KIR-binding antibodies and antigen-binding fragments thereof

Abstract

The present technology relates, in part, to antibodies or antigen-binding fragments thereof that bind to one or more members of the KIR protein family (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3) or portions thereof, as well as methods, systems, and kits for detecting KIR. In certain aspects, the present technology relates to antibodies or antigen-binding fragments thereof for use in determining KIR levels in a sample containing or suspected of containing KIR. In some aspects, the present technology relates to antibodies or antigen-binding fragments thereof for use in diagnosing or treating an individual having or suspected of having a disease or disorder associated with KIR.

Description

[0001] Related patent applications This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 640,988, filed May 1, 2024, entitled KIR-binding antibody and its antigen-binding fragment, with Evan TENG et al. listed as inventors, and designated by Attorney General BIOLEG-1002PROV. The entire contents of the aforementioned patent application are incorporated herein by reference for all purposes, including the entire text, tables, and figures. Technical Field

[0002] This technology relates in part to antibodies or antigen-binding fragments thereof that bind to one or more members of the KIR protein family (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and / or KIR3DL3) or portions thereof, and to methods, systems and kits for detecting KIR. In some aspects, this technology relates to antibodies or antigen-binding fragments thereof for use in determining KIR levels in samples containing or suspected of containing KIR. In some aspects, this technology relates to antibodies or antigen-binding fragments thereof for use in diagnosing or treating individuals who have or are suspected of having KIR-related diseases or conditions. Background Technology

[0003] Killer cell immunoglobulin-like receptors (KIRs) are a family of receptors expressed by natural killer cells and T lymphocytes. The KIR protein family is part of the transmembrane glycoprotein and immunoglobulin superfamily. KIR proteins comprise extracellular and intracellular structures. The extracellular KIR domain can be characterized as an immunoglobulin-like domain. The intracellular domain contains ITAM (activation) and / or ITIM (inhibition) regions, depending on the variant. KIR proteins range in size from approximately 30 kDa to approximately 50 kDa. Some KIR proteins interact with MHC class 1 molecules (HLA) on target cells to prevent NK cell-induced cell killing and lysis, while others react with HLA to lead to NK cell activation or inhibition (Pende, D. et al., FrontImmunol, (2019), Debska-Zielkowska, J. et al., Cells (2021)).

[0004] There is high homology among KIR receptors. Therefore, antibodies specifically recognizing certain members of the KIR family have not yet been developed. To generate antibodies specifically targeting certain members of the KIR family, a collection of monoclonal antibodies has been produced against certain KIR receptor family members, including, for example, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and KIR3DL3. The specificity of these antibodies has been validated using engineered cell lines and human peripheral blood samples. For example, the KIR2DS1-specific monoclonal antibody described herein distinguishes closely related KIR2DL1 from the KIR2DS1 protein; and the KIR2DL5-specific monoclonal antibody described herein targets a broader range of KIR2DL5 variants better than currently commercially available clones. In addition to monospecific KIR antibodies, multiple clones can identify shared epitopes of two or more KIRs (including KIR2DL2, KIR2DL3, and KIR2DS2). Such monoclonal antibodies can be used for, for example, immunophenotyping, diagnosis, and treatment. Summary of the Invention

[0005] In some respects, antibodies or antigen-binding fragments thereof that bind to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof are provided, said antibodies or antigen-binding fragments comprising (a) an immunoglobulin heavy chain variable domain comprising (i) a heavy chain complementarity-determining region 1 (CDRH1) comprising a CDRH1 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with a CDRH1 polypeptide provided in Table 3; (ii) a heavy chain complementarity-determining region 2 (CDRH2) comprising a CDRH2 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with a CDRH2 polypeptide provided in Table 3; and (iii) a heavy chain complementarity-determining region 3 (CDRH3) comprising a CDRH3 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with a CDRH3 polypeptide provided in Table 3; and (b) an immunoglobulin light chain variable domain comprising (i) a light chain complementarity-determining region 1 (ii) a light chain complementarity-determining region 2 (CDRL1), which contains the CDRL1 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the CDRL1 polypeptide provided in Table 3; and (iii) a light chain complementarity-determining region 3 (CDRL3), which contains the CDRL2 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the CDRL2 polypeptide provided in Table 3; and (iii) a light chain complementarity-determining region 3 (CDRL3), which contains the CDRL3 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the CDRL3 polypeptide provided in Table 3.

[0006] In some respects, isolated antibodies or antigen-binding fragments thereof that bind to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof are also provided, wherein the antibodies or antigen-binding fragments thereof block or are capable of blocking KIR ligand binding.

[0007] In some respects, isolated antibodies or antigen-binding fragments thereof that bind to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof are also provided, wherein the antibodies or antigen-binding fragments thereof enhance or are capable of enhancing KIR ligand binding.

[0008] In some respects, isolated antibodies or antigen-binding fragments thereof that bind to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof are also provided, wherein the antibodies or antigen-binding fragments thereof inhibit or are capable of inhibiting natural killer cell activity.

[0009] In some aspects, isolated antibodies or antigen-binding fragments thereof that bind to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof are also provided, wherein the antibodies or antigen-binding fragments thereof activate or are capable of activating natural killer cell activity.

[0010] In some aspects, isolated antibodies or antigen-binding fragments thereof that bind to one or more members of the killer cell immunoglobulin-like receptor (KIR) protein family or one or more portions thereof are also provided, wherein the antibody comprises: (i) an immunoglobulin heavy chain comprising a set of heavy chain complementarity-determining region (CDR) amino acid sequences CDRH1, CDRH2, and CDRH3; and (ii) an immunoglobulin light chain comprising a set of light chain CDR amino acid sequences CDRL1, CDRL2, and CDRL3, wherein the heavy chain and light chain CDR sets are each selected from the same group A, B, C, D, E, F, G, H, I, J, K, or L: Certain implementations are further described below in the description, examples, claims, and accompanying drawings. Attached Figure Description

[0011] The accompanying drawings illustrate certain embodiments of the present technology and are not limiting. For clarity and ease of illustration, the drawings are not drawn to scale, and in some cases, aspects may be enlarged or magnified to facilitate understanding of a particular embodiment.

[0012] Figure 1 This matrix shows a selection matrix of potential KIR2DL4-stained clones using the transfected Ba / F3 cell line. Positive clones are labeled “Y”, while negative clones are labeled “N”. The matrix shows the specificity of S23004D, S22010B, S22010H, and mAb-33 for a specific KIR (KIR2DL4 in this case).

[0013] Figure 2 Histograms showing clones tested against two positive cell lines (KIR2DL4-55 and KIR2DL4-71 transfected Ba / F3), which are positive for KIR2DL4.

[0014] Figure 3 Titration data for each clone against two KIR2DL4 (2DL4-55 and 2DL4-71) cell lines from 2 µg to 0.0625 µg are shown, with high mean fluorescence signals at each concentration. S23004D shows the highest fluorescence signal here.

[0015] Figure 4 Cross-blocking by KIR2DL4 antibodies is shown. Specifically, the results of epitope interaction assays are shown here, where one antibody is used to block another antibody to create a matrix. Changes resulting from no blocking are denoted as “signal,” loss of signal as “blocked,” and partial reduction as “partial.” Some interactions exhibiting increased signal are referred to as “higher.” This indicates that S23004D has epitopes different from other clones and / or exhibits significantly stronger binding.

[0016] Figure 5 The image shows primary peripheral blood lymphocytes stained using antibodies against CD56 (an NK marker) and KIR2DL4. In this case, the positive population is characterized by high CD56 and Y-axis positivity, demonstrating positive primary cell population identification.

[0017] Figure 6 This shows KIR2DL4 expression in an IL-2-stimulated PBMC population. Stimulation of KIR2DL4 resulted in a larger positive population. Control staining showed that S23004D identified the population better than other clones.

[0018] Figure 7 The figure shows the interaction between the test clone and the reference clone and the recombinant KIR2DL4 protein. Specifically, the figure shows the recombinant protein neutralization of the test clone, which shows reduced staining when blocked by the recombinant protein KIR2DL4. This indicates that the antibody is specific for KIR2DL4.

[0019] Figure 8 Some clones of novel anti-KIR binders are shown (single-specificity is indicated in bold, multi-specificity is indicated in non-bold).

[0020] Figure 9-18 This shows the data generated for the anti-KIR2DS1 clone using the method described in this paper.

[0021] Figure 19-28This shows the data generated for the anti-KIR3DL2 clone using the method described in this paper.

[0022] Figures 29-35 This shows the data generated for the anti-KIR2DS4 clone using the method described in this paper.

[0023] Figure 36-41 This shows the data generated for the anti-KIR3DL1 clone using the method described in this paper.

[0024] Figure 42-48 This shows the data generated for the anti-KIR2DL5 clone using the method described in this paper.

[0025] Figure 49 This shows that some clones cover a wider range of polymorphic variants compared to currently available clones for KIR2DL5. For example, clone S22010E is able to bind more polymorphic KIR2DL5 variants than the commercially available clone UP-R1, providing better coverage of donor-dependent KIR expression differentials. Top panel: Ba / F3 cells stained with clones S22010E and UP-R1 expressing different KIR2DL5 polymorphic variants. Bottom panel: SNP variants of KIR2DL5 at positions 141, 173, and 195.

[0026] Figures 50-54 This shows the data generated for the anti-KIR2DL5 clone using the method described in this paper.

[0027] Figures 55-59 This shows the data generated for the anti-KIR3DL3 clone using the method described in this paper.

[0028] Figures 60-63 This shows the data generated for the anti-KIR2DS5 clone using the method described in this paper.

[0029] Figure 64-101 This document displays data generated for anti-KIR2DL1, anti-KIR2DL2, anti-KIR2DS2, and / or anti-KIR2DL3 clones using the methods described herein.

[0030] Figure 102 Certain KIR-specific clones can characterize the KIR-positive NK population. The data shown were pre-gated on peripheral blood lymphocyte populations, and samples were stained with anti-CD56 APC and the indicated specific clones. Cells were analyzed by flow cytometry.

[0031] Figure 103A-103C This indicates that certain clones can affect KIR-ligand interactions. Figure 103A For KIR2DS1, HLA-C 04:01 is the reported KIR2DS1 ligand. The results indicate that KIR2DS1-binding clones S22019F and S22013A block HLA-C. 04:01 The tetramer binds to KIR2DS1. Figure 107 B: Regarding KIR3DL2, HLA-A 11:01 is the reported KIR3DL2 ligand. The results indicate that KIR3DL2-binding clone S22025B enhances HLA-A... 11:01 The tetramer binds to KIR3DL2. Figure 107 C: For KIR3DL3, HHLA2 is the reported KIR3DL3 ligand. The results indicate that the KIR3DL3-binding clone S23011E blocks the binding of HHLA2 to KIR3DL3.

[0032] Figure 104-121 The data shown characterizes the aforementioned anti-KIR clone relative to other commercially available antibodies.

[0033] Figure 122 This image shows representative images of activation in the KIR3DL1 cytotoxicity assay and representative effects of HLA or antibody addition.

[0034] Figure 123 The results show the CD107a activation changes from the KIR3DL1 cytotoxicity assay.

[0035] Figure 124 This study demonstrates the effect of KIR3DL1-binding antibody addition on the normalized CD107a activation ratio of NK cells in NK-K562 cocultures. NK cells in NK-K562 cocultures were separated and analyzed based on KIR3DL1 positivity. The CD107a activation ratio was calculated as the ratio of the percentage of CD107a-positive NK cells to the percentage of CD107a-negative NK cells. All samples were normalized to allotype control measurements (MOPC-21). Analysis was performed using Brown-Forsythe and Welch one-way ANOVA. = p-value < 0.0001, = p-value < 0.001. n = 7-21 samples / group.

