Immunohistochemistry and KIR3DL2-specific agents

Abstract

The present invention relates to antibodies and methods for detecting KIR3DL2 expression in paraffin-embedded tissue samples. Methods for producing antibodies, antibody fragments, and derivatives thereof that specifically bind to target antigens in paraffin-embedded tissue samples are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 170,603, filed April 5, 2021, which is incorporated by reference in its entirety, including any drawings.

[0002] Reference to sequence listing This application has been submitted with an electronic sequence listing, which is provided in a file entitled "KIR-12 PCT Sequences_ST25", created on March 30, 2022, and is 25.7KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION The present invention relates to a research and diagnostic tool for detecting KIR3DL2 in biological samples (e.g., paraffin-embedded tissue samples). The present invention also relates to a method of using said tool for detecting KIR3DL2-expressing cells. [Background technology]

[0004] background Killer immunoglobulin-like receptors (KIRs) are a family of receptors that, together with C-type lectin receptors (CD94-NKG2), are used by human NK cells and subsets of T lymphocytes to specifically recognize MHC class I molecules.

[0005] Among the members of the killer cell immunoglobulin (Ig)-like receptor (KIR) family, the killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2) has been investigated as a target for the treatment of CD4+ T cells expressing the KIR3DL2 receptor, particularly malignancies involving CD4+ T cells, including cutaneous T-cell lymphomas (CTCLs) such as mycosis fungoides and Sézary syndrome (see, for example, WO2010 / 081890 and WO02 / 50122). The KIR3DL2 receptor is also frequently expressed by tumor cells in other peripheral T-cell lymphomas (PTCLs) and at a lower frequency on normal lymphocytes.

[0006] Monoclonal antibodies have been developed that target the KIR3DL2 receptor and show increased activity in the treatment of KIR3DL2-expressing cellular malignancies, particularly CD4+ T cell malignancies (see, e.g., WO2014 / 044686).

[0007] To better understand the tumor environment, it is often desirable to detect the KIR3DL2 receptor present in tumor tissue. This can be done, for example, using frozen tissue samples. This is not only useful for research, but also helps to determine what type of treatment to use by detecting whether the tissue (e.g., tumor environment) is characterized by the presence of a protein that is the target of treatment (e.g., immunotherapy). This information helps to select treatments that can modulate the activity of the protein and / or the cells that express it.

[0008] Some antibodies suitable for detecting KIR3DL2 polypeptide in cell culture or frozen tissue samples are already known. Antibodies 12B11 and 19H12 are actually suitable for use in detecting KIR3DL2 expression on the surface of tumor cells (e.g. in vitro assays) because both can detect KIR3DL2-positive cells in detection assays. 12B11 is advantageous for immunohistochemical assays using frozen tissue sections, and 19H12 is advantageous for flow cytometry detection.

[0009] With access to frozen tissue samples being a limiting factor, new tests need to be developed to detect KIR3DL2 in other samples, such as tissue samples stored as formalin-fixed paraffin-embedded (FFPE) samples. Unfortunately, it is often not possible to find KIR3DL2-specific monoclonal antibodies that work effectively and specifically in FFPE sections. This is thought to be due to the effect of formalin fixation on the structure of the protein. The epitopes to which antibodies described as specific for recombinant proteins or cells bind are often also present on other proteins when used in FFPE, making the antibodies non-specific. In other cases, many epitopes on native cellular proteins are destroyed by formaldehyde (e.g., formalin) fixation, making antibodies identified using recombinant proteins or cells ineffective for staining FFPE sections.

[0010] Thus, there is a need for improved antibodies that specifically target KIR3DL2 for use in staining biological samples (eg, paraffin-embedded tissue sections).

[0011] Summary of the Invention The present invention relates inter alia to the study, detection and / or monitoring of KIR3DL2 polypeptide in biological samples, preferably formalin-fixed paraffin-embedded (FFPE) tissue samples. The present disclosure arises from the characterization of antibodies suitable for detecting KIR3DL2 in biological samples. The antibodies retain their specificity for such a given target antigen in FFPE protocols, and in particular, they bind to epitopes on KIR3DL2 polypeptide that remain present and specific after formalin fixation. The antibodies allow for highly specific detection of KIR3DL2 in IHC protocols without detecting KIR3DL1 and / or other KIR polypeptides (e.g., KIR3DS1). The resulting diagnostic antibodies can serve as reagents for consistent detection in FFPE samples from individuals.

[0012] The KIR3DL2-specific antibody described herein was discovered through the design and testing of synthetic peptides designed to mimic potential formalin-modified epitopes that may occur in KIR3DL2 protein.The antibody that binds to specific synthetic peptides was found to be selective for KIR3DL2 over KIR3DL1 in FFPE samples.Therefore, the study identified new epitopes or binding sites that exist or occur in formalin-treated KIR3DL2, and their corresponding sites or sequences in wild-type KIR3DL2 amino acid sequence.

[0013] Formalin-fixed paraffin-embedded (FFPE) tissue offers two main advantages over other immunological methods: (1) the tissue does not require special handling; and (2) the cytological and structural features are well recognized, which allows for improved histopathological interpretation. However, it has often been impossible to find a KIR3DL2-specific monoclonal antibody that works effectively and specifically in FFPE sections. Previously developed antibodies, when tested in FFPE samples, were not truly KIR3DL2-specific, since they also bind to KIR3DL1 polypeptides. The present inventors have developed an antibody that is suitable for specifically detecting KIR3DL2 polypeptides and for use in staining FFPE biological samples. The obtained antibody has been tested in various KIR3DL2-expressing cells (e.g., transfected cells, human tissues from human donors, or tumor tissues from patients) and found to maintain excellent performance in detecting the target antigen in FFPE tissue sections.

[0014] Thus, the disclosure provides an antibody or antibody fragment thereof capable of binding to a KIR3DL2 polypeptide in a biological sample, wherein the antibody or antibody fragment comprises a heavy chain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:21, and a light chain comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:22.

[0015] Another object of the present disclosure is an antibody or antibody fragment thereof capable of binding to a KIR3DL2 polypeptide, wherein the antibody or antibody fragment comprises heavy chain CDR1, 2 and 3 of a heavy chain variable region set forth in SEQ ID NO:21, and light chain CDR1, 2 and 3 of a light chain variable region set forth in SEQ ID NO:22.

[0016] In one embodiment, an antibody or antibody fragment is provided comprising: (i) a heavy chain comprising CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) having the sequences of SEQ ID NO:3 (HCDR1), SEQ ID NO:6 (HCDR2) and SEQ ID NO:9 (HCDR3); and (ii) a light chain comprising CDR1, 2 and 3 (LCDR1, LDR2, LCDR3) having the sequences of SEQ ID NO:12 (LCDR1), SEQ ID NO:15 (LCDR2) and SEQ ID NO:18 (LCDR3), wherein each CDR may optionally contain substitutions, deletions or insertions of 1, 2 or 3 amino acids.

[0017] In one embodiment, a monoclonal antibody or antibody fragment is provided that is a function-conservative variant of the antibody P3-R4D-H5. In one embodiment, an antibody or antibody fragment is provided that is a function-conservative variant of an antibody having a heavy chain variable region of SEQ ID NO:21, and light chain CDR1, 2 and 3 of a light chain variable region of SEQ ID NO:22.

[0018] In one embodiment, an isolated polypeptide is provided (e.g., an isolated polypeptide comprising or corresponding to an epitope on KIR3DL2 present following formalin treatment of a KIR3DL2-expressing cell), wherein the isolated polypeptide consists of the amino acid sequence of SEQ ID NO: 24. In another embodiment, an isolated polypeptide according to the present disclosure may be modified or fused to one or more heterologous polypeptides, or may be included in another polypeptide (e.g., a polypeptide comprising one or more non-KIR3DL2 amino acid sequences).

[0019] In one embodiment, an antibody or antibody fragment thereof that binds to such an isolated polypeptide is provided. In one embodiment, the binding of said antibody or antibody fragment thereof to said isolated polypeptide is determined by an ELISA test in which said antibody or antibody fragment thereof is contacted with a polypeptide comprising the amino acid sequence of SEQ ID NO: 24. In a particular embodiment, said polypeptide of the amino acid sequence of SEQ ID NO: 24 is bound to a solid support, for example, such a polypeptide can thus be fused to a linker peptide bound to a solid support.

[0020] In one embodiment, a monoclonal antibody or antibody fragment is provided that binds to a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of CEHFFLHREGISEDPSRLVG (SEQ ID NO: 23), CTPLTDTSVYTELPNAEPRS (SEQ ID NO: 24) and CPRAPQSGLEGVF (SEQ ID NO: 25). In one embodiment, the binding of said antibody or antibody fragment thereof to said polypeptide is determined by an ELISA test, in which said antibody or antibody fragment thereof is contacted with said polypeptide comprising the amino acid sequence of SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO: 25. In a particular embodiment, said polypeptide of amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 is bound to a solid support.

[0021] In one embodiment, a monoclonal antibody or antibody fragment that binds to a KIR3DL2 polypeptide is provided, wherein the antibody or antibody fragment binds to a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of CEHFFLHREGISEDPSRLVG (SEQ ID NO: 23), CTPLTDTSVYTELPNAEPRS (SEQ ID NO: 24) and CPRAPQSGLEGVF (SEQ ID NO: 25).

[0022] In one embodiment, a monoclonal antibody or antibody fragment is provided that binds to the same epitope on KIR3DL2 as an antibody having a heavy chain variable region of SEQ ID NO: 21, and light chain CDR1, 2 and 3 of a light chain variable region of SEQ ID NO: 22. Optionally, the antibody or antibody fragment binds to a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of CEHFFLHREGISEDPSRLVG (SEQ ID NO: 23), CTPLTDTSVYTELPNAEPRS (SEQ ID NO: 24) and CPRAPQSGLEGVF (SEQ ID NO: 25). In any embodiment herein, an antibody or antibody fragment thereof according to the present disclosure is capable of specifically binding to a KIR3DL2 polypeptide in a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0023] In another embodiment, an antibody or antibody fragment is provided that binds to an intracellular portion or epitope of a KIR3DL2 polypeptide in a biological sample. For example, the antibody or antibody fragment can be identified as binding to a cytoplasmic domain (or an epitope of such domain) of a KIR3DL2 polypeptide, optionally the cytoplasmic domain corresponding to residues 340-434 of SEQ ID NO:1. In one embodiment, the antibody or antibody fragment binds to a portion or epitope of a KIR3DL2 polypeptide corresponding to residues 399-417 of SEQ ID NO:1. In one embodiment, the antibody or antibody fragment binds to an intracellular portion or epitope of a KIR3DL2 polypeptide in a biological sample that is fixed in formalin and then dissected into pieces prior to contacting with said antibody or antibody fragment. In one embodiment, said intracellular portion or epitope of a KIR3DL2 polypeptide has the amino acid sequence TPLTDTSVYTELPNAEPRS (SEQ ID NO:31). In one embodiment, the antibody or antibody fragment binds to a polypeptide comprising or consisting of the amino acid sequence CTPLTDTSVYTELPNAEPRS (SEQ ID NO:24).

[0024] In a further embodiment, an antibody or antibody fragment is provided that binds to a KIR3DL2 polypeptide in a biological sample, and the antibody or antibody fragment does not substantially bind to a KIR3DL1 polypeptide (e.g., a KIR3DL1 allele *00101 comprising the amino acid sequence set forth in SEQ ID NO: 30). Preferably, the biological sample is a formalin-fixed, paraffin-embedded tissue sample.

[0025] In any of the embodiments herein, an antibody or antibody fragment can be identified as being capable of specifically binding to a KIR3DL2 polypeptide in a biological sample of cells that has been prepared as a paraffin-embedded cell pellet.

[0026] In one embodiment, an antibody or antibody fragment is capable of binding to (or, for example, staining) KIR3DL2-expressing cells that have been prepared as a paraffin-embedded cell pellet, and wherein the antibody or antibody fragment does not bind to (or, for example, stain) KIR3DL1-expressing cells that do not express KIR3DL2 and have been prepared as a paraffin-embedded cell pellet, and optionally further, the cells are fixed in formalin and then cut into sections before contacting with the antibody or antibody fragment thereof.

[0027] The present disclosure also provides an antibody or antibody fragment thereof for use in detecting the presence of KIR3DL2-expressing cells in a biological sample. In one embodiment, the biological sample is a tissue sample. In one embodiment, the biological sample is a fixed tissue sample. In yet another embodiment, the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In one embodiment, the detection of the presence of KIR3DL2-expressing cells by the antibody or antibody fragment thereof according to the present disclosure is performed by immunohistochemistry (IHC). Advantageously, the antibody or antibody fragment thereof for use in detecting KIR3DL2-expressing cells by immunostaining of paraffin-embedded tissue sections is specific and has advantageous affinity in FFPE, which allows accurate detection of KIR3DL2.

[0028] In another aspect, an in vitro method for detecting KIR3DL2-expressing cells in a sample is provided, the method comprising (i) providing a biological sample from an individual comprising cells, and (ii) detecting KIR3DL2-expressing cells with an antibody or antibody fragment thereof according to the present disclosure. In one embodiment, the biological sample is a tissue sample. In one embodiment, the biological sample is a fixed tissue sample. In yet another embodiment, the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In one embodiment, the step (ii) of detecting KIR3DL2-expressing cells with an antibody or antibody fragment thereof according to the present disclosure comprises contacting the sample with the disclosed antibody or antibody fragment, and detecting the formation of an immunological complex resulting from an immunological reaction between the antibody or antibody fragment thereof and the sample. In one embodiment, the detection of the formation of such an immunological complex between an antibody or antibody fragment thereof according to the present disclosure and the sample is performed by immunohistochemistry (IHC). In one embodiment, the detection of the formation of such an immunological complex between an antibody or antibody fragment thereof according to the present disclosure and the sample is performed by using a secondary antibody that specifically binds to the antibody or antibody fragment thereof according to the present disclosure. In one embodiment, paraffin-embedded tissue samples are fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to slides.

