and MICB polypeptides in paraffin-embedded tissue, comprising: a) providing cells that express a MICA polypeptide on their surface, providing cells that express a MICB polypeptide on their surface, and providing cells that do not express either a MICA polypeptide or a MICB polypeptide on their surface, and preparing a separate paraffin-embedded cell sample for each of said cells; b) providing a plurality of candidate antibodies; c) preparing or selecting antibodies from said plurality that bind to the paraffin-embedded cells of step a) that express MICA and the paraffin-embedded cells of step a) that express MICB without binding to the paraffin-embedded cells of step a) that express neither MICA nor MICB on their surface; A method comprising:

[0172] 35. The method of embodiment 34, further comprising the step of generating a derivative of the selected antibody.

[0173] 36. The method of embodiment 35, wherein producing the derivative comprises conjugating or covalently linking said antibody to a detectable moiety.

[0174] 37. The method of any one of embodiments 34 to 36, wherein the MICA polypeptide is a MICA*001 polypeptide.

[0175] 38.a) providing cells that express a MICA*001 polypeptide on their surface, providing cells that express a MICA*008 polypeptide on their surface, providing cells that express a MICB polypeptide on their surface, and providing cells that do not express a MICA polypeptide or a MICB polypeptide on their surface, and preparing a separate paraffin-embedded cell sample for each of said cells; b) providing a candidate antibody or a plurality of candidate antibodies; c) preparing or selecting an antibody from said plurality of candidate antibodies that tests the candidate antibody for binding to or binds to the paraffin-embedded cells of step a) that express MICA*001, the paraffin-embedded cells of step a) that express MICA*008, and the paraffin-embedded cells of step a) that express MICB, without binding to the paraffin-embedded cells of step a) that do not express MICA or MICB on their surface; 38. The method of any one of embodiments 34 to 37, comprising:

[0176] 39. An antibody obtained or produced according to the methods of embodiments 34 to 38.

[0177] 40. An antibody obtained or produced according to the method of embodiments 34 to 39, for use in a method for detecting MICA and / or MICB polypeptides in a sample from a human individual, optionally in a method according to any one of embodiments 10 to 29.