[0036] Figure 125 This image shows representative images of activation in the KIR2DS1 cytotoxicity assay and representative effects of HLA or antibody addition.

[0037] Figure 126 This study demonstrates the effect of KIR2DS1-specific antibody addition on the normalized CD107a activation ratio of NK cells in NK-K562 cocultures. NK cells in NK-K562 cocultures were separated and analyzed based on KIR2DS1 positivity. The CD107a activation ratio was calculated as the ratio of the percentage of CD107a-positive NK cells to the percentage of CD107a-negative NK cells. All samples were normalized to allotype control measurements (MOPC-21). Results were analyzed using Brown-Forsythe and Welch two-factor ANOVA. = p-value < 0.0001, = p-value < 0.001. n = 16 samples / group.

[0038] Figure 127 This image shows representative images of activation in the KIR2DS4 cytotoxicity assay and representative effects of HLA or antibody addition.

[0039] Figure 128 This study demonstrates the effect of KIR2DS4-specific antibody addition on changes in activated NK cells in NK-K562 co-cultures. NK cells in NK-K562 co-cultures were separated and analyzed based on CD107a positivity. Samples were normalized to an isotype control (MOPC-21) and statistically compared with the isotype control for CD107a+. The results were analyzed using a Turkey two-way ANOVA. = p-value < 0.001, = p-value < 0.01, ns = not significant. n = 11-22 samples / group.

[0040] Figure 129 The effect of KIR2DS4-specific antibody addition on TNFα and IFNγ secretion in NK-K562 cocultures was shown. The concentrations of TNFα and IFNγ in solution were analyzed in the NK-K562 coculture supernatant. Samples were normalized to the isotype control (MOPC-21) and statistical comparisons of analytes were performed with the isotype control. Results were analyzed using Brown-Forscythe and Welch one-way ANOVA. = p-value < 0.01, = p-value < 0.05, ns = not significant. n = 8-16 samples / group.

[0041] Figure 130 This study demonstrates the effect of KIR2DS4-specific antibody addition on K562 cell survival in NK-K562 cocultures. K562 cells in NK-K562 cocultures were separated and analyzed based on Apotracker Green positivity. Samples were normalized to the isotype control (MOPC-21) and the percentage of K562 cells was statistically compared with the isotype control. The results were analyzed using a Turkey two-way ANOVA. = p-value < 0.0001, ns = not significant. n = 11-22 samples / group.

[0042] Figures 131-132 This paper presents ligand binding interaction data generated for the anti-KIR2DL1 clone using the method described herein.

[0043] Figures 133-136 This paper presents ligand binding interaction data for anti-KIR2DL2, anti-KIR2DS2, and anti-KIR2DL3 clones generated using the methods described herein.

[0044] Figures 137-139 This paper presents ligand binding interaction data generated for the anti-KIR3DL1 clone using the method described herein.

[0045] Figures 140-142 This paper presents ligand binding interaction data for anti-KIR2DS4 clones generated using the methods described herein.

[0046] Figure 143 This paper presents ligand binding interaction data generated for the anti-KIR2DL5 clone using the method described herein.

[0047] Figure 144 Certain clones were shown to influence KIR-ligand interactions. Ba / F3 cells transfected with KIR2DL5A were incubated with CD155-FC, CD112-FC, or CD113-FC. Anti-FC antibodies were used to detect binding in flow cytometry. As shown, certain clones were added to the cells, followed by ligand incubation to determine whether the clones could influence ligand binding. CD155 has been previously reported as a ligand for KIR2DL5; CD112 and CD113, which are similar to CD155, were used as negative controls. The results showed that the KIR2DL5A-binding clone S22010G enhanced CD155 binding to KIR2DL5A.

[0048] Figures 145-147This paper presents ligand binding interaction data generated for the anti-KIR2DS1 clone using the method described herein.

[0049] Figures 148-150 This paper presents ligand binding interaction data generated for the anti-KIR3DL2 clone using the method described herein.

[0050] Figures 151-152 This paper presents ligand binding interaction data generated for the anti-KIR3DL3 clone using the method described herein. Detailed Implementation

[0051] This document provides killer cell immunoglobulin-like receptor (KIR) binding antibodies (e.g., antibodies binding KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and / or KIR3DL3) and their antigen-binding fragments, and nucleic acids encoding such antibodies, their components and their antigen-binding fragments. This document also provides vectors (e.g., recombinant expression vectors) and cells (e.g., recombinant cells) for expressing and generating said antibodies and antigen-binding fragments. In some embodiments, the antibody and its antigen-binding fragment can bind to KIRs under physiological and / or in vitro conditions. Methods for generating and using KIR-binding antibodies and their antigen-binding fragments are also provided, such as methods for detecting KIR in samples from individuals, including methods for laboratory / research purposes (e.g., flow cytometry, ELISA, and / or Western blotting), and / or methods for treating and / or preventing various diseases or conditions by using pharmaceutical compositions or other compositions containing such antibodies or their antigen-binding fragments, and by delivering pharmaceutical compositions or other compositions containing such antibodies or their antigen-binding fragments.

[0052] Anti-KIR reagent This document provides reagents for binding cytotoxic cell immunoglobulin-like receptors (KIRs) or portions thereof. For example, this document provides reagents for binding KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3 or portions thereof. Cytotoxic cell immunoglobulin-like receptors (KIRs) are also known as CD158. Reagents for binding KIRs or portions thereof may be referred to as anti-KIR reagents and may include anti-KIR antibodies, anti-KIR antibody fragments (e.g., antigen-binding fragments), and anti-KIR antibody derivatives. In some embodiments, the reagent is isolated (e.g., separated from components of its natural environment (e.g., animals, biological samples)). In some embodiments, the reagent is not naturally occurring (e.g., generated by human intervention). In some embodiments, the reagent is a humanized antibody or its antigen-binding fragment. In some embodiments, the reagent is a derivative of a humanized antibody that binds to a KIR.

[0053] In some embodiments, the reagent binds to KIR under laboratory conditions (e.g., in vitro, in flow cytometry, or in an ELISA). In some embodiments, the reagent binds to KIR under physiological conditions (e.g., in cells of a subject). In some embodiments, the reagent affects KIR signaling transduction. In some embodiments, the reagent blocks or is capable of blocking KIR signaling transduction. In some embodiments, the reagent enhances or is capable of enhancing KIR signaling transduction. In some embodiments, the reagent affects ligand binding to KIR. In some embodiments, the reagent blocks or is capable of blocking ligand binding to KIR. For example, the anti-KIR reagent of this article blocks HLA-C... 04:01 Binding to KIR2DS1. In another example, the anti-KIR reagent described herein can block the binding of HHLA2 to KIR3DL3. In some embodiments, the reagent enhances or is able to enhance the binding of the ligand to KIR. For example, the anti-KIR reagent provided herein can enhance the binding of CD155 to KIR2DL5A. In another example, the anti-KIR reagent provided herein can enhance HLA-A 11:01 binding to KIR3DL2. KIR ligand binding can be evaluated using any suitable assay, such as ligand binding assays, as in Example 2. Figure 103A-C and Figures 131-152 As shown in the image.

[0054] In some embodiments, the reagent affects immune cell activity. In some embodiments, the reagent affects natural killer cell activity. In some embodiments, the reagent inhibits natural killer cell activity. In some embodiments, the reagent activates natural killer cell activity. Natural killer cell activity can be assessed using any suitable assay, such as cytotoxicity assays (e.g., degranulation assays measured by the release of cytotoxic particles labeled by surface expression of CD107a), cytokine (e.g., TNFα, IFNγ) expression and / or secretion assays, and natural killer target cell survival assays (e.g., K562 cell survival assays), as in Example 2 and Figure 122-130 As shown in the image.

[0055] Generally, the anti-KIR reagents provided herein comprise at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, the anti-KIR reagents herein comprise two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Typically, each immunoglobulin heavy chain variable domain of the anti-KIR reagent comprises a first, second, and third heavy chain complementarity-determining region (CDR; CDRH1, CDRH2, CDRH3), and each immunoglobulin light chain variable domain of the anti-KIR reagent comprises a first, second, and third light chain CDR (CDRL1, CDRL2, CDRL3).

[0056] In some embodiments, the anti-KIR reagent provided herein comprises a heavy chain complementarity-determining region 1 (CDRH1), which comprises the CDRH1 peptide provided in Table 3 or a peptide having at least 80% identity with the CDRH1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH1, which comprises a peptide having at least 85% identity with the CDRH1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH1, which comprises a peptide having at least 90% identity with the CDRH1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH1, which comprises a peptide having at least 95% identity with the CDRH1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH1, which comprises a peptide having 100% identity with the CDRH1 peptide provided in Table 3.

[0057] In some embodiments, the anti-KIR reagent provided herein comprises heavy chain complementarity-determining region 2 (CDRH2), which comprises the CDRH2 peptide provided in Table 3 or a peptide having at least 80% identity with the CDRH2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH2, which comprises a peptide having at least 85% identity with the CDRH2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH2, which comprises a peptide having at least 90% identity with the CDRH2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH2, which comprises a peptide having at least 95% identity with the CDRH2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH2, which comprises a peptide having 100% identity with the CDRH2 peptide provided in Table 3.

[0058] In some embodiments, the anti-KIR reagent provided herein comprises a heavy chain complementarity-determining region 3 (CDRH3), which comprises the CDRH3 peptide provided in Table 3 or a peptide having at least 80% identity with the CDRH3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH3, which comprises a peptide having at least 85% identity with the CDRH3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH3, which comprises a peptide having at least 90% identity with the CDRH3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH3, which comprises a peptide having at least 95% identity with the CDRH3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRH3, which comprises a peptide having 100% identity with the CDRH3 peptide provided in Table 3.

[0059] In some embodiments, the anti-KIR reagent provided herein comprises a light chain complementarity-determining region 1 (CDRL1), which comprises the CDRL1 peptide provided in Table 3 or a peptide having at least 80% identity with the CDRL1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL1, which comprises a peptide having at least 85% identity with the CDRL1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL1, which comprises a peptide having at least 90% identity with the CDRL1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL1, which comprises a peptide having at least 95% identity with the CDRL1 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL1, which comprises a peptide having 100% identity with the CDRL1 peptide provided in Table 3.

[0060] In some embodiments, the anti-KIR reagent provided herein comprises a light chain complementarity-determining region 2 (CDRL2), which comprises the CDRL2 peptide provided in Table 3 or a peptide having at least 80% identity with the CDRL2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL2, which comprises a peptide having at least 85% identity with the CDRL2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL2, which comprises a peptide having at least 90% identity with the CDRL2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL2, which comprises a peptide having at least 95% identity with the CDRL2 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL2, which comprises a peptide having 100% identity with the CDRL2 peptide provided in Table 3.

[0061] In some embodiments, the anti-KIR reagent provided herein comprises a light chain complementarity-determining region 3 (CDRL3), which comprises the CDRL3 peptide provided in Table 3 or a peptide having at least 80% identity with the CDRL3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL3, which comprises a peptide having at least 85% identity with the CDRL3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL3, which comprises a peptide having at least 90% identity with the CDRL3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL3, which comprises a peptide having at least 95% identity with the CDRL3 peptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises CDRL3, which comprises a peptide having 100% identity with the CDRL3 peptide provided in Table 3.