[0029] Another aspect of the present disclosure is an in vitro method for evaluating the suitability of an individual with cancer for treatment with an immunotherapeutic agent, the method comprising (i) providing a biological sample from a patient, and (ii) detecting KIR3DL2-expressing cells in the sample using an antibody or antibody fragment according to the present disclosure, wherein detection of KIR3DL2-expressing cells indicates that the individual is suitable for treatment with the immunotherapeutic agent. In one embodiment, the biological sample is a tissue sample. In one embodiment, the biological sample is a fixed tissue sample. In yet another embodiment, the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In one embodiment, the immunotherapeutic agent for treating an individual with cancer is an agent that binds to a KIR3DL2 polypeptide. In one embodiment, the immunotherapeutic agent that binds to a KIR3DL2 polypeptide is an antibody that binds to a KIR3DL2 polypeptide and enhances cytotoxicity via ADCC against KIR3DL2-expressing cells. In a preferred embodiment, the antibody is LACUTAMAB. In a further embodiment, the paraffin-embedded tissue sample is fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide.

[0030] The present disclosure also provides a method of treating a disease in an individual, the method comprising: (i) providing a biological sample from the individual; (ii) detecting KIR3DL2-expressing cells in said sample using an antibody or antibody fragment thereof according to the present disclosure; and (iii) administering an immunotherapeutic agent to the individual upon detection of KIR3DL2-expressing cells. In one embodiment, the biological sample is a tissue sample. In one embodiment, the biological sample is a fixed tissue sample. In yet another embodiment, the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In one embodiment, the disease to be treated is cancer. In one embodiment, the cancer is a lymphoma, such as a lymphoma, such as a CD4+ T-cell lymphoma. In one embodiment, the CD4+ lymphoma is a cutaneous T-cell lymphoma (CTCL). In one embodiment, the CTCL is mycosis fungoides or Sézary syndrome. In a further embodiment, the CTCL is a transformed T-lymphoma. In another embodiment, the CD4+ lymphoma is a peripheral T-cell lymphoma (PTCL). In one embodiment, a paraffin-embedded biological sample is fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide.

[0031] Another aspect of the present disclosure relates to a kit comprising an antibody or antibody fragment according to the present disclosure and a labeled secondary antibody that specifically recognizes said antibody or antibody fragment according to the present disclosure. The present disclosure also provides an isolated nucleic acid encoding an antibody or antibody fragment according to the present disclosure.

[0032] Also provided are hybridomas or recombinant host cells producing the antibodies or antibody fragments according to the disclosure.

[0033] These and additional advantageous aspects and features of the present invention may be further described elsewhere herein. [Brief description of the drawings]

[0034] [Figure 1]FIG. 1 shows the optical density (450 nm) measured in an Elisa assay for several dilutions of clone P3-R4D-H5 soluble scFv selected from the scFv library against peptide 3 bound to BSA or against free peptide 3, or against BSA.

[0035] [Diagram 2] Figure 2 shows photographs of IHC staining obtained with anti-KIR3DL2 soluble scFv clones P3-R4D-H5, P3-R4D-F4, P3-R4D-C10, P3-R4D-C1, P3-R4D-B9, P3-R4D-B5 on KIR3DL2 and KIR3DL1 expressing cell pellets. Staining was performed at 5 μg / mL on a Leica Bond RX.

[0036] [Diagram 3] Figure 3 represents the photographs of IHC staining obtained with anti-KIR3DL2 clone P3-R4D-H5 or isotype control antibody on different CHO / CHO-mb-HuKIR3DL2 mixed FFPE cell pellets. Staining was performed at 5 μg / mL on a Leica Bond RX. Scale bar represents 25 μm. CHO does not express KIR3DL2, while CHO-mb-HuKIR3DL2 is KIR3DL2 expressing cells. As stated in the figures, Ab means antibody; FFPE means formalin fixed paraffin embedded; IC means isotype control, and IHC means immunohistochemistry.

[0037] [Figure 4] Figure 4 represents photographs of IHC staining obtained with anti-KIR3DL2 clone P3-R4D-H5 or isotype control antibody on different Raji / HuT mixed FFPE cell pellets. Staining was performed on a Leica Bond RX at 5 μg / mL. Scale bar represents 25 μm. Arrows represent KIR3DL2+ cells. As stated in the figures, Ab means antibody; FFPE means formalin fixed paraffin embedded; IC means isotype control and IHC means immunohistochemistry.

[0038] [Figure 5A] Figures 5A, 5B and 5C show photographs of IHC staining obtained with anti-KIR3DL2 clone 12B11 on KIR3DL2 expressing RAJI cells (and non-KIR3DL2 expressing RAJI cells as negative control).Several staining conditions were tested: citrate buffer pH 7, kit assumed + DAB; citrate buffer pH 7, kit assumed + tyramide-biotin + streptavidin-HRP + DAB; citrate buffer pH 6, kit assumed + DAB; EDTA pH 7 kit assumed + DAB; Tris-EDTA pH 9 kit assumed + DAB. [Figure 5B] Figures 5A, 5B and 5C show photographs of IHC staining obtained with anti-KIR3DL2 clone 12B11 on KIR3DL2 expressing RAJI cells (and non-KIR3DL2 expressing RAJI cells as negative control).Several staining conditions were tested: citrate buffer pH 7, kit assumed + DAB; citrate buffer pH 7, kit assumed + tyramide-biotin + streptavidin-HRP + DAB; citrate buffer pH 6, kit assumed + DAB; EDTA pH 7 kit assumed + DAB; Tris-EDTA pH 9 kit assumed + DAB. [Figure 5C] Figures 5A, 5B and 5C show photographs of IHC staining obtained with anti-KIR3DL2 clone 12B11 on KIR3DL2 expressing RAJI cells (and non-KIR3DL2 expressing RAJI cells as negative control).Several staining conditions were tested: citrate buffer pH 7, kit assumed + DAB; citrate buffer pH 7, kit assumed + tyramide-biotin + streptavidin-HRP + DAB; citrate buffer pH 6, kit assumed + DAB; EDTA pH 7 kit assumed + DAB; Tris-EDTA pH 9 kit assumed + DAB.

[0039] [Figure 6]Figure 6 is a graph showing the optical density measured on FFPE samples stained with several concentrations of anti-KIR3DL2 clone P3-R4D-H5 in IHC. Staining was performed on several FFPE samples: CHO-K1SV cells (CHO), CHO-K1SV-mb-HuKIR3DL1 cells (CHO-KIR3DL1), and CHO-K1SV-mb-HuKIR3DL2 cells (CHO-KIR3DL2).

[0040] [Figure 7] FIG. 7 represents images of KIR3DL2 chromogenic IHC staining on different CTCL biopsies from individuals with CTCL (e.g., mycosis fungoides or Sézary syndrome). FFPE sections were stained with clone P3-R4D-H5Ab at 5 μg / mL on a Leica Bond RX (left panel), and frozen sections were stained using anti-KIR3DL2 clone 12B11 at 10 μg / mL on a Ventana Benchmark XT (right panel). For each case, low and high magnifications are shown, and the percentage of KIR3DL2+ cells among mononuclear cells is indicated. Scale bars correspond to 1 or 2.5 mm for low magnification images (for left middle, left lower, right lower panels or right middle, left upper, right upper panels, respectively) and 50 μm for high magnification images. As stated in the figures, B means biopsy; CTCL means cutaneous T-cell lymphoma; FFPE means formalin-fixed paraffin-embedded; and IHC means immunohistochemistry.

[0041] [Figure 8]FIG. 8 represents images of KIR3DL2 chromogenic IHC staining on PTCL biopsies from individuals suffering from PTCL. FFPE sections were stained with clone P3-R4D-H5Ab at 5 μg / mL on a Leica Bond RX (left panel) and frozen sections were stained using anti-KIR3DL2 clone 12B11 at 10 μg / mL on a Ventana Benchmark XT (right panel). Low and high magnifications of two cases are shown, with the percentage of KIR3DL2+ cells among mononuclear cells as determined by a pathologist. Scale bars correspond to 2.5 mm for low magnification images and 50 μm for high magnification images. As noted in the figures, PTCL means peripheral T-cell lymphoma; FFPE means formalin-fixed paraffin-embedded; IHC means immunohistochemistry; and LN means lymph node.

[0042] [Figure 9] FIG. 9 depicts images of KIR3DL2 chromogenic IHC staining in FFPE samples from individuals with CTCL (mycosis fungoides). FFPE sections were stained with clone P3-R4D-H5Ab at 5 μg / mL on a Leica Bond RX. FIGs. 9A and 9C are IHC images of highly positive CTCL with strong KIR3DL2 signal. FIG. 9B is an IHC image of weakly positive CTCL. FIG. 9D is an IHC image of a recurrent CTCL (mycosis fungoides) sample with strongly positive and nearly negative areas of KIR3DL2.

[0043] [Figure 10] FIG. 10 depicts images of KIR3DL2 chromogenic IHC staining on FFPE samples from individuals with PTCL. FFPE sections were stained with clone P3-R4D-H5Ab at 5 μg / mL on a Leica Bond RX. FIG. 10A is an IHC image of a highly positive PTCL (not otherwise specified). FIG. 10B is an IHC image of a PTCL (not otherwise specified) with scattered positive tumor cells against a background of faint stromal cell positivity. FIGs. 10C and 10D are IHC images of a moderately KIR3DL2 positive PTCL (not otherwise specified) with localized variability within the sample in FIG. 10D.

[0044] Detailed Description of the Invention definition As used herein, "a" or "an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0045] When "comprising" is used, this can optionally be replaced with "consisting essentially of" and more optionally with "consisting of."

[0046] The term "antibody" herein is used 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. Various 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).

[0047] "Antibody fragments" include portions of full-length antibodies, such as antigen-binding regions or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (usually the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (usually the VH and CH1 domains), and dAb (usually 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 an antibody fragment and one or more isolated CDRs or functional paratopes, where the isolated CDRs or antigen-binding residues or polypeptides can be combined or linked to form a functional antibody fragment.

[0048] As used herein, the term "antigen-binding domain" refers to a domain that comprises a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, the domain may comprise a hypervariable region, optionally a VH and / or VL domain of an antibody chain, optionally at least a VH domain. In another embodiment, the binding domain may comprise one, two, or all three complementarity determining regions (CDRs) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.

[0049] As used herein, the term "antibody derivative" includes full length antibodies or fragments of antibodies, including, for example, at least the antigen binding region or the variable region thereof, in which one or more amino acids have been chemically modified, for example, by alkylation, PEGylation, acylation, ester formation or amide formation, etc. This includes, but is not limited to, PEGylated antibodies, cysteine ​​PEGylated antibodies, and variants thereof.

[0050] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. Hypervariable regions generally include amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain, and 31-35 (H1), 50-65 (H2) and 95-102 (H3) of 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) of the light chain variable domain, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196: 901-917). Typically, the numbering of amino acid residues in this region is done by the method described in Kabat et al., supra. Phrases such as "Kabat position," "variable domain residue numbering in Kabat," and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or the 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 or insertion of 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 CDRH2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence at the homology region 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 set forth below as a comparison in Table 1. 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 constitute a particular CDR, given the variable region amino acid sequence of an antibody. [Table 1]

[0051] As used herein, "framework" or "FR" residues 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 separated by the CDRs (FR1, FR2, FR3, and FR4).

[0052] 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. As used herein, "constant light chain" or "light chain constant region" refers to the region of an antibody encoded by the kappa (Ckappa) or lambda (Clambda) light chain. The constant light chain typically contains a single domain and, as defined herein, refers to positions 108-214 of Ckappa or Clambda, where the numbering is 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). As used herein, "constant heavy chain" or "heavy chain constant region" refers to the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes 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 thus includes positions 118 to 447, where numbering is according to the EU index.

[0053] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region alone or in the context of a polypeptide, multispecific polypeptide, or antibody, or any other embodiment as outlined herein.

[0054] As used herein, "single-chain Fv" or "scFv" refers to an antibody fragment that comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. Methods for generating scFvs are well known in the art. For a review of methods for generating scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0055] As used herein, "Fv" or "Fv fragment" or "Fv region" means a polypeptide comprising the VL and VH domains of a single antibody.

[0056] 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, 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 may vary, the human IgG heavy chain Fc region is usually defined to include residues C226, P230, or A231 to its carboxyl terminus, where numbering is according to the EU index. Fc may refer to this region alone or in the context of an Fc polypeptide, as described below. As used herein, "Fc polypeptide" or "Fc-derived polypeptide" refers to a polypeptide that comprises all or a portion of an Fc region. Fc polypeptides include, but are not limited to, antibodies, Fc fusions, and Fc fragments.

[0057] As used herein, "variable region" includes light chain (including kappa and lambda) and heavy chain immunoglobulin loci, respectively. The light or heavy chain variable region (VL or VH) is composed 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 is precisely defined, for example, as Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1983)) and as Chothia. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs that are primarily involved in binding to antigen.

[0058] The term "specifically binds to" means that the antibody or polypeptide can bind to a binding partner, such as a binding partner, preferably in a competitive binding assay. KIR3DL2 is assessed using either a recombinant form of the protein, an epitope therein, or the native protein present on the surface of isolated target cells. Competitive binding assays and other methods for measuring specific binding are further described below and are well known in the art.

[0059] As used herein, the term "affinity" refers to the strength of binding of an antibody or polypeptide to an epitope. The affinity of an antibody is determined by the dissociation constant K, defined as [Ab] x [Ag] / [Ab-Ag]. D 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, K A is 1 / K D The affinity of mAbs is defined as: 1. 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), all of 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 (such as analysis with a BIAcore® SPR analyzer).