[0178] Further aspects and advantages of the present invention are disclosed after the experimental section, which should be considered as illustrative and not limiting the scope of this application. For example, the present disclosure provides: [Section 1] An antibody or antibody fragment capable of specifically binding to a human MICA polypeptide and a human MICB polypeptide, comprising three CDRs of the heavy chain variable region sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region sequence of SEQ ID NO: 8, wherein the CDRs are determined according to Kabat numbering. [Section 2] An antibody or antibody fragment capable of specifically binding to human MICA and MICB polypeptides, wherein said binding is in a sample of cells expressing said MICA and / or MICB polypeptides and prepared as a paraffin-embedded cell pellet, said antibody or antibody fragment comprising a heavy chain variable domain comprising an amino acid sequence at least 80%, optionally at least 90%, identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable domain comprising an amino acid sequence at least 80%, optionally at least 90% identical to the amino acid sequence of SEQ ID NO:8. [Section 3] 3. The antibody or antibody fragment of paragraph 1 or 2, which is conjugated or covalently attached to a detectable moiety. [Section 4] 4. The antibody or antibody fragment of any one of items 1 to 3, which binds to a MICA polypeptide in a sample of MICA-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA- and MICB-negative cells, optionally Raji cells, prepared as a paraffin-embedded cell pellet. [Section 5] MICA prepared as a paraffin-embedded cell pellet * MICA in samples of 001-expressing cells * MICA bound to 001 polypeptide and prepared as a paraffin-embedded cell pellet * MICA in samples of 008-expressing cells * Item 5. The antibody or antibody fragment according to any one of Items 1 to 4, which further binds to a 008 polypeptide. [Section 6] Item 6. The antibody or antibody fragment according to any one of Items 1 to 5, which binds to a MICB polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet, but does not bind to MICA- and MICB-negative cells, optionally Raji cells, prepared as a paraffin-embedded cell pellet. [Section 7] The antibody or antibody fragment of any one of Items 1 to 6, which binds to BxPC-3 cells prepared as a paraffin-embedded cell pellet; optionally, when the antibody is provided at a low concentration (1 μg / mL), it is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet, and optionally, further, when the antibody is provided at low (1 μg / mL), medium (5 μg / mL), and high (10 μg / mL) concentrations, it is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet. [Section 8] Item 8. The antibody or antibody fragment of any one of Items 2 to 7, comprising three CDRs of the heavy chain variable region sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region sequence of SEQ ID NO: 8, wherein the CDRs are determined according to Kabat numbering. [Section 9] The antibody or antibody fragment of any one of Items 1 to 8, which competes with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8 for binding to human MICA polypeptide expressed by cells (e.g., MICA-expressing cells) prepared as a paraffin-embedded cell sample. [Section 10] 10. An in vitro method for detecting MICA and / or MICB polypeptides in a sample from a human individual, the method comprising: providing a paraffin-embedded sample from the individual; and detecting MICA polypeptides in the sample using the antibody or antibody fragment of any one of items 1 to 9. [Section 11] An in vitro method for detecting MICA and / or MICB polypeptides in a sample from a human individual, comprising providing a paraffin-embedded sample from said individual; and detecting MICA polypeptides prepared as a paraffin-embedded cell pellet. * Human MICA in samples of 001-expressing cells * MICA bound to 001 polypeptide and prepared as a paraffin-embedded cell pellet * Human MICA in samples of 008-expressing cells * and detecting a MICA polypeptide in a sample of MICB-expressing cells prepared as a paraffin-embedded cell pellet using an antibody or antibody fragment that binds to a human MICB polypeptide and that binds to a MICB-expressing cell sample prepared as a paraffin-embedded cell pellet, but that does not bind to MICA and MICB-negative cells, optionally Raji cells, prepared as a paraffin-embedded cell pellet. [Section 12] 12. The method of claim 11, wherein the antibody or antibody fragment binds to BxPC-3 cells prepared as a paraffin-embedded cell pellet; optionally, the antibody or antibody fragment is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet when the antibody is provided at a low concentration (1 μg / mL), and optionally, further, the antibody or antibody fragment is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet when the antibody is provided at low (1 μg / mL), medium (5 μg / mL), and high (10 μg / mL) concentrations. [Section 13] Item 13. The method according to Item 11 or 12, wherein the antibody or antibody fragment is the antibody or antibody fragment according to any one of Items 1 to 9. [Section 14] 13. The method of claim 11 or 12, wherein the antibody or antibody fragment is an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8; an antibody or antibody fragment that competes with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8 for binding to human MICA polypeptide expressed by cells prepared as a paraffin-embedded cell sample; or a function-conserving variant of an antibody or antibody fragment comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8. [Section 15] Item 15. The method according to any one of Items 10 to 14, wherein the step of detecting the MICA and / or MICB polypeptide comprises contacting the sample with the antibody or antibody fragment and detecting the formation of an immunological complex resulting from an immunological reaction between the antibody or antibody fragment and the sample. [Section 16] Item 16. The method according to any one of Items 10 to 15, wherein the sample is a tumor tissue. [Section 17] 17. The method of any one of items 10 to 16, wherein the individual has head and neck squamous cell carcinoma, lung cancer (e.g., NSCLC), mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, melanoma, urothelial cancer, pancreatic cancer, hepatocellular carcinoma (HCC), or endometrial cancer. [Section 18] Item 18. The method of any one of Items 10 to 17, wherein the individual has been treated with, is undergoing treatment with, or is a candidate for treatment with an anti-MICA antibody or antibody fragment. [Section 19] Item 19. The method according to any one of Items 10 to 18, wherein the individual has undergone pretreatment with a chemotherapeutic agent. [Section 20] 20. The method or composition according to any one of items 1 to 19, wherein the paraffin-embedded tissue sample is fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide. [Section 21] 21. The method or composition of any one of items 1 to 20, wherein the MICA and / or MICB polypeptide is detected using a secondary antibody that specifically binds to the antibody that binds to the MICA and / or MICB polypeptide. [Section 22] A method for predicting cancer progression in an individual having cancer, the method comprising: providing a paraffin-embedded tumor tissue sample from the individual; and detecting a MICA polypeptide in the sample according to the method of any one of Items 9 to 21 or using the antibody or antibody fragment of any one of Items 1 to 9. [Section 23] Detecting cells in the sample using antibodies includes: Obtaining a biological sample containing cells (e.g., as a biopsy); Fixing, embedding in paraffin, sectioning and deparaffinizing the sample, and optionally transferring the sample to a slide; contacting the section with the antibody; detecting the presence of bound antibody within said section; 23. The method according to any one of items 10 to 22, comprising: [Section 24] A kit comprising the antibody or antibody fragment according to any one of Items 1 to 9, optionally further comprising a labeled secondary antibody that specifically recognizes the antibody according to any one of Items 1 to 9. [Section 25] A nucleic acid or a set of nucleic acids encoding the antibody or antibody fragment according to any one of Items 1 to 9. [Section 26] A hybridoma or recombinant host cell that produces the antibody according to any one of Items 1 to 9 or that contains the nucleic acid according to Item 25. [Section 27] 1. A method for producing antibodies that specifically bind to human MICA and MICB polypeptides in paraffin-embedded tissue, comprising: a) providing cells that express a MICA polypeptide on their surface, providing cells that express a MICB polypeptide on their surface, and providing cells that do not express either a MICA polypeptide or a MICB polypeptide on their surface, and preparing a separate paraffin-embedded cell sample for each of said cells; b) providing a plurality of candidate antibodies; c) preparing or selecting antibodies from said plurality that bind to said paraffin-embedded cells of step a) that express MICA and to said paraffin-embedded cells of step a) that express MICB without binding to said paraffin-embedded cells of step a) that express neither MICA nor MICB on their surface; A method comprising: [Example]