[0062] In some embodiments, the anti-KIR reagent provided herein binds to KIR2DS1 or a portion thereof, which may be referred to herein as an anti-KIR2DS1 reagent. In some embodiments, the anti-KIR2DS1 reagent comprises: CDRH1 comprising the peptides provided in Table 3, CDRH2 comprising the peptides provided in Table 3, CDRH3 comprising the peptides provided in Table 3, CDRL1 comprising the peptides provided in Table 3, CDRL2 comprising the peptides provided in Table 3, and CDRL3 comprising the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR2DS1 reagent comprises CDRH1 comprising the peptide GYX1FTX2X3W, wherein X1 is S or T, X2 is T or R, and X3 is Y or N. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or T). In some embodiments, amino acid X1 may be substituted with S, T, C, N, A, or G. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or R). In some embodiments, amino acid X2 may be substituted with T, R, S, C, N, A, K, or H. Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for Y and / or N). In some embodiments, amino acid X3 may be substituted with Y, N, F, W, S, T, H, Q, or D. In some embodiments, amino acid X3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, the anti-KIR2DS1 reagent comprises CDRL2, which comprises a polypeptide X1TS, wherein X1 is Y or R. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for Y and / or R). In some embodiments, amino acid X1 may be substituted with Y, R, F, W, H, S, T, K, H, or Q. In some embodiments, the anti-KIR2DS1 reagent comprises CDRL3, which comprises a polypeptide QQGX1X2X3PX4T, wherein X1 is K or S, X2 is T or S, X3 is L or I, and X4 is Y or L. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for K and / or S). In some embodiments, amino acid X1 may be substituted with K, S, R, H, T, C, N, or G. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or S).In some embodiments, amino acid X2 may be substituted with T, S, C, N, A, or G. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for L and / or I). In some embodiments, amino acid X3 may be substituted with L, I, V, M, A, or F. In some embodiments, amino acid X3 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for Y and / or L). In some embodiments, amino acid X4 may be substituted with Y, L, F, W, H, S, T, I, V, M, or A.

[0063] In some embodiments, the anti-KIR reagent provided herein binds to KIR2DS4 or a portion thereof, which may be referred to herein as an anti-KIR2DS4 reagent. In some embodiments, the anti-KIR2DS4 reagent comprises: CDRH1 comprising the peptides provided in Table 3, CDRH2 comprising the peptides provided in Table 3, CDRH3 comprising the peptides provided in Table 3, CDRL1 comprising the peptides provided in Table 3, CDRL2 comprising the peptides provided in Table 3, and CDRL3 comprising the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR2DS4 reagent comprises CDRH1 comprising the peptide GYSFX1GYX2, wherein X1 is T or S, and X2 is T or Y. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for T and / or S). In some embodiments, amino acid X1 may be substituted with T, S, C, N, A, or G. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or Y). In some embodiments, amino acid X2 may be substituted with T, S, C, N, A, F, W, or H. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, the anti-KIR2DS4 reagent comprises CDRH2, which comprises the polypeptide IHPYNGX1T, wherein X1 is D or A. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for D and / or A). In some embodiments, amino acid X1 may be substituted with D, A, E, N, S, G, V, or L. In some embodiments, the anti-KIR2DS4 reagent comprises CDRH2, which contains the polypeptide ISYSX1X2T, wherein X1 is G or A, and X2 is S or T. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for G and / or A). In some embodiments, amino acid X1 may be substituted with G, A, V, S, L, P, or C. In some embodiments, amino acid X1 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for S and / or T). In some embodiments, amino acid X2 may be substituted with T, S, C, N, A, or G. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).In some embodiments, the anti-KIR2DS4 reagent comprises CDRH3, which contains the peptide ARX1X2GSX3X4X5AWFAY, wherein X1 is Q or R, X2 is Y or A, X3 is amino acid-free or S, X4 is amino acid-free or Y, and X5 is L or K. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 can be substituted with a conserved amino acid (i.e., conserved for Q and / or R). In some embodiments, amino acid X1 can be substituted with Q, R, N, E, H, S, T, or K. Amino acid X2 can be substituted with any amino acid. In some embodiments, amino acid X2 can be substituted with a conserved amino acid (i.e., conserved for Y and / or A). In some embodiments, amino acid X2 can be substituted with Y, A, F, W, H, S, T, G, V, or L. Amino acid X3 can be substituted with any amino acid. In some embodiments, amino acid X3 can be substituted with a conserved amino acid (i.e., conserved for S). In some embodiments, amino acid X3 can be substituted with S, T, C, N, or G. In some embodiments, amino acid X3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for Y). In some embodiments, amino acid X4 may be substituted with Y, F, W, H, S, or T. In some embodiments, amino acid X4 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, amino acid X4 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X5 may be substituted with any amino acid. In some embodiments, amino acid X5 may be substituted with a conserved amino acid (i.e., conserved for L and / or K). In some embodiments, amino acid X5 may be substituted with L, K, R, H, I, V, M, A, or F. In some embodiments, the anti-KIR2DS4 reagent comprises CDRH3, which contains the polypeptide X1X2SKYX3NSYAX4DY, wherein X1 is T or A, X2 is R or Q, X3 is A or S, and X4 is V or M. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for T and / or A). In some embodiments, amino acid X1 may be substituted with T, A, S, C, N, G, V, or L. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for R and / or Q). In some embodiments, amino acid X2 may be substituted with R, Q, K, H, N, E, S, or T. Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for A and / or S). In some embodiments, amino acid X3 may be substituted with A, S, G, V, L, T, C, or N.Amino acid X4 can be substituted with any amino acid. In some embodiments, amino acid X4 can be substituted with a conserved amino acid (i.e., conserved for V and / or M). In some embodiments, amino acid X4 can be substituted with V, M, I, L, A, or F. In some embodiments, amino acid X4 can be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the anti-KIR2DS4 reagent comprises CDRL2, which comprises a polypeptide GX1S, wherein X1 is A or S. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 can be substituted with a conserved amino acid (i.e., conserved for A and / or S). In some embodiments, amino acid X1 can be substituted with A, S, G, V, L, T, C, or N. In some embodiments, the anti-KIR2DS4 reagent comprises CDRL3, which comprises a polypeptide QNDHX1YPPT, wherein X1 is S or I. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., one that is conserved for S and / or I). In some embodiments, amino acid X1 may be substituted with S, I, T, C, N, G, L, V, M, A, or F.

[0064] In some embodiments, the anti-KIR reagent provided herein binds to KIR3DL1 or a portion thereof, which may be referred to herein as the anti-KIR3DL1 reagent. In some embodiments, the anti-KIR3DL1 reagent comprises: CDRH1 comprising the peptides provided in Table 3, CDRH2 comprising the peptides provided in Table 3, CDRH3 comprising the peptides provided in Table 3, CDRL1 comprising the peptides provided in Table 3, CDRL2 comprising the peptides provided in Table 3, and CDRL3 comprising the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR3DL1 reagent comprises CDRH1 comprising the peptide GYX1FX2X3YX4, wherein X1 is S or T, X2 is T or S, X3 is D or T or N, and X4 is T, Y, S, A, or W. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or T). In some embodiments, amino acid X1 may be substituted with S, T, C, N, A, or G. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or S). In some embodiments, amino acid X2 may be substituted with S, T, C, N, A, or G. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for D and / or T and / or N). In some embodiments, amino acid X3 may be substituted with D, T, N, E, S, C, A, Q, or H. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for T and / or Y and / or S and / or A and / or W). In some embodiments, amino acid X4 may be substituted with S, T, C, N, A, G, F, W, H, V, L, Y, or M. In some embodiments, the anti-KIR3DL1 reagent comprises CDRH2, which contains the polypeptide INPX1X2GX3T, wherein X1 is S or Y, X2 is S or N, and X3 is Y or G. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or Y). In some embodiments, amino acid X1 may be substituted with S, Y, T, C, N, G, F, W, or H. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X1 may be substituted with any amino acid.In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or N). In some embodiments, amino acid X1 may be substituted with S, N, T, C, N, G, Q, D, or H. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for Y and / or G). In some embodiments, amino acid X1 may be substituted with Y, G, A, S, P, C, F, W, H, or T. In some embodiments, the anti-KIR3DL1 reagent comprises CDRH2, which comprises the polypeptide INTX1X2GX3X4, wherein X1 is E or Y, X2 is T or Y, X3 is E or D, and X4 is P or A. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for E and / or Y). In some embodiments, amino acid X1 may be substituted with E, Y, F, W, H, S, T, D, Q, or N. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or Y). In some embodiments, amino acid X2 may be substituted with T, Y, F, W, H, S, C, N, or A. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for E and / or D). In some embodiments, amino acid X3 may be substituted with E, D, N, S, or Q. In some embodiments, amino acid X3 may be substituted with a negatively charged amino acid (e.g., E or D). Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for P and / or A). In some embodiments, amino acid X4 may be substituted with P, A, G, L, V, or S. In some embodiments, amino acid X4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the anti-KIR3DL1 reagent comprises CDRL1, which comprises the polypeptide ENIYSX1, wherein X1 is N or Y. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for N and / or Y). In some embodiments, amino acid X1 may be substituted with N, Y, F, W, H, S, T, Q, or D. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).In some embodiments, the anti-KIR3DL1 reagent comprises CDRL1, which comprises the polypeptide QSLLX1SX2NQKNY, wherein X1 is F or N, and X2 is S or G. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for F and / or N). In some embodiments, amino acid X1 may be substituted with F, N, Q, S, T, D, H, Y, W, L, M, I, or V. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for S and / or G). In some embodiments, amino acid X2 may be substituted with S, G, T, C, N, A, or P. In some embodiments, the anti-KIR3DL1 reagent comprises CDRL2, which comprises the polypeptide AAX1, wherein X1 is R or S. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for R and / or S). In some embodiments, amino acid X1 may be substituted with R, S, T, C, N, G, K, H, or Q. In some embodiments, the anti-KIR3DL1 reagent comprises CDRL2, which comprises a polypeptide X1AS, wherein X1 is W or G. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for W and / or G). In some embodiments, amino acid X1 may be substituted with W, G, A, S, P, C, F, Y, H, L, or M. In some embodiments, amino acid X1 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the anti-KIR3DL1 reagent comprises CDRL3, which comprises a polypeptide QHX1X1GTPLT, wherein X1 is F or H, and X2 is W or Y. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for F and / or H). In some embodiments, amino acid X1 may be substituted with F, H, Y, W, L, M, I, V, K, R, N, or Q. In some embodiments, amino acid X1 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for W and / or Y). In some embodiments, amino acid X2 may be substituted with W, Y, F, H, L, M, S, or T. In some embodiments, amino acid X2 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). In some embodiments, the anti-KIR3DL1 reagent comprises CDRL3, which comprises a polypeptide X1QSX2X3X4PYT, wherein X1 is S or Q, X2 is T or N, X3 is H or E, and X4 is V or D. Amino acid X1 may be substituted with any amino acid.In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or Q). In some embodiments, amino acid X1 may be substituted with S, Q, T, C, N, G, E, H, or K. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or N). In some embodiments, amino acid X2 may be substituted with T, N, S, C, A, Q, D, or H. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for H and / or E). In some embodiments, amino acid X3 may be substituted with H, E, D, Q, N, S, K, R, Y, or W. Amino acid X4 can be substituted with any amino acid. In some embodiments, amino acid X4 can be substituted with a conserved amino acid (i.e., conserved for V and / or D). In some embodiments, amino acid X4 can be substituted with V, D, I, L, A, M, E, N, or S.