[0060] Within the context of this invention, a "determinant" refers to a site of interaction or binding on a polypeptide.

[0061] The term "epitope" refers to an antigenic determinant, which is a region or area on an antigen to which an antibody or polypeptide binds. A protein epitope may include amino acid residues directly involved in binding as well as amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, i.e., amino acid residues within the "footprint" of an antibody. It is the simplest shape or smallest structural region on a complex antigen molecule that can bind to an antibody, receptor, etc. Epitopes can be linear or conformational. The term "linear epitope" is defined as an epitope that is composed of amino acid residues that are contiguous on a linear sequence of amino acids (primary structure). The term "conformational or structural epitope" is defined as an epitope that is composed of amino acid residues that represent isolated portions of a linear sequence of amino acids that are not all contiguous but are brought into close proximity to each other by folding of the molecule (secondary, tertiary, and / or quaternary structure). Conformational epitopes depend on three-dimensional structure. Thus, the term "structural" is often used interchangeably with "structural". Conformational epitopes depend on three-dimensional structure. Thus, the term "structural" is often used interchangeably with "structural."

[0062] As used herein, "amino acid modification" refers to the substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence. An example of an amino acid modification herein is a substitution. As used herein, "amino acid modification" refers to the substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence. As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a given 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 substantially the same amino acid sequence as a reference polypeptide, typically a native or "parent" polypeptide. A polypeptide variant may have one or more amino acid substitutions, deletions, and / or insertions at specific positions within the native amino acid sequence.

[0063] "Conservative" amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue that has 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0064] The term "identity" or "identical", when used in the context of the sequences of two or more polypeptides, refers to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences, with gap alignments (if any) accommodated by a particular mathematical model or computer program (i.e., an "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).

[0065] Preferred methods for determining identity are designed to give the greatest 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 the GCG program package, including 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)). 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.

[0066] An "isolated" molecule is one that is the predominant species in a composition where it is found with respect to the class of molecules to which it belongs (i.e., it constitutes at least about 50% of the species of molecules in the composition, and typically 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 of the polypeptide in the context of all peptide species present in the composition, or at least with respect to peptide species that are substantially active in the context of the proposed use.

[0067] The term "recombinant," when used with reference to, e.g., a cell, nucleic acid, protein, vector, etc., indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell that is so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0068] 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 an in vitro cell culture that have been harvested from the organism or an in vitro cell culture, fixed, embedded in paraffin, sectioned, deparaffinized, and transferred to a slide. Fixation and paraffin embedding can vary in many aspects, such as the fixation and embedding method used, the protocol followed, etc., and for the purposes of the present invention, it is understood to be a common practice that encompasses any such variant method as long as it involves fixing the tissue (such as formalin treatment), embedding in paraffin or equivalent material, sectioning, and transferring to a slide.

[0069] As used herein, the term "biological sample" or "sample" includes, but is not limited to, a bodily fluid (e.g., serum, lymph, blood), a cell sample, or a tissue sample (e.g., a tissue biopsy including bone marrow or mucosal tissue such as intestine, intestinal lamina propria, lung, etc.).

[0070] In the context of this specification, "treatment" or "treating" refers to preventing, alleviating, managing, curing or alleviating one or more symptoms or clinically relevant symptoms of a disease or disorder, unless the context is inconsistent. For example, "treatment" of an individual in whom symptoms or clinically relevant symptoms of a disease or disorder have not been identified is a preventative or prophylactic therapy, whereas "treatment" of an individual in whom symptoms or clinically relevant symptoms of a disease or disorder have been identified generally does not constitute a preventative or prophylactic therapy.

[0071] The term "KIR3DL2" (CD158k) refers to a disulfide-linked homodimer of three Ig domain molecules of approximately 140 kD described in Pende et al. (1996) J. Exp. Med. 184: 505-518, the disclosure of which is incorporated herein by reference. Several allelic variants of the KIR3DL2 polypeptide have been reported, each of which is encompassed by the term KIR3DL2. The amino acid sequence of mature human KIR3DL2 (allele *002) is shown below in SEQ ID NO:1, which corresponds to Genbank Accession No. AAB52520, in which the 21 amino acid residue leader sequence has been omitted. [Table 2]

[0072] The cDNA for KIR3DL2 (allele *002) is shown in Genbank accession number U30272. The amino acid sequence of human KIR3DL2 allele *003 is shown below and corresponds to Genbank accession number AAB36593. [Table 3]

[0073] Also encompassed are any nucleic acid or protein sequences that share one or more biological properties or functions with wild-type, full-length KIR3DL2, respectively, and share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide or amino acid identity.

[0074] As used herein, the term KIR3DL1 refers to another KIR3D receptor that shares relatively high amino acid identity with KIR3DL2, and various HLA ligands that bind to KIR3DL2 are also recognized by KIR3DL1. Such a KIR3DL1 polypeptide can be the KIR3DL1 allele *00101, which comprises the amino acid sequence set forth in SEQ ID NO:30.

[0075] Throughout this specification, whenever reference is made to "treatment" or "treatment of cancer" or the like in relation to an anti-KIR3DL2 binding agent (e.g., an antibody), it means: (a) a method of treating cancer, which method comprises administering (for at least one treatment) an anti-KIR3DL2 binding agent (preferably in a pharma- ceutically acceptable carrier material) to an individual, a mammal, particularly a human, in need of such treatment, in a dose (therapeutically effective amount) that allows for the treatment of cancer, preferably in a dose (amount) specified herein; (b) a method of treating cancer, for use in said treatment (particularly in a human), (c) a use of an anti-KIR3DL2 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, a method of using an anti-KIR3DL2 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer comprising mixing an anti-KIR3DL2 binding agent with a pharma- ceutical carrier, or a pharmaceutical preparation comprising an effective amount of an anti-KIR3DL2 binding agent suitable for the treatment of cancer; or (d) any combination of a), b), and c) in accordance with subject matter which is patentable in the country in which this application is filed.

[0076] Examples of antibodies that bind to human KIR3DL2 include, but are not limited to, antibody AZ158, antibody 19H12, antibody 2B12, and antibody 12B11. Further such antibodies are provided in PCT / EP2013 / 069302 and PCT / EP2013 / 069293 (both filed on September 17, 2013), the disclosures of which are incorporated herein by reference. AZ158 binds to human KIR3DL2 and human KIR3DL1 and KIR3DS1 polypeptides 2B12, and selectively binds to KIR3DL2 and does not bind to KIR3DL1 (or KIR3DS1). Antibody AZ158 can be used, for example, as a therapeutic agent administered to an individual to eliminate targets that express KIR3DL2, for example, by inducing ADCC and / or CDC. Antibodies 2B12, 19H12, and 12B11 are also suitable for use as therapeutics administered to individuals to eliminate KIR3DL2-expressing target cells. 19H12 and 12B11, as well as other antibodies disclosed in PCT / EP2013 / 069293, can be internalized intracellularly via KIR3DL2 and can be advantageously used as antibody-drug conjugates. 2B12 and other antibodies disclosed in PCT / EP2013 / 069302 do not induce KIR3DL2 internalization into tumor cells at all, thereby providing advantageous use when effector cell-mediated activity is required, for example to deplete antibodies that induce ADCC. PCT / EP2015 / 055224 discloses humanized antibodies. One such antibody that binds to human KIR3DL2 and is suitable for use as a therapeutic agent administered to an individual to eliminate KIR3DL2-expressing target cells is known under the trade name LACUTAMAB (see WHO Drug Information, Vol. 32, No. 4, 2018).

[0077] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art and refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR) recognize antibody bound to a target cell and subsequently cause lysis of the target cell. Nonspecific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

[0078] As used herein, "T cells" refers to a subpopulation of lymphocytes that mature in the thymus and display, among others, T cell receptors on their surface. T cells can be identified by certain characteristics and biological properties, such as the expression of certain surface antigens, including TCR, CD4 or CD8, and optionally CD4 and IL-23R, the ability of certain T cells to kill tumor or infected cells, the ability of certain T cells to activate other cells of the immune system, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0079] Generation of diagnostic antibodies The antibody or antibody fragment thereof specifically binds to KIR3DL2 in fixed samples, particularly in formalin-fixed paraffin-embedded (FFPE) tissue sections. The antibody can specifically bind to a target antigen in a biological sample (e.g., FFPE section) that contains KIR3DL2-expressing cells that have been prepared as paraffin-embedded cell pellets. The ability of the antibody to specifically bind to KIR3DL2 in paraffin-embedded tissue sections makes the antibody useful for many applications, particularly for detecting KIR3DL2 or KIR3DL2-expressing cells (e.g., cancer cells) and the level or distribution of KIR3DL2 or KIR3DL2-expressing cells for diagnostic or therapeutic purposes, as described herein. In certain embodiments, the antibody or antibody fragment thereof is used to determine the presence or level of KIR3DL2-expressing cells in or near cancer tissue in a sample (e.g., biopsy) taken from an individual, such as an individual with cancer. Optionally, in one embodiment, the detection of KIR3DL2 in a tissue sample determines the presence of KIR3DL2-expressing cells. Optionally, in another embodiment, upon detection of KIR3DL2 in a tissue sample (optionally upon detection of a predetermined level of KIR3DL2), the individual is determined to be suitable for treatment with a therapeutic antibody that binds to KIR3DL2.

[0080] Detection of the binding of the antibody to the target antigen can be carried out in any of many ways. For example, the antibody can be directly labeled with a detectable moiety, for example, a luminescent compound such as a fluorescent moiety, or a radioactive compound, gold, biotin (which allows subsequent binding to amplified avidin, for example avidin-AP), or an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). In another preferred embodiment, the binding of the antibody to the target antigen in the sample is evaluated indirectly, for example, by using a secondary antibody that binds to the primary anti-target antigen antibody and is itself preferably labeled with an enzyme, preferably with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); however, it is understood 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, for example, the EnVision system, in which the secondary antibody is conjugated to a polymer (e.g., dextran) that is conjugated to multiple copies of a detectable compound or enzyme, such as HRP or AP (see, e.g., Wiedorn et al. (2001) The Journal of Histochemistry & Cytochemistry, Volume 49(9): 1067-1071; Kammerer et al., (2001) Journal of Histochemistry and Cytochemistry, Vol. 49, 623-630, the entire disclosures of which are incorporated herein by reference).

[0081] KIR3DL2 polypeptide or one or more immunogenic fragments thereof can be used as an immunogen to generate antibodies, which can recognize epitopes within KIR3DL2 polypeptide on paraffin-embedded samples as described herein. Antibodies according to the present disclosure can recognize epitopes present on the extracellular domain (i.e. accessible to antibodies present outside the cell) or intracellular domain of KIR3DL2 polypeptide. Preferably, the recognized epitopes are present on the intracellular domain of KIR3DL2 polypeptide. Such epitopes present on the intracellular domain of KIR3DL2 polypeptide are accessible to antibodies in FFPE samples because the tissue is cut into sections before contacting said antibodies. In one embodiment, the epitope is an epitope that is specifically recognized by an antibody in paraffin-embedded cell pellet samples.

[0082] The antibodies of the present invention may be produced by various techniques known in the art. Typically, they are produced by immunization of a non-human animal, preferably a rabbit, with an immunogen comprising a KIR3DL2 polypeptide, preferably a human KIR3DL2 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 that comprises an epitope exposed on the surface of a cell expressing the KIR3DL2 polypeptide. Such a fragment typically contains at least about 7 contiguous amino acids of the mature polypeptide sequence, more preferably at least about 10 contiguous amino acids. The fragment is usually essentially derived from the extracellular domain of the KIR3DL2 receptor. Such fragments can typically be designed using tools such as IHC Peptide Profiler® technology (BIOTEM Corp.). In a preferred embodiment, immunization is achieved by injection of a pool of at least two immunogenic fragments, preferably at least three immunogenic fragments, derived from the KIR3DL2 polypeptide and obtained by the aforementioned tools. In one embodiment, immunogenic fragments derived from a KIR3DL2 polypeptide are defined by the sequences disclosed in the table below. [Table 4]

[0083] The step of immunizing a non-human mammal with a KIR3DL2 polypeptide or an immunogenic fragment thereof may be carried out by any method known in the art for stimulating the production of antibodies 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 entire disclosure of which is incorporated herein by reference). The immunogen is suspended or dissolved in a buffer, optionally with an adjuvant such as complete or incomplete Freund's adjuvant. Methods for determining the amount of immunogen, type of buffer, and amount of adjuvant are well known to those of skill in the art and are in no way limiting of the present invention. These parameters may vary depending on the immunogen, but are easily ascertained.

[0084] Similarly, the location and frequency of immunization sufficient to stimulate the production of antibodies is also well known in the art. In a typical immunization protocol, a non-human animal is injected intraperitoneally with an antigen on day 1 and again about one week later. This is followed by a recall injection of the antigen around day 21, optionally with an adjuvant such as incomplete Freund's adjuvant. The recall injection is given intravenously and may be repeated for several consecutive days. This is followed by a booster injection, usually without an adjuvant, on day 35, either intravenously or intraperitoneally. This protocol results in the production of antigen-specific antibody-producing B cells after about 45 days. Other protocols may also be used, so long as they result in the production of B cells expressing antibodies against the antigen used for immunization.

[0085] In another embodiment, lymphocytes from a non-immunized non-human mammal are isolated, expanded 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.