[0179] Example 1: Performance of BAMO1 antibody in FFPE samples MICA and its close relative MICB are highly polymorphic ligands of the NK cell-activating receptor NKG2D. MICA and MICB are induced on the cell surface by cellular stresses such as infection and tumor transformation. Indeed, MICA is specifically expressed on several highly prevalent solid tumors, including breast, colorectal, and lung. To identify therapeutic indications for anti-MICA / B therapeutic antibodies, it was necessary to evaluate MICA / B expression in different FFPE tumor samples by immunohistochemistry (IHC). The percentage of positive cells in the tumor and the level of cytoplasmic or membrane expression can help identify appropriate indications for anti-MICA / B therapeutic antibodies.

[0180] The purpose of this study was to test different approaches to attempt to improve the sensitivity of manual MICA / B immunostaining using available antibodies. The antibody BAMO1 (R&D Systems Inc.) was selected for testing because it has been described as capable of detecting MICA in formalin-fixed, paraffin-embedded tissue sections of human pancreas.

[0181] To evaluate the improvement of MICA / B staining sensitivity, cell lines with low levels of MICA / B expression were selected. Indeed, detecting even low expression by IHC has been challenging. To evaluate the antibodies for MICA / B detection in FFPE sections, MICA / B-positive and -negative cells were frozen-thawed, cultured, and then fixed and embedded in paraffin for IHC staining. Table 3 summarizes the cells used.

[0182] [Table 4]

[0183] Previous flow cytometry phenotyping studies allowed us to preselect different cell lines with low levels of MICA / B expression. To confirm the selection, the MICA / B expression levels on these cells were again assessed by flow cytometry. BxPC-3, Hs 700T, HT-29, and MIA PaCa-2 cells all expressed relatively low levels of MICA / B.