[0065] In some embodiments, the anti-KIR reagent provided herein binds to KIR3DL3 or a portion thereof, which may be referred to herein as an anti-KIR3DL3 reagent. In some embodiments, the anti-KIR3DL3 reagent comprises: CDRH1 comprising the peptides provided in Table 3, CDRH2 comprising the peptides provided in Table 3, CDRH3 comprising the peptides provided in Table 3, CDRL1 comprising the peptides provided in Table 3, CDRL2 comprising the peptides provided in Table 3, and CDRL3 comprising the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR3DL3 reagent comprises CDRH2 comprising the peptide IHYSGX1T, wherein X1 is T or S. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for T and / or S). In some embodiments, amino acid X1 may be substituted with T, S, C, N, A, or G. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, the anti-KIR3DL3 reagent comprises CDRL3, which contains the polypeptide QQWSSX1PLT, wherein X1 is D or N. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for D and / or N). In some embodiments, amino acid X1 may be substituted with D, N, E, S, Q, T, or H.

[0066] In some embodiments, the anti-KIR reagent provided herein combines KIR2DL2 or a portion thereof with KIR2DS2 or a portion thereof, and may be referred to herein as an anti-KIR2DL2 / KIR2DS2 reagent. In some embodiments, the anti-KIR2DL2 / KIR2DS2 reagent comprises: CDRH1 comprising the peptides provided in Table 3, CDRH2 comprising the peptides provided in Table 3, CDRH3 comprising the peptides provided in Table 3, CDRL1 comprising the peptides provided in Table 3, CDRL2 comprising the peptides provided in Table 3, and CDRL3 comprising the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR2DL2 / KIR2DS2 reagent comprises CDRH1, which comprises the peptide GX1SX2TSX3X4, wherein X1 is D or F, X2 is I or L, X3 is G or Y, and X4 is F or G. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for D and / or F). In some embodiments, amino acid X1 may be substituted with D, F, Y, W, L, M, I, V, E, N, or S. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for I and / or L). In some embodiments, amino acid X2 may be substituted with I, L, V, M, A, or F. In some embodiments, amino acid X2 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for G and / or Y). In some embodiments, amino acid X3 may be substituted with G, Y, F, W, H, S, T, A, P, or C. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for F and / or G). In some embodiments, amino acid X4 may be substituted with F, G, A, S, P, C, Y, W, L, M, I, or V. In some embodiments, amino acid X4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the anti-KIR2DL2 / KIR2DS2 reagent comprises CDRH2, which comprises a polypeptide IX1X2X3GST, wherein X1 is S or W, X2 is Y or T, and X3 is S or G. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or W). In some embodiments, amino acid X1 may be substituted with S, W, F, Y, H, L, M, T, C, N, or G. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for Y and / or T).In some embodiments, amino acid X2 may be substituted with Y, T, S, C, N, A, F, W, or H. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for S and / or G). In some embodiments, amino acid X3 may be substituted with S, G, T, C, N, A, or P. In some embodiments, the anti-KIR2DL2 / KIR2DS2 reagent comprises CDRL1, which comprises the polypeptide QSLLNSX1X2X3KNY, wherein X1 is F or R, X2 is N or T, and X3 is Q or R. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for F and / or R). In some embodiments, amino acid X1 may be substituted with F, R, Y, W, L, M, I, V, K, H, Q, or N. Amino acid X2 can be substituted with any amino acid. In some embodiments, amino acid X2 can be substituted with a conserved amino acid (i.e., conserved for N and / or T). In some embodiments, amino acid X2 can be substituted with N, T, S, C, A, Q, D, or H. In some embodiments, amino acid X2 can be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 can be substituted with any amino acid. In some embodiments, amino acid X3 can be substituted with a conserved amino acid (i.e., conserved for Q and / or R). In some embodiments, amino acid X3 can be substituted with Q, R, N, E, H, S, T, or K. In some embodiments, the anti-KIR2DL2 / KIR2DS2 reagent comprises CDRL2, which comprises a polypeptide X1AS, wherein X1 is F or W. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 can be substituted with a conserved amino acid (i.e., conserved for F and / or W). In some embodiments, amino acid X1 can be substituted with F, W, Y, H, L, M, or V. In some embodiments, amino acid X1 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, amino acid X1 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). In some embodiments, the anti-KIR2DL2 / KIR2DS2 reagent comprises CDRL3, which comprises a polypeptide X1QX2YNX3PX4T, wherein X1 is Q or K, X2 is Y or S, X3 is T or L, and X4 is V or W. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for Q and / or K). In some embodiments, amino acid X1 may be substituted with Q, K, N, E, H, S, T, or R. Amino acid X2 may be substituted with any amino acid.In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for Y and / or S). In some embodiments, amino acid X2 may be substituted with Y, S, T, C, N, G, F, W, or H. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for T and / or L). In some embodiments, amino acid X3 may be substituted with T, L, S, C, N, A, I, V, M, or F. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for V and / or W). In some embodiments, amino acid X4 may be substituted with V, W, I, L, A, M, F, Y, or H. In some embodiments, amino acid X4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).

[0067] In some embodiments, the anti-KIR reagent provided herein binds to KIR2DL3 or a portion thereof, KIR2DL2 or a portion thereof, and KIR2DS2 or a portion thereof, which may be referred to herein as an anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises: CDRH1 comprising the peptides provided in Table 3, CDRH2 comprising the peptides provided in Table 3, CDRH3 comprising the peptides provided in Table 3, CDRL1 comprising the peptides provided in Table 3, CDRL2 comprising the peptides provided in Table 3, and CDRL3 comprising the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH1, which comprises the peptide GX1TFX2X3YX4, wherein X1 is Y or F, X2 is T, K, or N, X3 is D or N, and X4 is A or D. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for Y and / or F). In some embodiments, amino acid X1 may be substituted with Y, F, W, H, S, T, L, M, I, or V. In some embodiments, amino acid X1 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or K and / or N). In some embodiments, amino acid X2 may be substituted with T, K, S, C, N, A, R, and H. Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for D and / or N). In some embodiments, amino acid X3 may be substituted with D, N, E, S, Q, T, or H. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for A and / or D). In some embodiments, amino acid X4 may be substituted with A, D, G, V, S, L, E, or N. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH1, which comprises the polypeptide GFTFX1X2X3X4, wherein X1 is N or K, X2 is T or N, X3 is N or Y, and X4 is A or D. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for N and / or K). In some embodiments, amino acid X1 may be substituted with N, K, Q, S, T, D, H, or R. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for T and / or N).In some embodiments, amino acid X2 may be substituted with T, N, S, C, A, Q, D, or H. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for N and / or Y). In some embodiments, amino acid X3 may be substituted with N, Y, Q, S, T, D, H, F, or W. In some embodiments, amino acid X3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for A and / or D). In some embodiments, amino acid X4 may be substituted with A, D, G, V, S, L, E, or N. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH1, which contains the polypeptide GX1TFX2X3X4A, wherein X1 is F or Y, X2 is N or T, X3 is T or D, and X4 is N or Y. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for F and / or Y). In some embodiments, amino acid X1 may be substituted with F, Y, W, H, S, T, L, M, I, or V. In some embodiments, amino acid X1 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for N and / or T). In some embodiments, amino acid X2 may be substituted with N, T, Q, S, D, H, C, or A. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for T and / or D). In some embodiments, amino acid X3 may be substituted with T, D, S, C, N, A, or E. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for N and / or Y). In some embodiments, amino acid X4 may be substituted with N, Y, F, W, H, S, T, Q, or D. In some embodiments, amino acid X4 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH1, which comprises the polypeptide GYX1FTDYX2, wherein X1 is S or T, and X2 is N or A. Amino acid X1 can be replaced by any amino acid.In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or T). In some embodiments, amino acid X1 may be substituted with S, T, C, N, A, or G. In some embodiments, amino acid X1 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for N and / or A). In some embodiments, amino acid X2 may be substituted with N, A, G, V, S, L, Q, T, D, or H. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH2, which comprises a polypeptide IX1X2X3NGX4T, wherein X1 is D or S, X2 is P or T, X3 is D or Y, and X4 is G or N. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for D and / or S). In some embodiments, amino acid X1 may be substituted with D, S, E, N, T, C, or G. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for P and / or T). In some embodiments, amino acid X2 may be substituted with P, T, S, C, N, A, G, L, or V. Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for D and / or Y). In some embodiments, amino acid X3 may be substituted with D, Y, E, N, S, F, W, H, or T. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for G and / or N). In some embodiments, amino acid X4 may be substituted with G, N, A, S, P, C, Q, T, D, or H. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH2, which contains a polypeptide IX1YDGX2X3X4, wherein X1 is S or T, X2 is S or Y, X3 is R or I, and X4 is I or T. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 can be substituted with a conserved amino acid (i.e., conserved for S and / or T). In some embodiments, amino acid X1 can be substituted with S, T, C, N, A, or G. In some embodiments, amino acid X1 can be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X2 can be substituted with any amino acid. In some embodiments, amino acid X2 can be substituted with a conserved amino acid (i.e., conserved for S and / or Y). In some embodiments, amino acid X2 can be substituted with S, Y, T, C, N, G, F, W, or H.In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for R and / or I). In some embodiments, amino acid X3 may be substituted with R, I, K, H, Q, N, L, V, M, A, or F. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for I and / or T). In some embodiments, amino acid X4 may be substituted with I, T, L, V, M, A, F, S, C, or N. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRH3, which contains the polypeptide ARX1X2X3X4YX5X6WFAY, wherein X1 is G or L, X2 is D or G, X3 is Y or N, X4 is G or N, X5 is L or G, and X6 is R or G. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 can be substituted with a conserved amino acid (i.e., conserved for G and / or L). In some embodiments, amino acid X1 can be substituted with G, L, I, V, M, A, F, S, P, or C. In some embodiments, amino acid X1 can be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). Amino acid X2 can be substituted with any amino acid. In some embodiments, amino acid X2 can be substituted with a conserved amino acid (i.e., conserved for D and / or G). In some embodiments, amino acid X2 may be substituted with D, G, A, S, P, C, E, or N. Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for Y and / or N). In some embodiments, amino acid X3 may be substituted with Y, N, F, W, H, S, T, Q, or D. In some embodiments, amino acid X3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for G and / or N). In some embodiments, amino acid X4 may be substituted with G, N, Q, S, T, D, H, A, P, or C. In some embodiments, amino acid X4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). Amino acid X5 may be substituted with any amino acid. In some embodiments, amino acid X5 may be substituted with a conserved amino acid (i.e., conserved for L and / or G). In some embodiments, amino acid X5 may be substituted with L, G, A, S, P, C, I, V, M, or F. In some embodiments, amino acid X5 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). Amino acid X6 may be substituted with any amino acid.In some embodiments, amino acid X6 may be substituted with a conserved amino acid (i.e., conserved for R and / or G). In some embodiments, amino acid X6 may be substituted with R, G, K, H, Q, N, A, S, P, or C. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRL1, which comprises a polypeptide QX1INX1Y, wherein X1 is D or N, and X2 is S or K. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for D and / or N). In some embodiments, amino acid X1 may be substituted with D, N, Q, S, T, H, or E. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for S and / or K). In some embodiments, amino acid X2 may be substituted with S, K, T, C, N, G, R, or H. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRL2, which contains the polypeptide KX1S, wherein X1 is V or E. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for V and / or E). In some embodiments, amino acid X1 may be substituted with V, E, I, L, A, M, D, Q, N, or S. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRL2, which contains the polypeptide X1AN, wherein X1 is R or N. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for R and / or N). In some embodiments, amino acid X1 may be substituted with R, N, Q, S, T, D, H, or K. In some embodiments, the anti-KIR2DL3 / KIR2DL2 / KIR2DS2 reagent comprises CDRL3, which comprises a polypeptide X1QX2X3EX4PYT, wherein X1 is Q or L, X2 is N or Y, X3 is N or D, and X4 is D or F. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for Q and / or L). In some embodiments, amino acid X1 may be substituted with Q, L, I, V, M, A, F, N, E, H, S, T, or K. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for N and / or Y). In some embodiments, amino acid X2 may be substituted with N, Y, F, W, H, S, T, Q, or D. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X3 can be replaced by any amino acid.In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for N and / or D). In some embodiments, amino acid X3 may be substituted with N, D, Q, S, T, H, or E. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for D and / or F). In some embodiments, amino acid X4 may be substituted with D, F, E, N, S, Y, W, L, M, I, or V.