[0086] B cells from splenocytes can be isolated from immunized non-human mammals, and total RNA can be extracted and quantified to construct scFv libraries. The construction of such libraries is common to those skilled in the art (e.g. Lennard S et al. (2002) Standard Protocols for the Construction of scFv Libraries. In Antibody Phage Display. Methods in Molecular Biology®, vol 178.). In detail, the RNAs encoding the variable domains of the gamma and kappa / lambda light chains can be de-amplified using specific primer sets, respectively. The VH and VL PCR products of the amplification can be pooled separately and cloned into a backup vector to generate two different sub-libraries (for heavy and light chains). The VL fragments are cloned into a phagemid vector, and then the VH fragments are inserted into a vector containing a VL repertoire. Usually, the vector format VH / VL-6His(HHHHHH)-FLAG(DYKDDDDK) is suitable for such construction. Typically, a suitable vector for constructing an scFv library in such disclosure is the M13K07 phage. Once constructed, the scFv library can be screened by common methods such as phage display. For this purpose, the phage-displayed scFv library can be subjected to several pannings using different strategies against KIR3DL2 polypeptides or fragments thereof. The clone DNA isolated by anti-KIR3DL2 peptides can be extracted and then sequenced. Such sequences can be inserted into a suitable vector to generate the corresponding scFv. Suitable expression vectors or expression systems are well known parts. For example, E. coli strains can be transformed and used to produce such scFv. The reactivity of such scFv against KIR3DL2 polypeptides can be evaluated to select the best clones.

[0087] According to any embodiment, DNA encoding an antibody that binds an epitope present on a KIR3DL2 polypeptide isolated from an scFv library is placed into a suitable expression vector for transfection into a suitable host cell, which is then used for recombinant production of the antibody or variants thereof, such as humanized versions of the monoclonal antibody, active fragments of the antibody, chimeric antibodies comprising an antigen recognition portion of the antibody, or versions comprising a detectable moiety.

[0088] DNA encoding the KIR3DL2-specific antibody of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). The isolated DNA is placed into an expression vector, which is then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to synthesize monoclonal antibodies in recombinant host cells. As described elsewhere herein, such DNA sequences can be modified for any of a number of purposes, such as humanizing the antibody, generating fragments or derivatives, or modifying the sequence of the antibody, for example, in the antigen-binding site, to optimize the binding specificity of the antibody.

[0089] According to one embodiment, an isolated polypeptide is provided that comprises or consists of the amino acid sequence of SEQ ID NO:24. Alternatively, the isolated polypeptide according to the present disclosure can be modified, fused to one or more heterologous polypeptides, or included in another polypeptide (e.g., a polypeptide that comprises one or more non-KIR3DL2 amino acid sequences). An antibody or antibody fragment thereof that binds to said polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO:24 is also provided. The binding of an antibody or antibody fragment thereof according to the present disclosure to said polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO:24 can be evaluated by any method known to the skilled artisan (e.g., ELISA, Biacore). In a preferred embodiment, such binding is evaluated by an ELISA test. The ELISA (enzyme-linked immunosorbent assay) test is a simple method that can detect the binding between an antibody and another antigen (such as a peptide). As an example, the detection of the binding of an antibody or antibody fragment thereof according to the invention to said polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 24 by an ELISA test can comprise the steps of: (i) coating the wells of a microtiter plate with said polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (ii) blocking all non-binding sites of the plate (for example by using BSA) to avoid false positive results; (iii) providing an antibody or antibody fragment thereof (for example a soluble scFv) to the wells; (iv) providing a second antibody specific to the first antibody or antibody fragment thereof, which antibody is bound to an enzyme; (v) providing a substrate suitable for reacting with the enzyme to produce a colored product, which indicates the binding of the antibody or antibody fragment thereof to the polypeptide. If desired, the polypeptide comprising the amino acid sequence of SEQ ID NO: 24 can be bound to a solid support and then proceeded to the ELISA test. Such a polypeptide can for example be fused to a linker polypeptide bound to a solid support.

[0090] The present disclosure provides a detection or screening method based on formalin-fixed paraffin-embedded samples (FFPE cell pellets), which reveals the KIR3DL2 epitopes present after formaldehyde or formalin treatment.Thus, in one aspect, the present disclosure provides a monoclonal antibody that specifically binds to KIR3DL2 polypeptide-expressing cells (e.g., cells that are made to express KIR3DL2) in samples that are stored as paraffin-embedded cell pellets and deparaffinized before analysis.Optionally, the antibody is further characterized by not binding or only binding poorly to KIR3DL2-negative cells (cells that do not express KIR3DL2) in paraffin-embedded cell pellets.Optionally, the antibody is further characterized by binding to KIR3DL2 polypeptide-expressing cells in paraffin-embedded tissue sections.

[0091] In one aspect, the present disclosure provides a monoclonal antibody or antibody fragment thereof that specifically binds to a human KIR3DL2 polypeptide, wherein the antibody specifically binds to the KIR3DL2 polypeptide in a biological sample that has been fixed using formaldehyde (e.g., formalin, paraformaldehyde).Formalin fixation can be used, inter alia, to prepare paraffin-embedded tissue sections that can then be deparaffinized and analyzed for the presence of a marker of interest, such as a KIR3DL2 polypeptide.

[0092] In one embodiment, a monoclonal antibody is provided that specifically binds to the human KIR3DL2 polypeptide expressed by cells that are preserved in paraffin, such as cells that are preserved as paraffin-embedded cell pellets.Optionally, cells are pelleted, formaldehyde-treated (e.g., formaldehyde, formalin, paraformaldehyde), and then paraffin-embedded.Optionally, the cells that express human KIR3DL2 polypeptide are present in biological samples that are deparaffinized before analysis.

[0093] In one embodiment, the antibody binds to an antigenic determinant present on KIR3DL2 in an FFPE cell pellet sample. The residue bound by the antibody can be identified as being present on the surface of the KIR3DL2 polypeptide, and optionally also in the KIR3DL2 polypeptide expressed by the cell.

[0094] In one embodiment, a method is provided for making or testing an antibody or antibody fragment thereof capable of binding to a KIR3DL2 polypeptide in a biological sample, comprising determining whether the antibody or antibody fragment binds to an amino acid sequence selected from the group consisting of CEHFFLHREGISEDPSRLVG (SEQ ID NO: 23), CTPLDTSVYTELPNAEPRS (SEQ ID NO: 24) and CPRAPQSGLEGVF (SEQ ID NO: 25).

[0095] In one embodiment, a method is provided for producing or testing an antibody or antibody fragment thereof capable of binding to a KIR3DL2 polypeptide in a biological sample, comprising determining whether the antibody or antibody fragment binds to the same epitope on KIR3DL2 as an antibody having a VH of SEQ ID NO:21 and a VL of SEQ ID NO:22.

[0096] In one embodiment, a method is provided for producing an antibody or antibody fragment thereof capable of binding to a KIR3DL2 polypeptide in a biological sample, comprising immunizing a non-human mammal with an amino acid sequence selected from the group consisting of CEHFFLHREGISEDPSRLVG (SEQ ID NO: 23), CTPLTDTSVYTELPNAEPRS (SEQ ID NO: 24) and CPRAPQSGLEGVF (SEQ ID NO: 25), isolating antibodies from the mammal that bind to KIR3DL2, and optionally further evaluating and / or selecting the antibodies therefrom for their ability to bind to a KIR3DL2 polypeptide in a biological sample (e.g., a FFPE sample). In one embodiment, a method of making an antibody or antibody fragment thereof capable of binding to a KIR3DL2 polypeptide in a biological sample is provided, comprising providing a plurality of antibodies and evaluating and / or selecting the antibodies for their ability to bind to an amino acid sequence selected from the group consisting of CEHFFLHREGISEDPSRLVG (SEQ ID NO:23), CTPLTDTSVYTELPNAEPRS (SEQ ID NO:24) and CPRAPQSGLEGVF (SEQ ID NO:25), and optionally further evaluating whether the antibody or antibody fragment thereof is capable of binding to a KIR3DL2 polypeptide in a biological sample (e.g., an FFPE sample). In one embodiment, an antibody or antibody fragment obtained by the method of making an antibody or antibody fragment of the present disclosure is provided.

[0097] In one aspect, an antibody or antibody fragment thereof capable of specifically binding to a KIR3DL2 polypeptide comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22 is provided (see Table 5 below). [Table 5]

[0098] In one embodiment, an antibody capable of specifically binding to a KIR3DL2 polypeptide comprises a heavy chain 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 having the amino acid sequence of SEQ ID NO:21.

[0099] In one embodiment, an antibody capable of specifically binding to a KIR3DL2 polypeptide comprises a light chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99%, or at least about 80%) to a light chain having the amino acid sequence of SEQ ID NO:22.

[0100] In either embodiment, an antibody capable of specifically binding to a KIR3DL2 polypeptide can be identified as comprising VH and VL frameworks (eg, FR1, FR2, FR3 and FR4) of human origin.

[0101] The antibody according to the invention may comprise an antigen binding region (e.g. light chain VH / VL) as defined herein, optionally fused to an immunoglobulin constant region of IgG type, optionally a constant region, optionally an IgG1, IgG2, IgG3 or IgG4 isotype, and optionally further comprising amino acid substitutions to reduce effector function (binding to Fcγ receptors).

[0102] In some embodiments, an antibody is provided that comprises heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) of the VH amino acid sequence of SEQ ID NO: 21, and light chain CDR1, 2 and 3 (LCDR1, LDR2, LCDR3) of the VL amino acid sequence of SEQ ID NO: 22. Optionally, the CDRs are determined according to the Kabat, Chothia, Abm, or IMGT numbering schemes.

[0103] In one embodiment, an antibody is provided comprising: (i) a heavy chain comprising CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) having the sequences of SEQ ID NO: 03 (HCDR1), SEQ ID NO: 06 (HCDR2) and SEQ ID NO: 09 (HCDR3), and (ii) a light chain comprising CDR1, 2 and 3 (LCDR1, LDR2, LCDR3) having the sequences of SEQ ID NO: 12 (LCDR1), SEQ ID NO: 15 (LCDR2), and SEQ ID NO: 18 (LCDR3) (see Tables 6 and 7 below). [Table 6] [Table 7]

[0104] Optionally, any one or more of the light or heavy chain CDRs may contain one, two, three, four or five or more amino acid modifications (eg, substitutions, insertions or deletions).

[0105] In one embodiment, an antibody capable of specifically binding to a KIR3DL2 polypeptide comprises: an HCDR1 comprising the amino acid sequence: TYAMS (SEQ ID NO:3), or a sequence of at least 4 consecutive amino acids thereof, where optionally, one or more of these amino acids may be replaced with a different amino acid; an HCDR2 comprising the amino acid sequence: IIGASGNTWYASWAKG (SEQ ID NO:6), or a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids thereof, where optionally, one or more of these amino acids may be replaced with a different amino acid; an HCDR3 comprising the amino acid sequence: FWAGYPSNAAATVSGMDP (SEQ ID NO:9), or a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive amino acids thereof, where optionally, one or more of these amino acids may be replaced with a different amino acid. an LCDR1 region comprising an amino acid; the amino acid sequence: TLSTGYSVGSYGIG (SEQ ID NO: 12), or a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be replaced with a different amino acid; an LCDR2 region comprising an amino acid sequence: YHTEEIKHQGS (SEQ ID NO: 15), 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 replaced with a different amino acid; and / or an LCDR3 region comprising an amino acid sequence: ATAHGSGSSFHVV (SEQ ID NO: 18), or a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11 or 12 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be deleted or replaced with a different amino acid.

[0106] A further object of the present invention also encompasses function-conservative variants of antibodies capable of specifically binding to the KIR3DL2 polypeptides of the present disclosure. A "function-conservative variant" is a variant in which a given amino acid residue in a protein (e.g., an antibody or antibody fragment) is altered without altering the overall conformation and function of the protein, including but not limited to, replacement of an amino acid with one having similar properties (e.g., polarity, hydrogen bond potential, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those indicated as conserved may differ within a protein, so that the percentage of protein or amino acid sequence similarity between any two proteins with similar functions may vary, for example, from 70% to 99%, as determined according to an alignment scheme such as the cluster method, where the similarity is based on the MEGALIGN algorithm. "Function-conservative variants" include polypeptides that have at least 60%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% amino acid identity with an antibody capable of specifically binding to a KIR3DL2 polypeptide as defined above, as determined by BLAST or FASTA algorithms, and have the same or substantially similar properties or functions as an antibody capable of specifically binding to a KIR3DL2 polypeptide as defined above.

[0107] Particular heavy chain, light chain, variable region and 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 1, 2, 3 or more amino acid substitutions, where 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 change the binding properties 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 are typically those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Particular amino acid sequences may contain insertions, deletions or substitutions of 1, 2, 3, 4 or more amino acids. When substitutions are made, preferred substitutions are conservative modifications. Families of amino acid residues with 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 substituted with other amino acid residues from the same side chain family, and the modified antibodies can be tested for retained function (i.e., the properties described herein) using the assays described herein.

[0108] In one embodiment, the antibody of the present invention is an antibody fragment that retains its binding and / or functional properties. (Unless otherwise stated or clearly contradicted by the context, the term "antibody" as used in this application encompasses) Fragments and derivatives of the antibody of the present invention, preferably anti-KIR3DL2 antibodies, can be produced by techniques known in the art. A "fragment" comprises a portion of an intact antibody, generally an antigen-binding site or a 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 a contiguous sequence of contiguous amino acid residues (herein referred to as "single-chain antibody fragments" or "single-chain polypeptides"), including, but not limited to, (1) a single-chain Fv molecule; (2) a single-chain polypeptide containing only a light chain variable domain, or a fragment thereof containing the three CDRs of a light chain variable domain and having no associated heavy chain portion; and (3) a single-chain polypeptide containing only a heavy chain variable region, or a fragment thereof containing the three CDRs of a heavy chain variable region and having no associated light chain portion; and multispecific antibodies formed from antibody fragments.