[0184] After confirming the cell phenotype (MICA / B positive) by flow cytometry, the cells were fixed and embedded in paraffin for IHC staining.

[0185] The results showed that MICA / B expression was clearly detected in MIA PaCa-2 cells using the BAMO1 antibody. Staining was positive in Hs700T and HT-29 cells, but not in all cells. MICA / B expression was not detected in BxPC-3 cells using the BAMO1 antibody. Although MICA / B expression was detected by flow cytometry, some cell lines showed no MICA / B staining by IHC. This suggested that the initial protocol was not sensitive enough to detect low levels of MICA / B expression. Therefore, these cells were selected to test the improvement of MICA / B staining sensitivity.

[0186] Several conditions were tested to optimize MICA / B IHC staining, and the following conditions were tested: Cleaning step: flow vs. bath Blockade of endogenous peroxidase before versus after the initial incubation Diaminobenzidine (DAB) incubation: 5 minutes vs. 20 minutes Tyramide-based amplification (TSA) Envision FLEX Kit (linker between primary and secondary HRP antibodies)

[0187] Using TSA, MICA / B expression was detected on MIA PaCa-2 cells, BxPC-3 cells, and, to a lesser extent, HT-29 cells. Unfortunately, these results were not reproducible (especially for BxPC3 cells, which stained inconsistently across repeated experiments), and staining intensity remained low.

[0188] QIFIKIT was used to obtain quantitative determination of MICA / B cell surface expression. Four experiments were performed using Qifikit on selected cell lines. The number of antigens found on the cell surface of different cell lines was comparable across experiments. The data shown in Table 2 are representative of four experiments. Based on cell surface antigenic sites, selected cells can be classified as follows: Mia PaCa-2 > BxPC3 > Hs700T > HT-29. Notably, QIFIKIT evaluates only cell surface antigens, not cytoplasmic antigens. Because BAMO1 was not consistently able to detect BxPC3 by IHC, we hypothesize that Hs700T cells were detected by IHC using antibody BAMO1, likely because they express more cytoplasmic MICA / B than BxPC-3.

[0189] [Table 5]

[0190] MICA / B IHC staining using BAMO1 on formalin-fixed, paraffin-embedded samples cannot be optimized. Furthermore, this antibody does not allow consistent staining of cells with low levels of cell surface MICA / B protein, which was considered problematic for detecting MICA / B expression by IHC in FFPE samples.

[0191] Example 2: Identification of antibodies that stain cells with low cell surface MICA / B in FFPE samples Because there were no satisfactory antibodies available for IHC on FFPE samples, mice were immunized and screened to identify antibodies that could consistently and specifically stain cells with low MICA / B expression.

[0192] Briefly, immunizations were performed using five Balb / c mice immunized with three different proteins (MICA*001, MICA*008, and MICB). Sera from five different animals were tested by IHC using C1Rneo, C1R MICA*008, and C1R MICB FFPE cell pellets. Three animals were selected for fusion because sera from these animals stained numerous MICA / B cells with strong staining intensity. After hybridoma culture and selection, 508 supernatants (undiluted) were tested by IHC using a manual protocol with two antigen retrieval conditions (pH 6 and 8) on FFPE cell pellets (a mixture of C1R MICA*008 and C1R MICB cells) and an automated protocol using a Ventana Discovery Ultra automaton and CC1 or CC2 pretreatment on Raji, BxPC-3, C1R MICA*001, C1R MICA*008, and C1R MICB FFPE cell pellets. The experiments are described in further detail below.