[0068] In some embodiments, the anti-KIR reagents provided herein are used in combination with KIR2DL1 or a portion thereof, KIR2DL2 or a portion thereof, KIR2DL3 or a portion thereof, KIR2DS1 or a portion thereof, and KIR2DS2 or a portion thereof, which may be referred to herein as anti-KIR2DL1 / KIR2DL2 / KIR2DL3 / KIR2DS1 / KIR2DS2 reagents. In some embodiments, the anti-KIR2DL1 / KIR2DL2 / KIR2DL3 / KIR2DS1 / KIR2DS2 reagent comprises: CDRH1 containing the peptides provided in Table 3, CDRH2 containing the peptides provided in Table 3, CDRH3 containing the peptides provided in Table 3, CDRL1 containing the peptides provided in Table 3, CDRL2 containing the peptides provided in Table 3, and CDRL3 containing the peptides provided in Table 3, or one or more variations thereof. For example, in some embodiments, the anti-KIR2DL1 / KIR2DL2 / KIR2DL3 / KIR2DS1 / KIR2DS2 reagent comprises CDRH3, which comprises the polypeptide ARX1X2X3X4X5YYGSSX6X7YX8MDY, wherein X1 is AA-free or R, X2 is AA-free or F, X3 is S or R, X4 is S or P, X5 is L or N, X6 is Y or F, X7 is D or AA-free, and X8 is V or A. Amino acid X1 can be substituted with any amino acid. In some embodiments, amino acid X1 can be substituted with a conserved amino acid (i.e., conserved for R). In some embodiments, amino acid X1 can be substituted with R, K, H, Q, N. In some embodiments, amino acid X1 can be substituted with a positively charged amino acid (e.g., K, R, H). Amino acid X2 can be substituted with any amino acid. In some embodiments, amino acid X2 can be substituted with a conserved amino acid (i.e., conserved for F). In some embodiments, amino acid X2 may be substituted with F, Y, W, L, M, I, or V. In some embodiments, amino acid X2 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, amino acid X2 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for S and / or R). In some embodiments, amino acid X3 may be substituted with S, R, T, C, N, G, K, H, or Q. Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for S and / or P). In some embodiments, amino acid X4 may be substituted with S, P, T, C, N, G, A, L, or V. Amino acid X5 may be substituted with any amino acid. In some embodiments, amino acid X5 may be substituted with a conserved amino acid (i.e., conserved for L and / or N).In some embodiments, amino acid X5 may be substituted with L, N, I, V, M, A, F, Q, S, T, D, or H. Amino acid X6 may be substituted with any amino acid. In some embodiments, amino acid X6 may be substituted with a conserved amino acid (i.e., conserved for Y and / or F). In some embodiments, amino acid X6 may be substituted with Y, F, W, H, S, T, L, M, I, or V. In some embodiments, amino acid X6 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X7 may be substituted with any amino acid. In some embodiments, amino acid X7 may be substituted with a conserved amino acid (i.e., conserved for D). In some embodiments, amino acid X7 may be substituted with D, E, N, or S. In some embodiments, amino acid X7 may be substituted with a negatively charged amino acid (e.g., D, E). Amino acid X8 may be substituted with any amino acid. In some embodiments, amino acid X8 may be substituted with a conserved amino acid (i.e., conserved for V and / or A). In some embodiments, amino acid X8 may be substituted with V, A, G, S, L, I, or M. In some embodiments, amino acid X8 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the anti-KIR2DL1 / KIR2DL2 / KIR2DL3 / KIR2DS1 / KIR2DS2 reagent comprises CDRL1, which comprises a polypeptide QX1VSX2D, wherein X1 is S or D, and X2 is N or T. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for S and / or D). In some embodiments, amino acid X1 may be substituted with S, D, T, C, N, G, or E. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for N and / or T). In some embodiments, amino acid X2 may be substituted with N, T, Q, S, D, H, C, or A. In some embodiments, amino acid X2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, the anti-KIR2DL1 / KIR2DL2 / KIR2DL3 / KIR2DS1 / KIR2DS2 reagent comprises CDRL2, which contains a polypeptide X1AS, wherein X1 is Y or W. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for Y and / or W). In some embodiments, amino acid X1 may be substituted with Y, W, F, H, S, T, L, or M. In some embodiments, amino acid X1 may be substituted with an aromatic amino acid (e.g., F, Y, W, H).In some embodiments, the anti-KIR2DL1 / KIR2DL2 / KIR2DL3 / KIR2DS1 / KIR2DS2 reagent comprises CDRL3, which contains the polypeptide QQX1X2SX3PX4T, wherein X1 is D or H, X2 is Y or F, X3 is S or T, and X4 is P or W. Amino acid X1 may be substituted with any amino acid. In some embodiments, amino acid X1 may be substituted with a conserved amino acid (i.e., conserved for D and / or H). In some embodiments, amino acid X1 may be substituted with D, H, K, R, N, Q, Y, W, E, or S. Amino acid X2 may be substituted with any amino acid. In some embodiments, amino acid X2 may be substituted with a conserved amino acid (i.e., conserved for Y and / or F). In some embodiments, amino acid X2 may be substituted with Y, F, W, H, S, T, L, M, I, or V. In some embodiments, amino acid X2 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). Amino acid X3 may be substituted with any amino acid. In some embodiments, amino acid X3 may be substituted with a conserved amino acid (i.e., conserved for S and / or T). In some embodiments, amino acid X3 may be substituted with S, T, C, N, A, or G. In some embodiments, amino acid X3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). Amino acid X4 may be substituted with any amino acid. In some embodiments, amino acid X4 may be substituted with a conserved amino acid (i.e., conserved for P and / or W). In some embodiments, amino acid X4 may be substituted with P, W, F, Y, H, L, M, A, G, or V. In some embodiments, amino acid X4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).

[0069] In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin heavy chain variable domain, which includes an immunoglobulin heavy chain variable domain polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the immunoglobulin heavy chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin heavy chain variable domain, which includes an immunoglobulin heavy chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin heavy chain variable domain, which includes a polypeptide having at least 90% identity with the immunoglobulin heavy chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin heavy chain variable domain, which includes a polypeptide having at least 95% identity with the immunoglobulin heavy chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin heavy chain variable domain, which includes a polypeptide having 100% identity with the immunoglobulin heavy chain variable domain polypeptide provided in Table 3.

[0070] In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin light chain variable domain, which includes an immunoglobulin light chain variable domain polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin light chain variable domain, which includes a polypeptide having at least 85% identity with the immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin light chain variable domain, which includes a polypeptide having at least 90% identity with the immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin light chain variable domain, which includes a polypeptide having at least 95% identity with the immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR reagent provided herein comprises an immunoglobulin light chain variable domain, which includes a polypeptide having at least 100% identity with the immunoglobulin light chain variable domain polypeptide provided in Table 3.

[0071] Percentage amino acid sequence identity relative to the reference VH, VL, and CDR peptide sequences in this paper generally refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference peptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve maximum percentage sequence identity, without considering any conserved substitutions as part of sequence identity. Alignments used for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

[0072] CDR Group In some embodiments, the anti-KIR reagent comprises an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, the anti-KIR reagent herein comprises two immunoglobulin heavy chain variable domains, each comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains, each comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). The CDR set may comprise any combination of the CDR amino acid sequences provided herein (i.e., CDRH1, CDRH2, CDRH3; and CDRL1, CDRL2, CDRL3). In some embodiments, the immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, and CDRH3 amino acid sequences, and the immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences selected from the groups provided in Table 3 herein. For an anti-KIR reagent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2, and CDRH3 amino acid sequences, and each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2, and CDRL3 amino acid sequences selected from the groups provided in Table 3.

[0073] In some implementations, all CDRs come from the same group. For example, for an anti-KIR reagent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may contain a set of CDRH1, CDRH2, and CDRH3 amino acid sequences from a single clone in Table 3, and each immunoglobulin light chain variable domain may contain a set of CDRL1, CDRL2, and CDRL3 amino acid sequences from the same clone in Table 3.

[0074] In some implementations, the CDRs come from different groups. For example, for an anti-KIR reagent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may contain a set of CDRH1, CDRH2, and CDRH3 amino acid sequences from the first clone in Table 3, and each immunoglobulin light chain variable domain may contain a set of CDRL1, CDRL2, and CDRL3 amino acid sequences from the second clone in Table 3. In another example, for an anti-KIR reagent comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, one immunoglobulin heavy chain variable domain may contain a set of CDRH1, CDRH2, and CDRH3 amino acid sequences from the first clone in Table 3, and the other immunoglobulin heavy chain variable domain may contain a set of CDRH1, CDRH2, and CDRH3 amino acid sequences from the second clone in Table 3; and one immunoglobulin light chain variable domain may contain a set of CDRL1, CDRL2, and CDRL3 amino acid sequences from the first clone in Table 3, and the other immunoglobulin light chain variable domain may contain a set of CDRL1, CDRL2, and CDRL3 amino acid sequences from the second clone in Table 3.

[0075] Competing reagents This document provides anti-KIR reagents that competitively bind to or are capable of competitively binding to one or more anti-KIR reagents described herein. Specifically, this document provides anti-KIR reagents that competitively bind to KIRs (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and / or KIR3DL3) with or are capable of competitively binding to KIRs (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and / or KIR3DL3) with or are capable of competitively binding to one or more anti-KIR reagents described herein. A reagent that competes with or is capable of competing with the anti-KIR reagent described herein may be referred to as a competing reagent. In some cases, a reagent (i.e., a competing reagent) may be considered to competitively bind a KIR when it binds to substantially the same KIR region as the anti-KIR reagent described herein. In some cases, a reagent (i.e., a competing reagent) may be considered to competitively bind a KIR when it binds to the exact same KIR region as the anti-KIR reagent described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)). In some cases, a reagent (i.e., a competing reagent) may be considered capable of competitively binding a KIR when, under suitable assay conditions, it binds to substantially the same KIR region as the anti-KIR reagent described herein. In some cases, a reagent (i.e., a competing reagent) may be considered capable of competitively binding a KIR when, under suitable assay conditions, it binds to the exact same KIR region as the anti-KIR reagent described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)).

[0076] In certain circumstances, a reagent (i.e., a competing reagent) may be considered to competitively bind to a KIR (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3) when a competing agent blocks the binding of one or more anti-KIR reagents described herein to a KIR under suitable assay conditions. Whether a competing agent blocks the binding of one or more anti-KIR reagents described herein to a KIR can be determined using a suitable competition assay or blocking assay, such as the blocking assay described in Example 4 herein. The competing reagent can block the binding of one or more anti-KIR reagents described herein to KIR by 50% or more in a competitive or blocking assay, and conversely, one or more anti-KIR reagents described herein can block the binding of a competing reagent to KIR by about 50% or more in a competitive or blocking assay. For example, the reagent (i.e., the competing reagent) can block the binding of one or more anti-KIR reagents described herein to KIR by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a competitive or blocking assay, and conversely, one or more anti-KIR reagents described herein can block the binding of a competing reagent to KIR by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a competitive or blocking assay.