[0109] FFPE sample preparation and staining The antibody has the special property of being able to efficiently and specifically bind to polypeptides (e.g., KIR3DL2 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 can be used.Furthermore, the antibody can bind to the target antigen in a sample in which therapeutic (e.g., function-neutralizing) antibodies are present at or before fixation.

[0110] The FFPE material in a biological sample from an individual is usually tissue, which is a piece of tissue that is first isolated from an animal specimen (such as a human individual) by dissection or biopsy. The tissue is then fixed to prevent decay or degeneration and to allow it to be clearly examined under a microscope for histological, pathological or cytological studies. Fixation is the process of fixing, killing and preserving tissue for the purpose of staining and observing it under a microscope. Post-fixation treatments make the tissue permeable to staining reagents and cross-link macromolecules, thus stabilizing and holding the tissue in place. This fixed tissue is then embedded in wax and cut into thin sections so that it can be stained with hematoxylin and eosin stains. Microtomy is then performed by cutting fine sections to study staining with antibodies under a microscope.

[0111] For example, it is understood that the antibodies of the present invention can be used with different suitable fixed cell or tissue preparations, and different specific fixation or embedding methods used. For example, the most common formaldehyde-based fixation procedures include formalin (e.g., 10%), but there are alternative methods such as paraformaldehyde (PFA), Bouin's solution (formalin / picric acid), alcohol, zinc-based solutions (see, for example, Lykidis et al., (2007) Nucleic Acids Research, 2007, 1-10, the disclosure of which is incorporated herein in its entirety) and others (see, for example, the HOPE method, Pathology Research and Practice, Volume 197, Number 12, December 2001, pp. 823-826(4), the disclosure of which is incorporated herein in its entirety). Similarly, although paraffin is preferred, other materials can 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 specimens, 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 PCT Patent Publication No. WO06074392, the entire disclosures of which are incorporated herein by reference.

[0112] In one embodiment of the present invention, the FFPE tissue is a human tissue (e.g., tumor tissue, tumor-adjacent tissue, normal tissue) in which the expression of KIR3DL2 is investigated. For example, the FFPE tissue can be any human tumor tissue in which the expression of KIR3DL2 is investigated. The tumor can be, for example, a squamous, bladder, stomach, kidney, head and neck, skin, breast, gastrointestinal tract, colon, esophagus, ovary, cervix, thyroid, intestine, liver, brain, pancreas, prostate, urogenital tract, lymphatic system, stomach, larynx and / or lung tumor. The FFPE tissue can be, for example, derived from a blood sample. In one embodiment, when KIR3DL2 is detected, the FFPE tissue can be a tumor or tumor-adjacent tissue obtained from an individual who has been treated with an anti-KIR3DL2 antibody, for example, who has been or is undergoing a course of treatment with such an antibody.

[0113] The antibody (e.g., anti-KIR3DL2 antibody) is incubated with the FFPE material for the detection of KIR3DL2 polypeptide. The term incubation step includes contacting the FFPE material with the antibody of the invention for different periods of time, depending on the type of material, antibody and / or antigen. The incubation process also depends on various other parameters, such as the detection sensitivity, the optimization of which follows routine procedures known to those skilled in the art. The addition of chemical solutions and / or the application of physical procedures, such as the effect of heat, can improve the accessibility of the target structures in the sample. As a result of the incubation, a specific incubation product is formed.

[0114] Suitable tests for detecting the formed antibody / antigen complex are known to those skilled in the art or can be routinely designed easily. Many different types of assays are known, examples of which are given below. The assay may be any assay suitable for using anti-KIR3DL2 mAb binding to detect and / or quantify KIR3DL2 expression, but preferably the latter is determined by a substance that specifically interacts with the anti-KIR3DL2 primary antibody.

[0115] Thus, for example, the sample (tissue or cells) to be examined is obtained by biopsy from body fluids, tumor tissue, or healthy tissue, and slices (e.g., 3 mm thick or less) are fixed using formalin or an equivalent fixation method (see above). Fixation times vary depending on the application, but can be from a few hours to 24 hours or more. After fixation, the tissue is embedded in paraffin (or an equivalent material), and very thin sections (e.g., 5 microns) are cut with a microtome and then mounted on a preferably coated slide. The slide is then dried, e.g., air-dried.

[0116] Tissue sections fixed and embedded on slides can be dried and stored indefinitely. For immunohistochemistry, slides are deparaffinized and then rehydrated. For example, they are exposed to a series of washes, first in xylene, then xylene with ethanol, and then decreasing percentages of ethanol in water.

[0117] Prior to antibody staining, tissues can be subjected to an enzymatic or heat-based antigen retrieval step to destroy 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.

[0118] Once the slides are rehydrated and antigen retrieval has ideally been performed, they can be incubated with the primary antibody. First, the slides are washed, for example with TBS, and then, following a blocking step, for example with serum / BSA, the antibody can be applied. The concentration of the antibody depends on its form (for example purified), its affinity, the tissue sample used, but a suitable concentration is, for example, 1-10 μg / ml. In one embodiment, the concentration used is 10 μg / ml. The incubation time can vary as well, but an overnight incubation is usually suitable. Following a post-antibody washing step, for example in TBS, the slides are then processed for detection of antibody binding.

[0119] The detection method used will depend on the antibody, tissue, etc. used, and may include, for example, detection of a luminescent or other visible or detectable moiety attached to a primary antibody, or may involve the use of a detectable secondary 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; PCT Patent Publication No. WO06074392, the entire disclosures of each of which are incorporated herein in their entirety.

[0120] Many direct or indirect detection methods are known and may be adapted for use. Direct labels include fluorescent or luminescent tags attached to antibodies, metals, dyes, radionuclides, etc. Antibodies labeled with iodine 125 (125I) may 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.

[0121] Indirect labeling includes various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, etc. Covalent binding of anti-3DL2 antibody to enzymes may be performed by different methods, such as coupling with glutaraldehyde. Both enzymes and antibodies are bound to glutaraldehyde via free amino groups, and the by-products of networked enzymes and antibodies are removed. In another method, when the enzyme is a glycoprotein, such as peroxidase, the enzyme is bound to the antibody via a sugar residue. The enzyme is oxidized by sodium periodate and directly binds to the amino group of the antibody. Other enzymes containing carbohydrates can also be bound to the antibody in this manner. Enzyme coupling may be performed by using a heterobifunctional linker, such as succinimidyl 6-(N-maleimido)hexanoate, to link the amino group of the antibody with the free thiol group of the enzyme, such as β-galactosidase. A horseradish peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which in the presence of hydrogen peroxide produces a soluble product detectable at 450 nm. An alkaline phosphatase detection system can be used with a chromogenic substrate such as p-nitrophenyl phosphate, which gives a soluble product that is readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which gives a soluble product detectable at 410 nm. A urease detection system can be used with substrates such as urea bromocresol purple.

[0122] In one embodiment, the binding of the primary antibody is detected by binding a labeled secondary antibody, preferably a secondary antibody covalently bound to an enzyme such as HRP or AP.In a particularly preferred embodiment, the signal generated by the binding of the secondary antibody is amplified using any of a number of methods for amplifying antibody detection.For example, the EnVision method is used (see, for example, U.S. Patent No. 5,543,332 and European Patent No. 594,772; Kammerer 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 bound to a polymer (e.g., dextran) that is itself bound to many copies of AP or HRP.

[0123] Use of antibodies in diagnosis, prognosis and therapy and methods thereof The antibody of the present invention is particularly effective for detecting KIR3DL2 polypeptide in biological samples. In a preferred embodiment, the antibody of the present invention is particularly effective for detecting KIR3DL2 polypeptide in tissue samples. In a further embodiment, the antibody of the present invention is particularly effective for detecting KIR3DL2 polypeptide in fixed tissue samples. In a preferred embodiment, the antibody of the present invention is particularly effective for detecting KIR3DL2 polypeptide in formalin-fixed paraffin-embedded tissue samples (FFPE) without non-specifically staining tissues or cells that do not express the target antigen polypeptide. Thus, the antibody is advantageous for use in the research, evaluation, diagnosis, prognosis and / or monitoring of diseases in which detection and / or localization of KIR3DL2 polypeptide and / or KIR3DL2-expressing cells is of interest.

[0124] Thus, methods of detecting, diagnosing, or monitoring cancer in an individual are provided, comprising contacting (e.g., in vitro) cancerous cells with an anti-KIR3DL2 antibody or antibody fragment thereof of the present disclosure and detecting a cancer cell-associated KIR3DL2 polypeptide. In related embodiments, the diagnostic method comprises immunohistochemistry (IHC). In certain embodiments, the biological sample is a tissue sample. In further embodiments, the biological sample is a fixed tissue sample. In preferred embodiments, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample. Those skilled in the art will further appreciate that such KIR3DL2 detection agents may be labeled or conjugated as effectors, markers, or reporters and detected using any of a number of standard imaging techniques. In other embodiments, the anti-KIR3DL2 antibody is not directly labeled and is detected using a detectable secondary agent (e.g., a labeled anti-rabbit antibody). In certain embodiments, the present disclosure provides methods for identifying or selecting individuals for administration of a treatment (e.g., chemotherapy, immunotherapy), comprising diagnosing the individual using any of the anti-KIR3DL2 compositions and detection methods of the present invention, and adjusting the course of treatment based on the results. The present disclosure further provides methods for selecting individuals with KIR3DL2-expressing cancers for administration of agents that enhance anti-tumor response regimens (e.g., chemotherapeutic agents, immunotherapeutic agents such as anti-KIR3DL2 monoclonal antibodies) for preventing or treating the cancer. In certain embodiments, the present disclosure provides methods for monitoring (e.g., the effectiveness of) a therapy (e.g., a therapeutic anti-KIR3DL2 antibody) for preventing or treating cancer.

[0125] The antibodies described herein can be used to detect the presence of KIR3DL2-expressing cells, e.g., cancerous cells (e.g., cancerous CD4 T cells, cancerous CD8 T cells), preferably in vitro. Such methods typically include contacting a biological sample from an individual with an antibody according to the present disclosure and detecting the formation of an immune complex resulting from an immune reaction between the antibody and the biological sample. In certain embodiments, the biological sample is a tissue sample. In further embodiments, the biological sample is a fixed tissue sample. In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample. The complex can be detected directly by labeling the antibody according to the present disclosure, or indirectly by adding a molecule (such as a secondary antibody) that reveals the presence of the antibody according to the present invention. For example, labeling can be achieved by binding the antibody to a radioactive or fluorescent tag. These methods are well known to those skilled in the art. Thus, the present invention also relates to the use of antibodies according to the present disclosure for the preparation of diagnostic compositions that can be used to detect the presence of KIR3DL2-expressing cells (e.g., cancerous cells, cancerous CD4 T cells, cancerous CD8 T cells), optionally to detect the presence of a pathology in which KIR3DL2-expressing cells are present, and optionally to characterize cancer or other pathologies in vivo or in vitro.

[0126] In some embodiments, the antibody of the present disclosure is useful for predicting the progression of cancer.Cancer prognosis, prognosis of cancer or cancer progression includes providing any one or more of the following outlooks or predictions (prognosis): the survival time of a subject susceptible to cancer or diagnosed with cancer, the progression-free survival time of a subject susceptible to cancer or diagnosed with cancer, the response rate to treatment of a subject or group of subjects susceptible to cancer or diagnosed with cancer, and / or the duration of response, the extent of response, or survival 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 can be determined according to standard tumor response criteria conventions, for example, "Response Evaluation Criteria in Solid Tumors" (RECIST) v1.1, detailed by Eisenhauer, EA, et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1), Eur J Cancer 2009:45:228-247, the disclosure of which is incorporated herein by reference.

[0127] In some embodiments, a biological sample from an individual with cancer can be characterized or evaluated using the antibodies disclosed herein to evaluate KIR3DL2 polypeptide and / or KIR3DL2-expressing cells in the biological sample. In certain embodiments, the biological sample is a tissue sample. In further embodiments, the biological sample is a fixed tissue sample. In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample. In one embodiment, the individual has not been treated (e.g., via ADCC) with an agent (e.g., a therapeutic antibody) that binds to KIR3DL2 polypeptide and enhances cytotoxicity against KIR3DL2-expressing cells. Alternatively, the individual has been or is undergoing a course of treatment with a therapeutic antibody that binds to KIR3DL2 polypeptide, e.g., an anti-KIR3DL2 monoclonal antibody such as racutamab.

[0128] The methods of the present disclosure can be useful for determining whether an individual has a cancer characterized by KIR3DL2-expressing cells. Such methods can be useful for monitoring an individual's disease, as well as for determining and / or providing an optimal course of treatment for the individual.

[0129] In one embodiment, the present disclosure provides a method for detecting KIR3DL2-expressing cells in an individual with cancer, the method includes providing a biological sample from the individual, where the sample optionally comprises tumor tissue or cancerous cells, and using the monoclonal antibody of the present disclosure to detect KIR3DL2 polypeptide in the sample.In a particular embodiment, the biological sample is a tissue sample.In a further embodiment, the biological sample is a fixed tissue sample.In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample.In one embodiment, the detection of KIR3DL2 polypeptide indicates the presence of KIR3DL2-expressing cells in the tissue, where the KIR3DL2-expressing cells are optionally cancerous cells.

[0130] In one embodiment, the individual has undergone or is undergoing a course of treatment with a chemotherapeutic agent.

[0131] In one embodiment, the individual is eligible (eg, the individual is a candidate or potential candidate) for a course of treatment with an agent that binds to a KIR3DL2 polypeptide (eg, a therapeutic antibody).

[0132] In one embodiment, the individual has undergone or is undergoing a course of treatment with a therapeutic antibody that specifically binds to a KIR3DL2 polypeptide and / or a KIR3DL2-expressing cell.