[0193] Different cell lines were frozen, thawed, and subcultured. Raji, C1R-neo, BxPC-3, C1R MICA*001, C1R MICA*008, and C1R MICB were cultured in Roswell Park Memorial Institute medium (RPMI) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, 1% non-essential amino acids, and 1% sodium pyruvate. Raji, C1R-neo, C1R MICA*001, C1R MICA*008, and C1R MICB were grown in suspension. BxPC-3 cells are adherent and were detached using 2 mM PBS-EDTA. C1R MICA*001, C1R MICA*008, and C1R MICB were selected with 1.8 mg / ml geneticin. At the end of the culture period and after 6 passages for Raji cells, 7 or 8 passages for BXPC3, 2 passages for C1R-neo, 2 passages for C1R MICA*001, 3 or 4 passages for C1R MICA*008, and 3 or 4 passages for C1R MICB, cells were fixed in formalin and embedded in paraffin.

[0194] Before embedding, cells were stained with anti-MICA / B monoclonal Ab (mAb; clone 19E9, see International Publication No. WO 2013 / 117647) (PE conjugate), anti-MICA monoclonal Ab (mAb; clone 20C6, see International Publication No. WO 2013 / 117647) (PE conjugate), and a commercially available anti-MICB monoclonal Ab (mAb; 236511 R&D) (PE conjugate) and analyzed by flow cytometry to assess MICA / B, MICA, and MICB expression. No MICA / B expression was observed on Raji cells, and endogenous MICA / B expression was very low in C1R-neo cells. Low MICA expression was observed on BxPC-3 cells. Finally, C1R MICA*001 and C1R MICA*008 cells were strongly MICA-positive, and C1R MICB cells were strongly MICB-positive.

[0195] The characteristics of the cell lines used in this study are summarized as follows: -Raji cells (organism: Homo sapiens, human / cell type: B lymphocyte / tissue: lymphoblast / disease: Burkitt lymphoma / origin: ATCC), no MICA / B expression -C1R-neo cells (organism: Homo sapiens, human / cell type: B lymphoblast; Epstein-Barr virus transformed / tissue: peripheral blood lymphoma / ATCC ref. CRL-2369), with very low endogenous expression of MICA / B -BxPC-3 cells (organism: Homo sapiens, human / tissue: pancreas / disease: adenocarcinoma lymphoma / ATCC ref. CRL-1687), low endogenous MICA expression -C1R MICA*001 cells: C1R-neo cells transfected with human MICA*001 (high MICA*001 expression level) -C1R MICA*008 cells: C1R-neo cells transfected with human MICA*008 (high MICA*008 expression level) -C1R MICB: C1R-neo cells transfected with human MICB*002 (high MICB expression level)

[0196] In parallel with flow cytometric phenotyping, cell lines were fixed in formalin and embedded in paraffin. Briefly, 20 × 106–40 × 106 cells were fixed in 4% formalin for 1 h. Cells were washed twice in PBS and resuspended in Histogel. Cell pellets were dehydrated and embedded in paraffin. Several FFPE cell pellets were prepared for each cell line. A mixture of cells (C1R MICA*008 (15.106 cells) and C1R MICB (15.106 cells)) was also embedded in paraffin. FFPE cell pellets were sectioned and MICA / B stained by IHC. Briefly, 5 μm-thick sections were incubated, deparaffinized, and subjected to an antigen retrieval step. They were incubated with immune or non-immune sera, hybridoma supernatants, chain combination Abs, or purified and commercially available Abs, followed by a signal amplification step. Finally, enzymatic revelation was performed using 3,3'-diaminobenzidine (DAB). For IHC staining with mouse serum, staining interpretation was performed by attributing the percentage of stained cells in pellet sections and an intensity score using the following criteria: "-": negative staining; "+": weak positive staining; "++": moderate intensity IHC signal, and "+++": strong positive staining.

[0197] Five Balb / c mice were initially immunized (two intraperitoneal injections) with a mixture of MICA / B recombinant proteins (MICA*001, MICA*008, and MICB), and the sera were tested by IHC. Preimmune and immune sera were tested by IHC on a mixture of C1R neo cells and C1R MICA*008 + C1R MICB FFPE cell pellets at three different dilutions (1 / 1000, 1 / 5000, and 1 / 10000) and three different antigen retrieval conditions (pH 6, pH 8, and pH 9).