[0077] In certain circumstances, a reagent (i.e., a competing reagent) may be considered to competitively bind to a KIR (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3) when the competing agent binds to a KIR with an affinity similar to that of one or more anti-KIR reagents described herein. In certain circumstances, a reagent (i.e., a competing reagent) may be considered capable of competitively binding to a KIR when, under suitable assay conditions, the competing agent binds to a KIR with an affinity similar to that of one or more anti-KIR reagents described herein. In some embodiments, a reagent (i.e., a competing reagent) is considered to competitively bind to a KIR when the competing agent binds to a KIR with an affinity of at least about 50% of that of one or more anti-KIR reagents described herein. For example, a reagent (i.e., a competing reagent) may be considered to competitively bind to a KIR when the competing agent binds to a KIR with an affinity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-KIR reagents described herein. A competing reagent may contain any of the characteristics described herein for anti-KIR reagents.

[0078] This document also provides anti-KIR reagents that bind to or are capable of binding to the same epitopes as one or more anti-KIR reagents described herein. Specifically, this document provides anti-KIR reagents that compete with one or more anti-KIR reagents described herein for binding to the same epitopes on a KIR (e.g., epitopes on KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3). Such reagents that bind to the same epitopes may be referred to as epitope competitors. In some cases, epitope competitors may bind to the exact same KIR region as the anti-KIR reagents described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)). In some cases, epitope competitors block the binding of one or more anti-KIR reagents described herein to a KIR. Epitope competitive agents can block the binding of one or more anti-KIR reagents described herein to KIR by about 50% or more in a competitive assay, and conversely, one or more anti-KIR reagents described herein can block the binding of epitope competitive agents to KIR by 50% or more in a competitive assay. In some cases, epitope competitive agents bind to KIR with an affinity similar to that of one or more anti-KIR reagents described herein. In some embodiments, epitope competitive agents bind to KIR with an affinity of at least about 50% of the affinity of one or more anti-KIR reagents described herein. For example, epitope competitive agents can bind to KIR with an affinity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-KIR reagents described herein. Epitope competitive agents may contain any of the features described herein for anti-KIR reagents.

[0079] Antibody In some embodiments, the anti-KIR agent is a KIR-binding antibody or an antigen-binding fragment thereof (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and / or KIR3DL3 binding antibodies or antigen-binding fragments thereof). Antibodies as described herein typically comprise one or more polypeptides encoded substantially by immunoglobulin genes or segments of immunoglobulin genes. Immunoglobulin genes typically include κ, λ, α, γ, δ, ε and μ constant region genes and immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. The antibodies described in this article can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, or IgE.

[0080] Antibodies can be monoclonal. Monoclonal antibodies generally refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical (e.g., assessed at the level of the Ig heavy chain and / or light chain amino acid sequence) and / or bind to the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or appearing during the generation of the monoclonal antibody population) (such variants are typically present in smaller quantities). In contrast to polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier monoclonal indicates the antibody characteristic of being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be generated by any particular method. For example, monoclonal antibodies used according to this technology can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0081] Antibody structural units can exist in tetramer form. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one light chain (approximately 25 kD) and one heavy chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0082] Antibodies typically include one or more variable regions or variable domains, which are portions of the amino acid sequence in the antibody light and heavy chains that include complementarity-determining regions (CDRs, such as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3) and framework regions (FRs). The variable domains of the heavy and light chains are typically named VH and VL, respectively. Variable domains are included in antigen-binding fragments such as Fab, F(ab')2, Fv, and scFv and participate in specific antigen recognition. Complementarity-determining regions (CDRs) are three hypervariable regions in each chain that interrupt the four framework regions established by the variable regions of the light and heavy chains. CDRs are primarily responsible for binding epitopes of the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (sequentially numbered starting from the N-terminus) and are also usually identified by the chain in which a particular CDR is located. Therefore, VH CDR3 is located in the variable domain of the antibody heavy chain in which it is located, while VL CDR1 is CDR1 from the variable domain of the antibody light chain in which it is located. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is a combined framework region of the constituent light and heavy chains, is used to locate and arrange the CDRs in three-dimensional space.

[0083] Antibodies typically possess a heavy chain variable domain comprising an amino acid sequence represented by the formula: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, where FRH1-4 represent four heavy chain framework regions and CDRH1-3 represent three hypervariable regions of the antibody's variable heavy chain domain. In some embodiments, FRH1-4 is derived from a common sequence (e.g., the most common amino acid in the type, subclass, or subgroup of the heavy or light chain of a human immunoglobulin) or from a single human antibody framework region or a combination of different framework region sequences.

[0084] Antibodies also typically have a light chain variable domain comprising an amino acid sequence represented by the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, where FRL1-4 represent four frame regions and CDRL1-3 represent three hypervariable regions of the antibody's variable light chain domain. In some embodiments, FRL1-4 is derived from a common sequence (e.g., the most common amino acid in the type, subclass, or subgroup of the heavy or light chain of a human immunoglobulin) or from a single human antibody frame region or a combination of different frame region sequences.

[0085] In some embodiments, the antibody is monovalent (an antibody having one antigen-binding site). In some embodiments, the antibody is bivalent (an antibody having two antigen-binding sites). In some embodiments, the antibody is multivalent, such as trivalent (an antibody having three antigen-binding sites) or tetravalent (an antibody having four antigen-binding sites). In some embodiments, the antibody is monospecific (binds to one antigen or epitope). In some embodiments, the antibody is multispecific (binds to more than one antigen or epitope; for example, bispecific, trispecific, etc.).

[0086] The anti-KIR antibodies described herein may include VH-VL dimers, comprising single-chain antibodies (antibodies existing as a single polypeptide chain), such as single-chain Fv antibodies (sFv or scFv), wherein variable heavy chain and variable light chain domains are linked together (directly or via peptide linkers) to form a continuous polypeptide. Single-chain Fv antibodies are typically covalently linked VH-VL, which can be expressed from nucleic acids including VH- and VL-coding sequences linked directly or via peptide-coding linkers. Although VH and VL are linked to each other as single polypeptide chains, the VH and VL domains are typically non-covalently associated. The VH and VL domains together typically include six complementarity-determining regions (CDRs) (three from each of the heavy and light chains), which facilitate antigen binding of amino acid residues and provide antigen-binding specificity to the antibody. In some cases, a single variable domain (or only half of an Fv containing the three CDRs specific to the antigen) has the ability to recognize and bind antigens. dsFv is an Fv with modified intermolecular disulfide bonds that stabilize the VH-VL pair.

[0087] Anti-KIR antibodies as described herein may include antigen-binding fragments generated by altering intact antibodies. For example, pepsin digests an antibody at the C-terminus of a disulfide bond in its hinge region to produce F(ab)'2, a dimer of Fab, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bond in its hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of its hinge region. Although various antigen-binding fragments are defined according to the digestion of intact antibodies, antigen-binding fragments can be synthesized de novo by chemical methods or by using recombinant DNA methods. Therefore, an antibody can refer to an antigen-binding fragment generated by altering an intact antibody or synthesized using recombinant DNA methods.

[0088] The anti-KIR antibodies described herein may include antibody fragments or antigen-binding fragments thereof that retain binding specificity. Antibody fragments or antigen-binding fragments thereof may include a portion of a complete antibody (e.g., the antigen-binding region of a complete antibody and / or the variable region of a complete antibody). Antibody fragments or antigen-binding fragments thereof include, but are not limited to, Fv fragments, disulfide-linked Fv(dsFv), Fab fragments, Fab' fragments, and F(ab')2 fragments, as described above. Antibody fragments or antigen-binding fragments thereof also include, but are not limited to, Fd fragments (i.e., antibody fragments containing a variable domain (VH) and a constant region domain (CHI) of the antibody heavy chain), Fd' fragments (i.e., antibody fragments containing a heavy chain portion of the F(ab')2 fragment), biantibodies (i.e., dimer scFv), linear antibodies, and single-chain antibody molecules (e.g., single-chain Fv (scFv) or single-chain Fab (scFab)).

[0089] In some embodiments, the anti-KIR antibody or its antigen-binding fragment is a chimeric antibody. A chimeric antibody may include an immunoglobulin molecule in which a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that provides novel properties to the chimeric antibody. A chimeric antibody may also include an immunoglobulin molecule in which a variable region or a portion thereof is altered, replaced, or exchanged with a variable region or a portion thereof having different or altered antigen specificity, or with a corresponding sequence from another species or another antibody type or subclass.

[0090] In some embodiments, the anti-KIR antibody or its antigen-binding fragment comprises one or more human frame regions. In some embodiments, the anti-KIR antibody or its antigen-binding fragment is humanized. A humanized antibody generally refers to an immunoglobulin molecule in which a CDR derived from a donor antibody (e.g., mouse, rabbit) is grafted onto a human frame sequence. Humanized antibodies may also include donor-derived residues in the frame sequence. Humanized antibodies may also include at least a portion of the human immunoglobulin constant region. Humanized antibodies may also include residues not present in the recipient antibody and the introduced CDR or frame sequence. Humanization can be performed using methods known in the art.

[0091] In some embodiments, the anti-KIR antibody or its antigen-binding fragment contains one or more variations (e.g., amino acid substitutions, deletions, and / or insertions). An example method for identifying certain residues or regions in the antibody that are preferred sites for amino acid substitutions is alanine scan mutagenesis. Here, a residue or a group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced by neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid sites confirmed to be functionally sensitive to the substitution are then refined by introducing further or other variants at or for the substitution site. Thus, although the sites where amino acid sequence variations are introduced are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scans or random mutagenesis are performed at the target codon or region, and expressed antibody variants can be screened for the desired activity. Amino acid sequence insertions may include N-terminal and / or C-terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion of an N-terminal methionine residue or the antibody with an epitope tag. Other insertion variants include the fusion of an enzyme or peptide that increases the antibody's serum half-life with the N- or C-terminus of the antibody.

[0092] Sites for amino acid substitutions may include sites in the hypervariable region and sites in the framework region. Amino acid substitutions may include conserved or non-conserved substitutions. Examples of substitutions are listed below.

[0093] Ala (A): val; leu; ile Arg (R): lys; gln; asn Asn (N): gln; his; asp; lys; arg Asp (D): glu; asn Cys (C): ser; ala Gln (Q): asn; glu Glu (E): asp; gln Gly (G): ala His (H): asn; gln; lys; arg Ile (I): leu; val; met; ala; phe; leucine Leu (L): leucine; ile; val; met; ala; phe Lys (K): arg; gln; asn Met (M): leu; phe; ile Phe (F): leu; val; ile; ala; tyr Pro (P): ala Ser (S):thr Thr (T): ser Trp (W): tyr; phe Tyr (Y): trp; phe; thr; ser Val (V): ile; leu; met; phe; ala; leucine Significant alterations to the biological properties of an antibody can be achieved by selecting substitutions that have significantly different effects on maintaining: (a) the structure of the polypeptide backbone in the substitution region, such as a folded or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chains.

[0094] Naturally occurring residues can be grouped according to the common side-chain properties listed below.

[0095] (1) Hydrophobic: Leucine, met, ala, val, leu, ile; (2) Neutral hydrophilic: cys, ser, thr; (3) Acidic: asp, glu; (4) Alkaline: asn, gln, his, lys, arg; (5) Residues affecting chain orientation: gly, pro; and (6) Fang ethnic group: trp, tyr, phe.

[0096] Non-conservative permutations typically require swapping members of one of these categories with members of another category.

[0097] Any cysteine ​​residues that do not participate in maintaining the correct conformation of the antibody can be substituted, for example, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability (e.g., in the case that the antibody is an antibody fragment, such as an Fv fragment).