[0133] In one embodiment, a cancer or tumor (or an individual having such a cancer or tumor) characterized by KIR3DL2 and / or KIR3DL2-expressing cells can be identified as suitable for (e.g., would benefit from) treatment with a chemotherapeutic or immunotherapeutic agent. For example, an immunotherapeutic agent may be an agent that acts directly or indirectly on KIR3DL2-expressing cells by inducing cytotoxicity (e.g., via ADCC). Such agents may be useful for treating individuals having cancer or tumor tissue characterized by detectable and / or elevated levels of KIR3DL2 expression.

[0134] In one embodiment, the present disclosure provides an in vitro method for the diagnosis, prognosis, monitoring and / or characterization of cancer in an individual in need thereof, the method comprising providing a biological sample from the individual and detecting a KIR3DL2 polypeptide (e.g., KIR3DL2) in the sample using a monoclonal antibody that specifically binds to a human KIR3DL2 polypeptide, preferably in a fixed tissue sample, optionally in a paraffin-embedded tissue sample, where detection of the KIR3DL2 polypeptide indicates that the individual is susceptible to (e.g., benefited from) treatment with a chemotherapeutic or immunotherapeutic agent. An immunotherapeutic agent may be an agent that acts directly or indirectly on KIR3DL2-expressing cells by inducing cytotoxicity (e.g., via ADCC). Such agents may be useful for treating individuals with cancer or tumor tissue characterized by detectable and / or elevated levels of KIR3DL2 expression. In one embodiment, the biological sample is a tissue sample. In one embodiment, the biological sample is a fixed tissue sample, preferably a chemically fixed tissue sample. In one embodiment, the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue.

[0135] In one embodiment, the individual is undergoing or has undergone a course of treatment with a therapeutic antibody, such as lacutema, that binds to a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) and induces cytotoxicity (e.g., via ADCC). A biological sample from the individual can be evaluated for expression and / or levels of KIR3DL2. If KIR3DL2 can be detected, then the individual may be considered suitable for continued or further treatment with a therapeutic antibody that binds to a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) and induces cytotoxicity (e.g., via ADCC), and / or treatment with an additional or different therapeutic agent. Thus, 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 biological sample from an individual treated with a therapeutic antibody such as lacutemab that binds to a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) and induces cytotoxicity (e.g., via ADCC), and detecting a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) in the sample using a monoclonal antibody that specifically binds to a human KIR3DL2 polypeptide in a fixed tissue sample, optionally a paraffin-embedded tissue sample, wherein detection of a KIR3DL2 polypeptide indicates that the individual is suitable for (e.g., will benefit from) treatment with a chemotherapeutic or immunotherapeutic agent.

[0136] In one embodiment, an individual having cancerous cells or tumor tissue characterized by expression of a KIR3DL2 polypeptide can be treated with an anti-cancer agent, e.g., a chemotherapeutic agent, an immunotherapeutic agent, an agent that binds to a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) and enhances cytotoxicity (e.g., via ADCC) against the KIR3DL2-expressing cell, such as racutamab. Such agents may be useful in treating individuals having tumors or tumor tissue characterized by detectable and / or elevated levels of KIR3DL2 expression.

[0137] In one embodiment, the present disclosure provides a method for the treatment or prevention of cancer in an individual in need thereof, the method comprising: (i) providing a biological sample from an individual and detecting KIR3DL2-expressing cells in the sample; (ii) administering an immunotherapeutic agent to the individual based on a determination that the biological sample has an increased level of KIR3DL2-expressing cells, optionally compared to a reference level.

[0138] In certain embodiments, the biological sample is a tissue sample. In further embodiments, the biological sample is a fixed tissue sample. In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample.

[0139] In one embodiment, the present disclosure provides a method for the treatment or prevention of cancer in an individual in need thereof, the method comprising: (i) providing a biological sample from an individual, detecting KIR3DL2-expressing cells in the sample using an antibody of the present disclosure, and (ii) administering an immunotherapeutic agent to the individual based on a determination that the biological sample has an increased level of KIR3DL2-expressing cells, optionally compared to a reference level.

[0140] In certain embodiments, the biological sample is a tissue sample. In further embodiments, the biological sample is a fixed tissue sample. In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample.

[0141] In one embodiment, the present disclosure provides a method for the treatment or prevention of cancer in an individual in need thereof, the method comprising: (i) providing a biological sample from an individual, detecting KIR3DL2-expressing cells in the sample using an antibody of the present disclosure, and (ii) based on a determination that the biological sample has an increased level of KIR3DL2-expressing cells, optionally compared to a reference level, administering to the individual an immunotherapeutic agent, such as lacutemab, that binds to a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) and enhances cytotoxicity (e.g., via ADCC) against the KIR3DL2-expressing cell.

[0142] In certain embodiments, the biological sample is a tissue sample. In further embodiments, the biological sample is a fixed tissue sample. In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample.

[0143] In any aspect, detecting KIR3DL2 polypeptide in a sample using an antibody can include contacting a biological sample from an individual with the antibody and detecting the formation of an immunological complex resulting from the immunological reaction between the antibody and the biological sample.In certain embodiments, the biological sample is a tissue sample.In further embodiments, the biological sample is a fixed tissue sample.In a preferred embodiment, the biological sample is a formalin-fixed and / or paraffin-embedded tissue sample.

[0144] An agent that binds to KIR3DL2 (e.g., KIR3DL2 on the surface of a tumor cell) may be a depleting anti-KIR3DL2 antibody that binds to KIR3DL2 (e.g., a KIR3DL2-binding antibody or antibody fragment, optionally fused to a cytotoxic agent such as a toxin), a chimeric antigen receptor (e.g., a CAR-T cell receptor) comprising a KIR3DL2-binding antibody fragment, an effector cell (e.g., an NK cell or a T cell) expressing a chimeric antigen receptor on its surface), a polypeptide fused to an Fc domain, an immunoadhesin, etc. Examples of antibody agents (therapeutic antibodies) are disclosed in PCT Publication WO2014 / 044686.

[0145] Optionally, the antibody is characterized by an EC50 in a 51Cr release assay against HuT78 oncolysis by PBMCs from healthy volunteers of less than 100 ng / ml, optionally between 1 and 100 ng / ml, optionally between 1 and 50 ng / ml, optionally between 25 and 75 ng / ml, optionally about 50 ng / ml. The antibody is optionally characterized by an EC50 in a 51Cr release assay against HuT78 oncolysis by PBMCs from healthy volunteers that is comparable to the EC50 of an anti-KIR3DL2 antibody disclosed herein (e.g., has an EC50 lower than or within 1 or 0.5 logs of the EC50 of an antibody having a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4, comprises an Fc domain of a wild-type or modified human IgG1 isotype, and mediates ADCC).

[0146] In one embodiment, the therapeutic anti-KIR3DL2 antibody used according to the present disclosure is or comprises the heavy and light chain amino acid sequences of racutamab (see WHO Drug Information, Vol. 32, No. 4, 2018). racutamab is a humanized antibody having Kabat heavy chain CDRs 1, 2 and 3 of the heavy chain variable region of SEQ ID NO: 26 and Kabat light chain CDRs 1, 2 and 3 of the light chain variable region of SEQ ID NO: 27 (see Table 8 below). The CDRs can be determined by an appropriate numbering scheme, e.g., Kabat numbering. In one embodiment, racutamab can be characterized as comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27. [Table 8]

[0147] In one embodiment, racutamab can be characterized as comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:28 and a light chain comprising the amino acid sequence of SEQ ID NO:29 (see Table 9 below). [Table 9]

[0148] In one embodiment, the anti-KIR3DL2 therapeutic antibody comprises the heavy and light chain CDRs of antibodies 11E1, 8C7, 3C12 and / or 6E1 disclosed in PCT Publication WO2014 / 044681 or WO2014 / 044686.

[0149] Also provided is a diagnostic or prognostic kit, for example of cancer, comprising the antibody or antibody fragment thereof according to the present disclosure. Optionally, the kit comprises the antibody or antibody fragment of the present disclosure, and a labeled secondary antibody that specifically recognizes said antibody or antibody fragment of the present disclosure. Optionally, the kit comprises the antibody of the present invention for use as a diagnostic or prognostic agent, and an immunotherapeutic agent. In one embodiment, the immunotherapeutic agent is an agent that binds to a KIR3DL2 polypeptide (e.g., a KIR3DL2-expressing cell) and enhances cytotoxicity (e.g., via ADCC) against KIR3DL2-expressing cells, such as racutamab. The kit may further comprise a means for detecting immunological complexes resulting from the immunological reaction between a biological sample and an antibody, in particular a reagent that allows detection of said labeled antibody.

[0150] The methods of the invention may be useful in the study, evaluation, diagnosis, prognosis, and / or monitoring of a range of cancers.

[0151] Such methods are suitable for detecting, assessing suitability for treatment, and / or treating individuals with TCL, individuals susceptible to TCL, or individuals who have experienced TCL. In one embodiment, the TCL is progressive or advanced TCL (e.g., stage IV, or more generally, above stage II). In one embodiment, the individual suffers from relapsed or refractory disease. In one embodiment, the individual has a poor prognosis for disease progression (e.g., poor prognosis for survival), a poor prognosis for response to treatment, or disease progression or recurrence after previous treatment with a previous therapy.

[0152] In one embodiment, the TCL is an aggressive T-cell neoplasm. In one embodiment, the TCL is an aggressive non-cutaneous TCL. In another embodiment, the TCL is an aggressive cutaneous TCL, optionally a primary cutaneous CD4+ small / medium T-cell lymphoma or a primary CD8+ small / medium T-cell lymphoma. In one embodiment, the TCL is a cutaneous T-cell lymphoma (CTCL). In one embodiment, the TCL is a peripheral T-cell lymphoma (PTCL), optionally a non-cutaneous PTCL. PTCL and PTCL-NOS may optionally be specified as a disease other than cutaneous T-cell lymphoma.

[0153] Cutaneous T-cell lymphoma (CTCL) (see image below) is a group of lymphoproliferative disorders characterized by the localization of neoplastic T-lymphocytes in the skin. Collectively, CTCL are classified as a type of non-Hodgkin's lymphoma (NHL). The World Health Organization-European Organisation for Research and Treatment of Cancer (WHO-EORTC) classification of CTCL is reported in Willemze et al. (2005) Blood 105:3768-3785. The WHO-EORTC divides CTCL into patients with indolent clinical behaviour and those with aggressive subtypes. The third category is that of precursor hematologic neoplasms that are not T-cell lymphomas (CD4+ / CD56+ hematocutaneous neoplasms, blastic natural killer (NK) cell lymphomas, or primary cutaneous neoplasms of B-cell origin). CTCLs that may exhibit indolent clinical manifestations include mycosis fungoides (MF) and its variants, primary cutaneous CD30+ lymphoproliferative disorders (primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, etc.), subcutaneous panniculitis-like T-cell lymphoma (provisional), and primary cutaneous CD4+ small to medium-sized pleomorphic T-cell lymphoma (provisional). CTCLs that exhibit aggressive clinical behavior include Sézary syndrome (SS), adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal type, primary cutaneous peripheral T-cell lymphoma, unspecified, primary cutaneous invasive epidermotropic CD8+ T-cell lymphoma (provisional), and cutaneous gamma / delta positive T-cell lymphoma (provisional). The methods disclosed herein can be used to treat each of these conditions.

[0154] The most common CTLCs are MF and SS. Their features are reviewed in Willemze et al. (2005) Blood 105:3768-3785, the disclosure of which is incorporated herein by reference. In most cases of MF, the diagnosis is made by its clinical features, medical history, histomorphological and cytomorphological findings. An additional diagnostic criterion to distinguish CTCL from inflammatory skin diseases is the demonstration of a predominant T-cell clone in skin biopsy specimens by molecular assays (Southern blot, polymerase chain reaction (PCR), etc.). Genetic testing may also be considered. Classical mycosis fungoides is divided into three stages: (1) macules (atrophic or nonatrophic): nonspecific dermatitis, macules on the lower trunk and buttocks; minimal / no pruritus; (2) plaques: plaques with severe itching, lymphadenopathy, and (3) tumors: prone to ulceration. Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin, lymphadenopathy, palmar and / or plantar keratosis, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly. Criteria for the diagnosis of Sézary syndrome usually include absolute Sézary cell counts demonstrated by molecular genetic or cytogenetic methods, immunophenotypic abnormalities, and loss of T cell antigens and / or T cell clones in peripheral blood.

[0155] CTCL stages include I, II, III, IV according to the TNM classification, and also include peripheral blood involvement, if appropriate. Involvement of peripheral blood mycosis fungoides or Sézary syndrome (MF / SS) cells correlates with more advanced skin stage, lymph node and visceral involvement, and shorter survival. MF and SS have formal staging systems proposed by the International Society for Cutaneous Lymphoma (ISCL) and the European Organisation for Research and Treatment of Cancer (EORTC). See Olsen et al., (2007) Blood. 110(6):1713-1722; and Agar et al. (2010) J. Clin. Oncol. 28(31):4730-4739, the disclosures of which are incorporated herein by reference.

[0156] In one embodiment, the TCL is a peripheral T-cell lymphoma (PTCL), optionally a non-cutaneous PTCL. PTCL and PTCL-NOS may optionally be specified as diseases other than cutaneous T-cell lymphoma, Sézary syndrome, and mycosis fungoides, which are considered separate pathologies. In one embodiment, the PTCL is a nodal (e.g., primarily or primarily nodal) PTCL. Predominantly nodal PTCL includes, among others, PTCL-NOS (peripheral T-cell lymphoma, not otherwise specified), anaplastic large cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma (AITL), e.g., the PTCL may be an aggressive, non-cutaneous, primarily nodal PCTL (the disease may also present with extranodal manifestations).