[0198] No staining was observed when preimmune serum was used. When serum was diluted 1 / 5000 or 1 / 10000, the mixture of C1R MICA*008 and C1R MICB gave weak and heterogeneous staining, so we decided to perform a third intraperitoneal injection to enhance the immune response. After this third injection, we again tested serum from five mice by IHC, and we observed an overall stronger reactivity after the third injection. The best results were obtained with serum from three mice (strong staining intensity of C1R MICA*008 and a higher number of C1R MICB-stained cells). In comparison, serum from another mouse stained less intensely for C1R MICA*008 and C1R MICB cells; in another mouse, when used at 1 / 5000 and 1 / 10000, fewer C1R MICA*008 cells were stained, and when used at 1 / 5000 and 1 / 10000, no C1R MICB cell staining was obtained. Three mice with the best results were selected for the final boost (intravenous injection of mixed MICA / B recombinant protein). The animals were euthanized, and their spleens were removed and used as a source of cells for fusion with myeloma cells. After culturing in methylcellulose semisolid medium, hybridoma colonies were picked and cultured in 27 different 96-well plates. Next, ELISA was performed to select only mouse IgG-secreting hybridomas. Following this test, another ELISA was performed to test previously identified positive hybridomas (IgG secretion) to exclude anti-tag hybridomas. At the end, 508 hybridomas were maintained and expanded, generating 1.5 mL of supernatant / hybridoma.

[0199] We first tested the supernatants by IHC on a mixture of C1R MICA*008 + C1R MICB FFPE cell pellet sections using different antigen unmasking conditions (pH 6, pH 8). Among the 508 supernatants, 46 samples (11 of which were positive for both pH 6 and pH 8) were selected as giving the best results (strongly positive and uniform staining) on ​​the mixture of C1R MICA*008 + C1R MICB cells.

[0200] We retested 46 supernatants that stained on a mixture of C1R MICA*008 and C1R MICB cells by IHC on C1R-neo, BxPC-3, CR1 MICA*001, C1R MICA*008, and C1R MICB FFPE cell pellet sections on a Ventana using CC1 or CC2 pretreatment. Six supernatants (including 12C9) were selected and prepared as mouse antibodies with a mouse gamma 1 chain (mIgG1 isotype) because they gave the strongest staining on all positive cells tested and no or weak staining on C1R-neo cells.

[0201] In parallel, 66 supernatants that were partially positive in a mixture of C1R MICA*008 + C1R MICB cells were retested on Raji, BxPC-3, CR1 MICA*001, C1R MICA*008, and C1R MICB FFPE cell pellet sections on a Ventana. Three supernatants were selected and prepared because they gave the strongest staining and were specific for MICA or MICB (one MICA-specific and two MICB-specific).

[0202] After transient gene transfer, distinct rearrangements were obtained for each antibody selected and tested by IHC on Raji, BxPC-3, C1R MICA*001, C1R MICA*008, and C1R MICB FFPE cell pellet sections on a Ventana Discovery Ultra automaton using CC1 Discovery Cell Conditioning 1 (CC1) or RiboCC (CC2) pretreatment conditions. Six antibodies gave positive staining in all tested cells, with no staining in Raji cells. These antibodies were selected and generated as mouse antibodies with a mouse γ1 (gamma 1) chain (mIgG1 isotype).

[0203] The generated and purified antibodies were tested by IHC on Raji, BxPC-3, C1R MICA*001, C1R MICA*008, and C1R MICB FFPE cell pellet sections at three different concentrations (1, 5, and 10 μg / mL) on a Ventana automaton using CC1 or CC2 pretreatment conditions.