[0098] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development will have improved biological properties relative to the parent antibodies from which they originate. A convenient way to generate such substitution variants is through phage display affinity maturation. In short, several hypervariable sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The resulting antibody variants are displayed monovalently from filamentous phage particles as fusions of the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity). To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly promote antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and adjacent residues are substitution candidates. Once such variants are generated, the variant group is screened, and antibodies with superior properties can be selected for further development in one or more relevant assays.

[0099] Another type of amino acid variant of an antibody alters its original glycosylation pattern. For example, one or more carbohydrate moieties present in the antibody can be deleted, and / or one or more glycosylation sites absent in the antibody can be added. Antibody glycosylation is typically N-linked and / or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moieties to a side chain of asparagine residues. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the most common recognition sequences for the enzymatic attachment of carbohydrate moieties to asparagine side chains. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used. Glycosylation sites can be added to the antibody (for N-linked glycosylation sites) by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences. It can also be altered by adding or substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites).

[0100] Fc polypeptide In some embodiments, the antibody or its antigen-binding fragment includes a fragment crystallizable region (Fc region), also known as an Fc polypeptide. An Fc polypeptide is a portion of each of the two heavy chains in the antibody and can interact with certain cell surface receptors and components of the complement system. Fc polypeptides typically include CH2 and CH3 domains, which are immunoglobulin constant region domain polypeptides. In some embodiments, the Fc polypeptide in the antibodies described herein may be a wild-type Fc polypeptide, such as the human IgG1 Fc polypeptide. In some embodiments, the antibodies described herein may comprise a wild-type Fc polypeptide having the following sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 1).

[0101] In some embodiments, the antibody or its antigen-binding fragment comprises a wild-type Fc polypeptide variant having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity with the wild-type Fc polypeptide (e.g., SEQ ID NO: 1) and having at least one amino acid substitution relative to the wild-type Fc polypeptide sequence (e.g., SEQ ID NO: 1).

[0102] In some embodiments, the Fc peptide includes one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions relative to a comparable wild-type Fc region). Antibodies containing modified Fc peptides typically exhibit altered phenotypes compared to antibodies containing wild-type Fc peptides. For example, antibodies containing modified Fc peptides may have altered serum half-life, altered stability, altered susceptibility to cellular enzymes, and / or altered effector functions (e.g., as measured in NK-dependent or macrophage-dependent assays).

[0103] In some embodiments, the Fc peptide in the antibody or its antigen-binding fragment may include amino acid substitutions that modulate effector function. In some embodiments, the Fc peptide in the antibody described herein may include amino acid substitutions that reduce or eliminate effector function. Illustrative Fc peptide amino acid substitutions that reduce effector function include, but are not limited to, substitutions in the CH2 domain, such as at positions 4 and 5 (position numbers relative to the sequence of SEQ ID NO: 1). For example, in some embodiments, one or both Fc peptides in the antibody described herein may contain L4A and L5A substitutions.

[0104] Additional Fc peptide amino acid substitutions that modulate effector function include, for example, a substitution at position 99 (position number relative to the sequence of SEQ ID NO: 1). For example, in some embodiments, one or both Fc peptides of the antibodies described herein may contain a P99G substitution. In some embodiments, one or both Fc peptides of the antibodies described herein may have L4A, L5A, and P99G substitutions.

[0105] In some embodiments, the Fc polypeptide includes an Fc polypeptide modified (relative to wild-type Fc polypeptide) to activate Fc. γ R (e.g., Fc) γ RIIA or Fc γ The affinity of RIIIA relative to inhibitory Fc γ R (e.g., Fc) γ One or more modifications to the ratio of RIIB: When the affinity ratio of the modified Fc peptide is greater than 1, the antibodies described herein can be specifically used to provide therapeutic or preventative treatment of diseases, symptoms, or infections, or to improve their symptoms, where the modification requires the use of Fc peptides. γ R-mediated enhancement of effector cell function, such as in cancer or infectious diseases. In cases where the affinity ratio of the modified Fc region is less than 1, the antibodies described herein can be particularly useful for providing therapeutic or preventative treatment of diseases or conditions, or for improving their symptoms, where Fc activation is required. γ R-mediated reduction in the efficacy of effector cell functions, such as in autoimmune or inflammatory diseases. Examples of single, double, triple, tetra, and pentaamino acid substitutions in Fc peptides with affinity ratios greater than 1 or less than 1 are given below. antigen Antibodies can bind to one or more antigens. An antigen generally refers to a molecule, compound, or complex that is recognized by an antibody, meaning it can be specifically bound by the antibody. The term antigen is used interchangeably with immunogen, antibody target, target analyte, etc. An antigen can refer to any molecule that can be specifically recognized by an antibody, such as peptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated or glycosylated peptides). The term antigen does not necessarily indicate that the molecule is immunogenic in every background, but generally indicates that it can be targeted by an antibody.

[0106] Antibodies typically bind to epitopes on antigens. An epitope is a local site on an antigen that is recognized and bound by an antibody. An epitope may comprise several amino acids or portions of several amino acids, such as 5 or 6 or more, or 20 or more amino acids or portions of these amino acids. In some cases, epitopes include non-protein components, such as those derived from carbohydrates, nucleic acids, or lipids. In some cases, epitopes are three-dimensional portions. Thus, for example, in the case where the target is a protein, an epitope may comprise continuous amino acids or amino acids from different portions of a protein that are close together by protein folding (e.g., discontinuous epitopes). The same is true for other types of target molecules that form three-dimensional structures. Epitopes typically comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids in a unique spatial conformation. In some cases, epitopes comprise 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0107] The terms "specific to," "specific binding," etc., generally refer to a molecule (e.g., antibody or antigen-binding fragment) that binds to a target with at least twice the affinity of a non-target compound, such as at least 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, or 100 times the affinity of the non-target compound. For example, an antibody that specifically binds to a target will typically bind to the target with at least twice the affinity of the non-target compound. Specificity can be determined using standard methods, such as solid-phase ELISA immunoassay. The term "binding" with respect to an antibody target (e.g., antigen, analyte, immune complex) generally indicates that the antibody binds to a majority of the antibody target in a pure population (assuming a suitable molar ratio). For example, an antibody binding to a given antibody target typically binds to at least about 2 / 3 of the antibody target in solution (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the antibody target in solution).

[0108] Modified reagents / antibodies In some embodiments, the reagent / antibody contains one or more modifications. For example, an immunoconjugate containing the antibody described herein may be conjugated with a cytotoxic agent such as a toxin (e.g., an enzyme-active toxin of bacterial, fungal, plant, or animal origin or a fragment thereof), a radioactive isotope (e.g., a radioconjugate), or a cytotoxic drug. Such conjugates are sometimes referred to as antibody-drug conjugates or ADCs. The conjugate can be prepared using any suitable bifunctional protein conjugating agent, such as N-succinimide-3-(2-pyridinedithio)propionate (SPDP), imine thiacyclopentane (IT), bifunctional derivatives of imine esters (e.g., hexamethylenediimide dimethyl HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis-(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and biactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0109] In some embodiments, the reagents / antibodies disclosed herein can be formulated as immunoliposomes. Liposomes containing antibodies can be prepared using suitable methods known in the art. Liposomes with enhanced cycle times can be produced. For example, liposomes can be produced by a reverse evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through a filter with defined pore sizes to obtain liposomes with the desired diameter. The Fab' fragment of the antibody provided herein can be coupled to the liposome via, for example, a disulfide exchange reaction. Other active ingredients can be contained within the liposomes.

[0110] In some embodiments, the enzyme or other peptide may be covalently bound to the reagent / antibody disclosed herein using suitable techniques, such as the use of heterobifunctional cross-linking agents. In some embodiments, fusion proteins comprising at least an antigen-binding region of an antibody provided herein linked to at least the functionally active portion of the enzyme may be constructed using recombinant DNA techniques.

[0111] In some implementations, it may be desirable to use an antigen-binding fragment, rather than an intact antibody, to increase penetration into target tissues and cells, for example. In such cases, it may be desirable to modify the antigen-binding fragment to increase its serum half-life. This can be achieved, for example, by incorporating a rescue receptor-binding epitope into the antigen-binding fragment, such as by mutating a suitable region in the antigen-binding fragment, or by incorporating the epitope into a peptide tag (which is then fused to the antigen-binding fragment at either end or in the middle, for example, through DNA or peptide synthesis).

[0112] In some embodiments, the antibody or its antigen-binding fragment comprises, is conjugated with, or hybridizes with one or more oligonucleotides. In some embodiments, the oligonucleotide contains a barcode sequence (e.g., a sample barcode sequence). In some embodiments, the oligonucleotide further contains a binding site and / or anchor for a primer. In some embodiments, a detectable marker or label (e.g., a radioisotope or fluorophore) is conjugated to the oligonucleotide. In some embodiments, the oligonucleotide is a polymer sequence. The terms oligonucleotide and polynucleotide are used interchangeably to refer to single-stranded polymers of nucleotides from about 2 to about 500 nucleotides in length. Oligonucleotides can be synthetic, enzymatically prepared (e.g., by polymerization), or prepared using a "split-merge" method. Oligonucleotides may include ribonucleotide monomers (i.e., may be oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). Oligonucleotides may include combinations of both deoxyribonucleotide monomers and ribonucleotide monomers in the oligonucleotide (e.g., random or ordered combinations of deoxyribonucleotide monomers and ribonucleotide monomers). Oligonucleotides may include one or more functional parts that are attached (e.g., covalently or non-covalently) to another structure.

[0113] An anchor typically refers to a polymer, such as a polynucleotide or oligonucleotide, designed to hybridize with a complementary oligonucleotide sequence. In some embodiments, the anchor is designed to generate a double-stranded construct oligonucleotide sequence. In some embodiments, the anchor is located at the 3' end of the construct oligonucleotide sequence. In some embodiments, the anchor is located at the 5' end of the construct oligonucleotide sequence. Each anchor can be specific to its intended complementary sequence.

[0114] In some embodiments, the primer binding site is a functional component of an oligonucleotide, which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for oligonucleotide amplification. The primer binding site may be formed from a polymer of DNA, RNA, PNA, modified bases, or combinations thereof, or polyamides, etc. In some embodiments, the primer binding site is about 10 such monomeric components, such as nucleotide bases. In some embodiments, the primer binding site is at least about 5 to 100 monomeric components, such as nucleotides. In some embodiments, the primer binding site may be a universal sequence suitable as an annealing site for various amplification techniques. Amplification techniques include, but are not limited to, DNA-polymerase-based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and polymerization by any number of DNA polymerases (e.g., T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase), and RNA-polymerase-based amplification systems (e.g., T7-, T3-, and SP6-RNA polymerase amplification), nucleic acid sequence-based amplification (NASBA), self-sustaining sequence replication (3SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (I), branching amplification methods, and RNA-seq.

[0115] In some embodiments, modifications are introduced into the antibody (e.g., within the polypeptide chain, or at the N-terminus or C-terminus), for example, to extend the in vivo half-life, such as through PEGylation or incorporation of a long-chain polyethylene glycol polymer (PEG). Introducing PEG or a long-chain polymer of PEG increases the effective molecular weight of the polypeptide, for example, to prevent rapid filtration into urine. In some embodiments, lysine residues in the sequence are coupled to PEG directly or via a linker. Such a linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group, for attachment to a suitably modified PEG chain. An alternative method for introducing the PEG chain is to first introduce a Cys residue at the C-terminus or at a solvent-exposed residue (e.g., by replacing an Arg or Lys residue). The Cys residue then attaches site-specifically to a PEG chain containing, for example, maleimide functionality. Methods for incorporating PEG or a long-chain polymer of PEG are known in the art.