[0157] In one embodiment, the PTCL is an extranodal (e.g., primarily extranodal) PTCL. For example, the PTCL may be an aggressive non-cutaneous extranodal PTCL.

[0158] In one embodiment, the PTCL is adult T-cell leukemia or lymphoma (ATL), for example, HTLV+ATL.

[0159] In one embodiment, the PTCL is extranodal NK / T-cell lymphoma, nasal type. In one embodiment, the PTCL is enteropathy-associated T-cell lymphoma.

[0160] In one embodiment, the PTCL is hepatosplenic T-cell lymphoma, optionally hepatosplenic αβ T-cell lymphoma, optionally hepatosplenic γδ T-cell lymphoma.

[0161] In one embodiment, the PTCL is anaplastic large cell lymphoma (ALCL), optionally ALK+ALCL, optionally ALK-ALCL. ALK+ALCL usually has a good prognosis using conventional therapy (5-year survival rate 93%), while ALK-ALCL has a poor prognosis (37%). In one embodiment, the PTCL is angioimmunoblastic T-cell lymphoma (AITL), optionally cutaneous AITL, optionally primary cutaneous CD4+ small / medium T-cell lymphoma or primary CD8+ small / medium T-cell lymphoma, optionally non-cutaneous AITL.

[0162] In one embodiment, the PTCL is an intestinal lymphoma, such as intestinal ALCL.

[0163] In one embodiment, the PTCL is T-cell prolymphocytic leukemia.

[0164] In one embodiment, the PTCL is PTCL-NOS (peripheral T-cell lymphoma, not otherwise specified). PTCL-NOS (also called PCTL-U or PTCL-unspecified) is an aggressive lymphoma, predominantly nodal, but extranodal metastases are common. The majority of nodal cases are CD4+ and CD8-. Most patients with PTCL-NOS show lymph node metastases; however, many extranodal sites (liver, bone marrow, gastrointestinal, skin, etc.) may also be involved. Studies generally report a 5-year overall survival rate of approximately 30%-35% with standard chemotherapy. In the past, a number of distinct entities have been reported that correspond to recognizable subtypes of T-cell neoplasms, such as Lennert's lymphoma, T-zone lymphoma, pleomorphic T-cell lymphoma, and T-immunoblastic lymphoma, but evidence that these correspond to distinctive clinicopathological entities is still lacking. For this reason, the recent World Health Organization (WHO) classification of hematopoietic and lymphoid neoplasms has combined them into a single broad category, PTCL-NOS / U. PTCL-NOS may therefore be specified to exclude certain distinctive clinicopathologic entities such as T-cell prolymphocytic leukemia, ATL / adult T-cell leukemia, extranodal NK / T-cell leukemia, nasal type, EATL / enteropathic T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, ALCL / anaplastic large cell lymphoma, and / or AITL / angioimmunoblastic T-cell lymphoma.

[0165] The diagnostic criteria for PTCL can be, for example, those of standard medical guidelines according to the World Health Organization (WHO) classification system (see, for example, World Health Organization. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France: IARC Press, 2008). See also, for example, Foss et al. (2011) Blood 117:6756-6767, the disclosure of which is incorporated herein by reference.

[0166] In one embodiment, TCL is characterized by tumors or tumor cells that express significant and / or detectable KIR3DL2 polypeptide on their surface. Advantageously, KIR3DL2 expression is determined by a method according to the present disclosure.

[0167] Nucleic acids encoding the antibodies or antibody fragments of the disclosure are also provided. In one embodiment, the nucleic acids are isolated and / or recombinant (e.g., including essentially pure nucleic acids) nucleic acids comprising sequences encoding the antibodies or antibody fragments of the disclosure.

[0168] Hybridomas or recombinant host cells that produce the antibody fragments of the disclosure are provided. Thus, the hybridomas or recombinant host cells of the disclosure can contain a nucleic acid that encodes an antibody or antibody fragment of the disclosure.

[0169] Working Example Example 1: Generation of monoclonal antibodies for KIR3DL2 immunohistochemistry (IHC) staining in formalin-fixed paraffin-embedded (FFPE) samples Vaccination of rabbits Immunization of rabbits to generate anti-KIR3DL2 antibodies was performed using four different strategies: (1) Immunization of two rabbits (rabbits #121 and #122) with native recombinant KIR3DL2 protein (2) Immunization of two rabbits (rabbits #278 and #372) with the recombinant extracellular domain of KIR3DL2 protein processed according to the first IHC-like protocol (IHC-A). (3) Immunization of two rabbits (rabbits #373 and #374) with the recombinant extracellular domain of KIR3DL2 protein treated according to a second IHC-like protocol (IHC-B). (4) Three peptides (peptide 1: CEHFFLHREGISEDPSRLVG (SEQ ID NO: 23), peptide 3: CTPLTDTSVYTELPNAEPRS (SEQ ID NO: 24), and peptide 4: CPRAPQSGLEGVF (SEQ ID NO: 25)) were designed using IHC PeptideProfiler® technology (BiotemCorp., France). Peptide 1 is an epitope derived from the extracellular domain of the KIR3DL2 receptor, whereas peptides 3 and 4 are epitopes derived from the intracellular domain of the KIR3DL2 receptor.

[0170] Such peptides were generated and conjugated to a carrier protein (immunogenic peptides to KLH, screening peptides to BSA and / or free).

[0171] Rabbits were injected with antigen (subcutaneous route) on days 1, 21, and 35. Blood was collected on day 45. Sera were screened by protein and peptide ELISA, and IHC on FFPE tissue microarrays.

[0172] Rabbits 121 and 122 show a similar profile, but according to the titers reached, rabbit 121 seems to be the better candidate for library construction. In IHC, good reactivity is observed, although there is no clear specificity for KIR3DL2. Rabbits 278 and 372 show a similar profile, but according to the titers reached, rabbit 278 seems to be the better candidate for library construction. In IHC, good reactivity is observed, although there is no clear specificity for KIR3DL2. Rabbits 373 and 374 show a similar profile, but according to the titers reached, rabbit 374 seems to be the better candidate for library construction. In IHC, good reactivity is observed, although there is no clear specificity for KIR3DL2. Rabbits 375 and 376 show a similar profile, but according to the titers reached, rabbit 376 seems to be the better candidate for library construction. In IHC, good reactivity / specificity is observed for KIR3DL2.

[0173] Taken together, these data led to the selection of one animal immunized with the pool of peptides (rabbit #376) and one animal immunized with the recombinant extracellular domain of KIR3DL2 protein treated with IHC-like procedure B (rabbit #374), which showed strong Ab titers by ELISA and more intense IHC staining. Whereas sera obtained after immunization with the pool of peptides showed staining in KIR3DL2-transfected CHO cells but not in non-transfected CHO cells, sera obtained after immunization with the recombinant extracellular domain of KIR3DL2 protein treated with IHC-like procedure B stained both KIR3DL2 and KIR3DL1-transfected cells. The latter was chosen as a second choice due to potential cross-reactivity against KIR3DL1.

[0174] Construction of scFV library Splenectomy was performed on selected rabbits. Spleens were processed for RNA extraction in the molecular biology laboratory. Total RNA was then quantified. RNAs encoding the variable domains of the gamma and kappa / lambda light chains were de-amplified using specific primer sets, respectively. The quality of the amplification was controlled by electrophoresis using a 1% TBE / 0.8% agarose gel. A SmartLadder SFMW-1800-04 (Eurogentec) was used as a reference during electrophoresis. The VH and VL PCR products of the amplification were pooled separately and cloned into a backup vector to generate two different sub-libraries (one for heavy chain and one for light chain). The first step of the library construction consisted of cloning the VL fragments into a phagemid vector, then inserting the VH fragments into a vector containing the VL repertoire. The vector format VH / VL-6His(HHHHHH)-FLAG(DYKDDDDK) has been selected for the construction.

[0175] The final scFv library constructed from rabbits immunized with KIR3DL2 protein treated with IHC-like procedure B contained 2.4 × 10 8The final scFv library constructed from rabbits immunized with the pool of three peptides consisted of 1.7 × 108 independent clones with a full-size insert rate of 94% (by colony PCR) and was finally packaged into M13K07 phage. After reactive clone selection and sequence analysis of the candidates, 24 clones were produced in E. coli as soluble scFVs and purified. Of these candidates, 16 were obtained from immunization of rabbits with recombinant extracellular domain of KIR3DL2 protein treated with IHC-like procedure B (rabbit #374) and 8 were obtained from immunization of rabbits with the pool of peptides (rabbit #376).

[0176] For these candidates, the reactivity of the scFVs was evaluated by ELISA. Strong reactivity was observed against KIR3DL2 protein, but not against peptides 1, 3, and 4 of three of the 16 scFvs recovered from immunization of rabbits with recombinant extracellular domain of KIR3DL2 protein treated with IHC-like procedure B. Of the eight scFvs recovered from immunization of rabbits with pools of peptides, two anti-peptide 1 scFvs (P1-R7A-C11 and P1-R7A-G1) showed high reactivity against target peptide 1 bound or free to BSA. Reactivity was also observed against the extracellular domain of KIR3DL2. Six anti-peptide 3 scFvs showed reactivity against peptide 3 (stronger for peptide bound to BSA compared to the free one), and no reactivity was observed against BSA. The reactivity of clone P3-R4D-H5 against peptide 3, free or bound to BSA, evaluated by ELISA, is shown in Figure 1. Clone P3-R4D-H5 appears to have stronger reactivity to peptide 3 conjugated to BSA than to that without peptide 3.

[0177] The reactivity of these 24 candidates was then assessed by IHC using KIR3DL2- and KIR3DL1-transfected CHO cells. Sixteen clones (rabbit #374) identified from immunization with KIR3DL2 protein treated with IHC-like procedure B showed strong signals in both KIR3DL2- and KIR3DL1-transfected CHO cells.

[0178] Of the eight candidates selected from immunization of rabbits with a pool of peptides (rabbit #376), two were specific for peptide 1 (P1-R7A-G1 and P1-R7A-C11), and six were specific for peptide 3 (P3-R4D-F4, P3-R4D-C1, P3-R4D-H5, P3-R4D-C10, P3-R4D-B5, and P3-R4D-B9). Both anti-peptide 1 clones showed strong staining in KIR3DL2-transfected cells and lower signals in KIR3DL1-transfected cells. According to the ELISA data, clone P1-R7A-C11 seemed to be a better candidate than clone P1-R7A-G1, and the former candidate was selected. As shown in Figure 2, all anti-peptide 3 candidates showed strong reactivity to KIR3DL2-transfected cells and lower reactivity to KIR3DL1-transfected cells. The strongest differential staining between KIR3DL2 and KIR3DL1 transfected cells was obtained with clone P3-R4D-H5, demonstrating its specificity for the KIR3DL2 polypeptide. Differential staining was also obtained with clones P3-R4D-C10, P3-R4D-B5, and P3-R4D-B9. The following clones were selected because they gave the strongest differential staining between KIR3DL2 and KIR3DL1 transfected cells: P3-R4D-H5, P3-R4D-C10, P3-R4D-B5, and P3-R4D-B9.

[0179] In conclusion, based on ELISA and IHC data, P1-R7A-C11, P3-R4D-H5, P3-R4D-C10, P3-R4D-B5, and P3-R4D-B9 were selected and produced in rabbit IgG format.

[0180] Reformatting and IgG production Selected scFvs were reformatted in full rabbit IgG antibodies: P1-R7A-C11 (rabbit IgG / K), P3-R4D-H5 (rabbit IgG / lambda), P3-R4D-C10 (rabbit IgG / lambda), P3-R4D-B5 (rabbit IgG / lambda) and P3-R4D-B9 (rabbit IgG / lambda). Sequences encoding the heavy chain variable domain (VH) and light chain variable domain (VL) (shown in the table below) were optimized for expression in mammalian cells and synthesized. The corresponding synthetic genes were cloned in a vector system containing the rabbit constant regions of the IgG heavy chain and lambda or kappa light chain. After being verified by sequencing, the vectors were amplified to prepare low-endotoxin plasmid DNA. [Table 10]

[0181] Transient transfections were performed using Chinese Hamster Ovary cells at Biotem. Supernatants were collected on the last day of culture (before purification) and antibody titers were measured using a ForteBio Protein A biosensor. Supernatants were finally purified by Protein A affinity chromatography.

[0182] Selection of anti-KIR3DL2 antibody candidates for IHC on FFPE samples IHC testing was performed on a VentanaBenchmarkUltra automated slide stainer, which is widely used by pathologists and is compatible with the development of companion diagnostics.

[0183] First, clones P1-R7A-C11, P3-R4D-H5, P3-R4D-C10, P3-R4D-B5, and P3-R4D-B9 selected in rabbit IgG format were tested to evaluate their specificity on FFPE cell pellets and normal skin in tissue microarray format. Two clones (P3-R4D-H5 and P1-R7A-C11) were selected as more specific Abs against KIR3DL2 after IHC evaluation and were kept for additional testing. These two clones were tested at several concentrations (10, 7.5, 5, and 2.5 μg / mL) on FFPE cell pellets in tissue microarray format that also included normal skin and FFPE human tissues (two lymph nodes, colon, liver, and CTCL). Compared to clone P1-R7A-C11, clone P3-R4D-H5 had lower nonspecific staining in KIR3DL2-negative cells and FFPE tissue. Moreover, stronger membrane staining intensity was obtained in HuT78 cells with endogenous KIR3DL2 expression compared to that obtained in CHO-mb-HuKIR3DL1 or CHO cells. Clone P3-R4D-H5 gave the best results in FFPE samples.