[0204] The three antibodies tested (including 12C9) gave strong membrane staining in MICA and MICB transfected cells and no staining in Raji cells. Using the staining conditions tested, two of the antibodies showed no or very weak staining on BxPC-3 at low concentrations (1 μg / mL) and inhomogeneous staining at medium (5 μg / mL) and high (10 μg / mL) concentrations. Only 12C9 showed the ability to stain BxPC-3 FFPE cell pellet selection at the three concentrations tested.

[0205] The amino acid sequences of the heavy and light chain variable regions of 12C9 are shown below (Kabat CDRs underlined).

[0206] Heavy chain variable region (VH) of 12C9: [ka]

[0207] 12C9 light chain variable region (VL): [ka]

Claims

1. An antibody or antibody fragment capable of specifically binding to a human MICA polypeptide and a human MICB polypeptide, comprising three CDRs of the heavy chain variable region sequence of SEQ ID NO: 7 and three CDRs of the light chain variable region sequence of SEQ ID NO: 8, wherein the CDRs are determined according to Kabat numbering.

2. The antibody or antibody fragment described in claim 1, wherein the antibody or antibody fragment is capable of specifically binding to human MICA and human MICB polypeptides, and the binding is in a sample of cells that express the MICA and / or MICB polypeptides and have been prepared as a paraffin-embedded cell pellet.

3. 3. The antibody or antibody fragment of claim 1 or 2, conjugated or covalently attached to a detectable moiety.

4. The antibody or antibody fragment of any one of claims 1 to 3, which binds to BxPC-3 cells prepared as a paraffin-embedded cell pellet; optionally, when the antibody is provided at a low concentration (1 μg / mL), it is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet, and further, when the antibody is provided at low (1 μg / mL), medium (5 μg / mL), and high (10 μg / mL) concentrations, it is capable of staining BxPC-3 cells prepared as a paraffin-embedded cell pellet.

5. 10. An in vitro method for detecting MICA and / or MICB polypeptides in a sample from a human individual, comprising providing a paraffin-embedded sample from said individual and detecting MICA and / or MICB polypeptides in said sample using an antibody or antibody fragment according to any one of claims 1 to 4.

6. The method of claim 5, wherein detecting the MICA and / or MICB polypeptide comprises contacting the sample with the antibody or antibody fragment and detecting the formation of an immunological complex resulting from an immunological reaction between the antibody or antibody fragment and the sample.

7. An in vitro method for detecting cells expressing a MICA and / or MICB polypeptide in a sample from a human individual, comprising: - obtaining a biological sample containing cells; - Fixing, embedding in paraffin, sectioning and deparaffinizing the sample and optionally transferring the resulting sections to slides; - contacting the obtained section with the antibody or antibody fragment according to any one of claims 1 to 4; detecting the presence of bound antibody or antibody fragment within said section; A method comprising:

8. The method of claim 7, wherein detecting the presence of a bound antibody or antibody fragment includes detecting the formation of an immunological complex resulting from an immunological reaction between the antibody or antibody fragment and the section.

9. 9. The method of any one of claims 5 to 8, wherein the sample is tumor tissue and the individual has head and neck squamous cell carcinoma, lung cancer, mesothelioma, breast cancer, estrogen-positive breast cancer, estrogen-negative breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, melanoma, urothelial cancer, pancreatic cancer, hepatocellular carcinoma (HCC), or endometrial cancer.

10. 10. The method of any one of claims 5 to 9, wherein the sample is fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide.

11. A method described in any one of claims 5 to 10, comprising detecting the antibody or antibody fragment using a secondary antibody that specifically binds to the antibody or antibody fragment.

12. A kit comprising the antibody or antibody fragment of any one of claims 1 to 4, optionally further comprising a labeled secondary antibody that specifically recognizes the antibody of any one of claims 1 to 4.

13. A nucleic acid or a set of nucleic acids encoding the antibody or antibody fragment according to any one of claims 1 to 4.

14. A recombinant host cell producing an antibody according to any one of claims 1 to 4 or comprising a nucleic acid or set of nucleic acids according to claim 13.

Images