[0184] Example 2: Use of KIR3DL2-specific antibodies in IHC staining of cell pellets. KIR3DL2 staining on FFPE cell pellets using clone P3-R4D-H5 Tests were performed with CHO-mb-HuKIR3DL2 and HuT78ATCCTIB-161 cells, as these cells overexpress KIR3DL2. After 7 days of culture (3 passages), CHO (KIR3DL2 negative cells) were mixed with CHO-mb-HuKIR3DL2 cells to generate 8 mixtures of KIR3DL2 negative and positive cells at different ratios. After 17 days of culture (7 passages), Raji and HuT cells were mixed by serial dilution to generate 8 mixtures of KIR3DL2 negative and positive cells at different ratios. FFPE cell pellets were prepared with 20 million cells per pellet. Cell lines were fixed in formalin for 1 hour. Cells were then washed with 1XPBS and resuspended in molten histogel. After solidification of the histogel at +5±3°C, the cell pellets were placed in standard cassettes and dehydrated in a tissue processor. The cell pellets were then embedded in paraffin. After paraffin solidification, the FFPE cell pellet blocks were stored at room temperature (RT). Sections were dewaxed at 72°C for 30 min and an epitope retrieval step was performed with epitope retrieval buffer at +100°C for 20 min. Sections were incubated with primary antibody (clone P3-R4D-H5 or isotype control) for 20 min. Sections were then rinsed and incubated with post-primary antibody (rabbit anti-mouse) for 8 min and then with horseradish peroxidase (HRP) polymer (anti-rabbit HRP) for 8 min. Confirmation of staining was performed using 3,3'-diaminobenzidine (DAB) for 10 min.

[0185] IHC staining was performed with LEICABONDRX on 5 sections / block of 8CHO / CHO-mb-HuKIR3DL2 mixed FFPE cell pellets and 1 section / block using 5 μg / mL of anti-KIR3DL2 clone P3-R4D-H5. The same FFPE cell pellets were analyzed using the same concentration of IC (isotype control). As shown in Figure 3, the presence of very few KIR3DL2+ cells (up to 2.75% as determined by flow cytometry) was detectable. As expected, the number of cells stained with anti-KIR3DL2 antibody clone P3-R4D-H5 increased with the percentage of KIR3DL2 transfected cells in the pellet. No staining was observed with the isotype control.

[0186] Furthermore, on 5 sections / blocks of 8 Raji (human KIR3DL2 negative cells) / HuT (HuT78, ATCC reference TIB-161, human KIR3DL2 positive cells) mixed FFPE cell pellets, anti-KIR3DL2 clone P3-R4D-H5 was used at 5 µg / mL on 1 section / block of the same pellet, using the same concentration of isotype control. As shown in Figure 4, here too the presence of very few KIR3DL2+ cells (2.42% measured by flow cytometry) was detectable. As expected, the number of cells stained with anti-KIR3DL2 antibody clone P3-R4D-H5 increased with the percentage of HuT cells in the pellet. No staining was seen with the isotype control. Taken together, these data on mixed pellets containing either transfected (CHO-mb-HuKIR3DL2) or endogenously expressing (HuT) cells suggest that clone P3-R4D-H5 is a specific anti-KIR3DL2 antibody for IHC on FFPE samples, allowing the discrimination of cells with and without endogenous KIR3DL2 expression at the expected percentage values ​​and even in the presence of very low frequencies of positive cells.

[0187] Comparison of clone P3-R4D-H5 and 12B11KIR3DL2 staining The same type of experiment described above was performed on mixed frozen pellets and FFPE cell pellets using clone 12B11, the best known tissue staining anti-KIR3DL2 antibody. Preparation of FFPE cell pellets was the same as above. Different unmasking and amplification conditions were performed (e.g., buffer, buffer pH, addition of tyramide-biotin). For frozen samples, sections were rehydrated, incubated with 0.3% H2O2 for 10 min, washed three times with PBS1X squeeze bottle, and incubated with protein block for 30 min. The protein block was then removed and sections were incubated with primary antibody (anti-KIR3DL2 12B11 antibody or mouse IgG1 isotype control) for 1 h at room temperature. Sections were rinsed three times for 5 min in PBS1X and then incubated with HRP-conjugated secondary Ab for 30 min at room temperature (EnVision kit from Dako). Sections were then washed three times for 5 min in PBS1X. Finally, staining was confirmed using DAB for 5 min. For IHC staining of frozen samples using LEICABONDRX, the protocol was the same as that used for FFPE samples without the dewaxing and epitope retrieval steps.

[0188] Clone P3-R4D-H5 allowed the staining of endogenous KIR3DL2 expressing cell lines (HuT cells) on FFPE pellet sections. Clone 12B11 (reference antibody for KIR3DL2 tissue staining) was able to stain HuT cell lines in frozen samples after protocol optimization. However, further analysis by digital pathology showed that the staining accuracy was low in frozen samples stained with clone 12B11, resulting in inferior staining quality compared to the staining observed in FFPE cell pellet samples. In conclusion, anti-KIR3DL2 clones P3-R4D-H5 and 12B11 for IHC of FFPE and frozen samples, respectively, are KIR3DL2-specific antibodies that allow the detection of low numbers of KIR3DL2 expressing cells. However, the quality of staining of FFPE samples is better than that of frozen samples.

[0189] As shown in Figure 5A, B and C, IHC with clone 12B11 Ab (reference antibody for KIR3DL2 tissue staining on frozen samples) under some conditions does not allow expression of KIR3DL2 on FFPE cell pellets containing KIR3DL2-positive cells. Therefore, clone 12B11 cannot be used for KIR3DL2 IHC staining of FFPE samples.

[0190] Specificity of clone P3-R4D-H5 for KIR3DL2 and KIR3DL1 in IHC The same kind of experiment as above (see R3DL2 staining of FFPE cell pellets with clone P3-R4D-H5) was performed on FFPE cell pellets consisting of CHO cells (CHO), human KIR3DL2 and KIR3DL1 negative cells; human KIR3DL1 positive and KIR3DL2 negative cells, CHO-mb-HuKIR3DL1 cells (CHO-KIR3DL1); and human KIR3DL2 positive and KIR3DL1 negative cells, CHO-mb-HuKIR3DL2 cells (CHO-KIR3DL2). After IHC staining with several concentrations of anti-KIR3DL2 antibody clone P3-R4D-H5, the optical density of the FFPE sections was measured by HistoQuantif. As shown in Figure 6, anti-KIR3DL2 clone P3-R4D-H5 binds to KIR3DL2 expressing cells, but no substantial binding was observed to KIR3DL1 expressing cells. Thus, clone P3-R4D-H5 is specific for the KIR3DL2 polypeptide in FFPE samples.

[0191] Example 3: Use of KIR3DL2-specific antibodies in IHC staining of cell pellets prepared from biopsies Staining of CTCL individual biopsies Clones P3-R4D-H5 and 12B11 were used for staining of FFPE and corresponding frozen CTCL biopsies, respectively. Staining assessment of FFPE and frozen sections of biopsies of individuals with mycobacterium mycosis or Sézary syndrome was performed by a pathologist on scanned slides. For each FFPE block, 3 μm thick sections were prepared, placed on superfrosted glass slides and dried for at least 1 h at +45 + / - 3 °C in a ventilated oven. Representative images of KIR3DL2 staining on FFPE and frozen sections are shown in Figure 7. The percentage of KIR3DL2+ cells in mononuclear cells was estimated by a pathologist in a semiquantitative manner. The percentage of KIR3DL2+ cells in mononuclear cells was estimated by a pathologist for FFPE CTCL samples stained with anti-KIR3DL2 clone P3-R4D-H5 and for corresponding frozen samples stained with anti-KIR3DL2 clone 12B11.

[0192] By comparing the percentage of KIR3DL2+ cells estimated in clone 12B11 and clone P3-R4D-H5 in the corresponding CTCL biopsies, we observed that clone P3-R4D-H5 usually had a higher percentage of stained cells, reflecting the fact that staining assessment of frozen samples is more difficult and the accuracy of the poor quality of frozen samples was lower in these biopsies (frozen samples are more sensitive to temperature changes compared to FFPE samples).

[0193] Further IHC staining of mycosis fungoides tumor samples (CTCL) with anti-KIR3DL2 clone P3-R4D-H5 by the above method is shown in Figures 9A, 9B, 9C and 9D. Figures 9A and 9C show tumor samples with high expression (strong signal) of KIR3DL2, while Figure 9B shows a weakly positive tumor sample. Figure 9D shows a recurrent mycosis fungoides tumor sample that encompasses a strong KIR3DL2 positive area and a nearly KIR3DL2 negative area.

[0194] Staining of PTCL individual biopsies Clones P3-R4D-H5 and 12B11 were used to stain FFPE and matched frozen PTCL biopsies, respectively. Staining assessment of FFPE and frozen sections of biopsies of individuals with PTCL was performed by a pathologist on scanned slides. Representative images of KIR3DL2 staining on FFPE and frozen sections are shown in Figure 8. The percentage of KIR3DL2+ cells among mononuclear cells was estimated by a pathologist for frozen PTCL samples stained with anti-KIR3DL2 clone 12B11 and corresponding FFPE samples stained with anti-KIR3DL2 clone P3-R4D-H5 or isotype control.

[0195] The results show that clone 12B11 and clone P3-R4D-H5 allow staining of KIR3DL2+ cells on frozen and FFPEPTCL samples, respectively.

[0196] Further IHC staining of PTCL tumor samples with anti-KIR3DL2 clone P3-R4D-H5 by the above method is shown in Figures 10A, 10B, 10C and 10D. Figure 10A shows a PTCL-NOS tumor sample that is highly KIR3DL2 positive, while Figures 10C and 10D show a PTCL-NOS tumor sample with moderate KIR3DL2 positivity (with local variability within the sample in Figure 10D). Figure 10B shows a PTCL-NOS tumor sample with scattered KIR3DL2 positive tumor cells against a background of faint stromal cell KIR3DL2 positivity.

[0197] All references cited in this specification, including publications, patent applications, and patents, are incorporated by reference in their entireties herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein (to the maximum extent permitted by law), regardless of whether an incorporation of a particular document made elsewhere in this specification is individually provided.

[0198] Unless otherwise indicated, all exact values ​​provided herein represent the corresponding approximations (e.g., all exact exemplary values ​​provided for a particular factor or measurement can also be considered to provide the corresponding approximate measurement, modified by "about" where appropriate). When "about" is used in connection with a numerical value, this can be specified as including a value corresponding to + / - 10% of the specified numerical value.

[0199] Description of any aspect or embodiment of the invention herein using terms such as "comprising," "having," "including," or "containing" in reference to one or more elements is intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" that particular element, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood to also describe a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0200] Any examples provided herein, or the use of exemplary language (e.g., "etc.") are merely intended to facilitate a better understanding of the invention and are not intended to impose limitations on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Claims

1. A monoclonal antibody or antibody fragment thereof capable of specifically binding to a KIR3DL2 polypeptide in a biological sample, wherein the antibody or antibody fragment comprises heavy chain CDR1, 2 and 3 of the heavy chain variable region of SEQ ID NO:21, and light chain CDR1, 2 and 3 of the light chain variable region of SEQ ID NO:

22.

2. The antibody or antibody fragment described in claim 1, comprising a heavy chain having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:21, and a light chain having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:

22.

3. 3. The antibody or antibody fragment thereof of claim 1 or 2, wherein the antibody or antibody fragment thereof is capable of specifically binding to a KIR3DL2 polypeptide in a formalin-fixed paraffin-embedded (FFPE) tissue sample.

4. An antibody or antibody fragment thereof described in claim 1, comprising an amino acid sequence of SEQ ID NO: 21 and a light chain having an amino acid sequence of SEQ ID NO:

22.

5. 2. The antibody or antibody fragment thereof of claim 1 for use in detecting the presence of KIR3DL2-expressing cells in a biological sample.

6. 6. The antibody or antibody fragment for use according to claim 5, wherein the sample is formalin-fixed paraffin-embedded (FFPE) tissue.

7. An in vitro method for detecting KIR3DL2-expressing cells in a sample, the method comprising: (i) providing a biological sample from an individual containing cells; and (ii) detecting KIR3DL2-expressing cells with an antibody or antibody fragment thereof as defined in claim 1.

8. 8. The method of claim 7, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) tissue.

9. 11. An in vitro method for assessing the suitability of an individual having cancer for treatment with an immunotherapeutic agent, the method comprising: (i) providing a biological sample from the individual; and (ii) detecting KIR3DL2-expressing cells in the sample using an antibody or antibody fragment thereof described in claim 1, wherein detection of KIR3DL2-expressing cells indicates that the individual is suitable for treatment with an immunotherapeutic agent.

10. 10. The method of claim 9, wherein the immunotherapeutic agent is an agent that binds to a KIR3DL2 polypeptide.

11. An agent that binds to a KIR3DL2 polypeptide for use in treating a disease in an individual, the treatment comprising: (i) providing a biological sample from the individual; detecting KIR3DL2-expressing cells in the sample using an antibody or antibody fragment thereof described in claim 1 or according to the method of claim 7 or 8; and (ii) administering an immunotherapeutic agent to the individual upon detection of KIR3DL2-expressing cells.

12. The method of claim 11, wherein the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

13. The agent for use according to claim 11, wherein the disease is cutaneous T-cell lymphoma (CTCL) or peripheral T-cell lymphoma (PTCL).

14. An isolated nucleic acid or set of nucleic acids encoding the antibody or antibody fragment of claim 1.

15. 15. A hybridoma or recombinant host cell producing the antibody or antibody fragment of claim 1 or comprising the nucleic acid of claim 14.

16. An isolated nucleic acid or set of nucleic acids encoding the antibody or antibody fragment described in claim 4.

17. A hybridoma or recombinant host cell producing the antibody or antibody fragment of claim 4 or containing the nucleic acid of claim 16.

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