[0116] Covalent modification of antibodies is also within the scope of this technology. For example, modifications can be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of antibodies can be introduced into the molecule by reacting target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues. Examples of covalent modifications of antibodies include linking the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene oxides.

[0117] Markings and solid supports In some embodiments, the anti-KIR reagent / antibody contains a detectable biomarker or label. In some embodiments, the anti-KIR reagent / antibody is conjugated to a detectable biomarker or label.

[0118] For example, for research and diagnostic applications, anti-KIR reagents / antibodies may be labeled with a detectable portion. Any suitable biomarker, label, or portion may be associated with or conjugated to the anti-KIR reagents / antibodies described herein. In some embodiments, the anti-KIR reagents / antibodies are made with one or more radioisotopes, such as... 35 S, 14 C 125 l、 3 H and 131 I-labeling. Antibodies can be labeled with radioisotopes using techniques known in the art, and radioactivity can be measured, for example, using scintillation counting. In some embodiments, the anti-KIR reagent / antibody is labeled with one or more fluorescent markers, such as rare earth chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl sulfonyl, lissamine, phycoerythrin, Texas Red, and Brilliant Violet™. Fluorescent labeling can be conjugated to the antibody using methods known in the art. Quantitative fluorescence can be achieved using, for example, flow cytometry, imaging microscopy, or a fluorometer.

[0119] In some embodiments, the anti-KIR reagent / antibody is labeled with one or more enzyme-substrate tags. The enzyme catalyzes a chemical change in the chromogenic substrate, which can be measured using various techniques. For example, the enzyme catalyzes a color change in the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence changes are known in the art. For example, a chemiluminescent substrate undergoes electronic excitation through a chemical reaction, which can then emit light that can be measured (e.g., using a chemiluminescence analyzer) or transfer energy to a fluorescent acceptor. Examples of enzyme labeling include luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Techniques for conjugating enzymes to antibodies are known in the art.

[0120] In some cases, labels are indirectly coupled to reagents / antibodies. For example, antibodies can be coupled to biotin, and any of the labels described above can be coupled to avidin, or vice versa. Biotin selectively binds to avidin, so labels can be coupled to antibodies in this indirect manner. Alternatively, to achieve indirect coupling between labels and antibodies, antibodies can be coupled to small haptens (e.g., digoxigenin), and labels described above can be coupled to anti-hapten antibodies (e.g., anti-digoxigenin antibodies). Thus, indirect coupling between labels and antibodies can be achieved.

[0121] In some implementations, the anti-KIR reagent / antibody does not need to be labeled, and its presence can be detected, for example, using a labeled antibody that binds to the anti-KIR antibody.

[0122] In some embodiments, the anti-KIR reagent / antibody described herein is immobilized on a solid support or matrix. In some embodiments, the anti-KIR reagent / antibody described herein is immobilized on a solid support in a non-diffusion manner (e.g., the anti-KIR reagent / antibody does not detach from the solid support). The solid support or matrix can be any physically separable solid to which the anti-KIR reagent / antibody can be directly or indirectly attached, including but not limited to surfaces provided by microarrays and pores, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports may also include, for example, chips, columns, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled-aperture glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylic resins, polystyrene, copolymers of styrene or other materials, polybutene, polyurethane, TEFLON™, polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride). Polyvinyl difluoroethylene (PVDF), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon, silica gel and modified silicon), Sephadex®, Sepharose®, carbon, metals (e.g., steel, gold, silver, aluminum, silicon and copper), inorganic glasses, conductive polymers (including polymers such as polypyrrole and polyindole); microstructured or nanostructured surfaces, such as nucleic acid tiled arrays, nanotubes, nanowires or nanoparticle-modified surfaces; or porous surfaces or gels, such as methacrylates, acrylamides, sugar polymers, cellulose, silicates or other fibrous or chain polymers. In some embodiments, the solid support or matrix may be coated with a passive or chemically derived coating having a variety of materials, including polymers such as dextran, acrylamides, gelatin or agarose. Beads and / or particles may be free or connected to each other (e.g., sintered). In some embodiments, the solid support or matrix may be an aggregate of particles. In some embodiments, the particles may contain silica, and the silica may contain silicon dioxide. In some embodiments, the silica may be porous, and in some embodiments, the silica may be non-porous. In some embodiments, the particles further comprise a reagent that imparts paramagnetic properties to the particles. In some embodiments, the reagent comprises a metal, and in some embodiments, the reagent is a metal oxide (e.g., iron or iron oxide, wherein the iron oxide contains Fe). 2+ and Fe 3+(A mixture of substances). Anti-KIR reagents / antibodies can be linked to the solid support via covalent bonds or non-covalent interactions, and can be linked to the solid support directly or indirectly (e.g., via intermediate reagents, such as spacer molecules or biotin).

[0123] Nucleic acids, vectors, host cells, and recombinant methods This article provides isolated nucleic acids encoding anti-KIR reagents (e.g., anti-KIR antibodies), vectors and host cells containing said nucleic acids, and recombinant technologies for producing said reagents or antibodies.

[0124] This document provides nucleic acids (e.g., isolated nucleic acids) comprising nucleotide sequences encoding anti-KIR reagents or antibodies or fragments thereof. In some embodiments, the nucleic acid encodes an immunoglobulin heavy chain variable domain of the anti-KIR reagent provided herein. In some embodiments, the nucleic acid encodes an immunoglobulin light chain variable domain of the anti-KIR reagent provided herein. In some embodiments, the nucleic acid encodes both an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of the anti-KIR reagent provided herein. In some embodiments, the nucleic acid comprises the nucleotide sequences provided in Table 3. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence provided in Table 3. For example, the nucleic acid may comprise a nucleotide sequence encoding a CDR amino acid sequence of any of the CDRs provided in Table 3. The nucleic acid may comprise a nucleotide sequence encoding an immunoglobulin heavy chain variable domain amino acid sequence of any of the immunoglobulin heavy chain variable domains provided in Table 3. The nucleic acid may comprise a nucleotide sequence encoding an immunoglobulin light chain variable domain amino acid sequence of any of the immunoglobulin light chain variable domains provided in Table 3.

[0125] To generate anti-KIR reagents or antibodies through recombination, nucleic acids encoding anti-KIR reagents or antibodies can be isolated and inserted into a reproducible vector for further cloning and / or expression. In some cases, anti-KIR reagents or antibodies can be generated through homologous recombination. DNA encoding anti-KIR reagents or antibodies can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody. Any suitable vector can be used. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0126] Suitable host cells for cloning and / or expressing DNA in vectors include prokaryotic cells, yeast cells, and higher eukaryotic cells. For this purpose, suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia coli (e.g., *Escherichia coli*), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., *Salmonella typhimurium*), Serratia (e.g., *Serratia marcescens*), and Shigella, as well as Bacillus (e.g., *Bacillus subtilis* and *Bacillus licheniformis*), Pseudomonas (e.g., *Pseudomonas aeruginosa*), and Streptomyces. In some cases, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable hosts for cloning and / or expressing anti-KIR reagent / antibody-encoding vectors. *Saccharomyces cerevisiae* or *Bacillus baker's yeast* are commonly used among lower eukaryotic host microorganisms. Many other genera, species, and strains are readily available and used herein, such as *Schizosaccharomyces*; hosts of the *Kluyveromyces* genus, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wilfordii* (ATCC 24,178), *Kluyveromyces valti* (ATCC 56,500), *Kluyveromyces drosophila* (ATCC 36,906), *Kluyveromyces thermostrophicus*, and *Kluyveromyces maculae*; *Yersinia* genus (EP 402,226); *Pichia pastoris* (EP 183,070); *Candida* genus; *Trichoderma reesei* (EP 244,234); *Neurospora crassa*; *Schwannium* genus, such as *Schwannium serratum*; and filamentous fungi, such as hosts of *Neurospora*, *Penicillium*, *Cyclophorus*, and *Aspergillus*, such as *Aspergillus nidus* and *Aspergillus niger*.

[0127] Suitable host cells for expressing anti-KIR reagents / antibodies can be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, along with corresponding permissive insect host cells from hosts such as the fall armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm (silkworm moth). Various viral strains used for transfection are publicly available, such as the L-1 variant of the alfalfa looper NPV and the Bm-5 strain of the silkworm NPV. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0128] Suitable host cells for expressing anti-KIR reagents / antibodies may also include vertebrate cells (e.g., mammalian cells). Vertebrate cells can proliferate in culture (tissue culture). Examples of available mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumors (MMT). 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0129] Host cells can be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media, which are modified where appropriate to induce promoters, select transformants, and / or amplify genes encoding desired sequences. Host cells used to produce the reagents / antibodies described herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma), are suitable for culturing these host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™), trace elements (defined as inorganic compounds typically present in micromolar final concentrations), and glucose or an equivalent energy source. It may also include any other necessary supplements at appropriate concentrations, which are known to those skilled in the art. Certain culture conditions, such as temperature, pH, etc., may include conditions previously used for selecting the host cells for expression.

[0130] When using recombinant technologies, reagents / antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris (host cell or lysed fragments) is removed, for example by centrifugation or ultrafiltration. Reagent / antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. Protein A can be used to purify human heavy chain-based antibodies. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domains present in the antibody. Protein G can be used for mouse isotypes and human IgG3. Other techniques for protein purification, such as fractionation on ion-exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin SEPHAROSE™ chromatography, anion or cation exchange resin chromatography (e.g., polyaspartic acid columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, can also be used, depending on the antibody to be recovered.

[0131] Drug formulation, administration and route of administration This document provides therapeutic compositions comprising the anti-KIR reagent / antibody described herein and pharmaceutically acceptable excipients. This document provides antibodies and related compositions that can be used, for example, to block KIR ligand binding. In some embodiments, this document provides antibodies and related compositions that can be used to enhance KIR ligand binding. In some embodiments, this document provides antibodies and related compositions that can be used to inhibit natural killer cell activity. In some embodiments, this document provides antibodies and related compositions that can be used to activate natural killer cell activity. Serum KIR levels can be associated with the prognosis of patients with certain diseases or conditions. Therefore, anti-KIR antibodies can be used to detect KIR levels in certain patients.

[0132] In some embodiments, anti-KIR antibodies or antigen-binding fragments thereof may be formulated in pharmaceutical compositions for various purposes, including the treatment of diseases or conditions. Pharmaceutical compositions comprising one or more antibodies may be administered to patients in need using a pharmaceutical device, and according to one embodiment of the present technology, a kit including such a device is provided. Such devices and kits may be designed for routine administration of the pharmaceutical compositions herein, including self-administration.

[0133] This document provides therapeutic compositions comprising the anti-KIR reagent / antibody described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer. Therapeutic formulations of anti-KIR antibodies can be prepared for storage by mixing a reagent or antibody of desired purity with a physiologically and / or pharmaceutically acceptable carrier, excipient, or stabilizer, in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0134] The formulations described herein may also contain more than one active compound as needed for a specific indication being treated, preferably those active compounds that have complementary activities and do not adversely affect each other. Such molecules are suitably present in a combination of amounts effective for the intended purpose. Formulations intended for in vivo administration are generally sterile. This can be achieved, for example, by filtering through, for example, a sterile filter membrane.

[0135] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix containing the reagent / antibody, a solid hydrophobic polymer, in the form of a molded article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can achieve molecular release over 100 days, some hydrogels release proteins over shorter time periods.

[0136] For therapeutic applications, the antibodies described herein can be administered in pharmaceutically acceptable dosage forms, such as those discussed above, including those that can be administered to humans via intravenous bolus or continuous infusion over a period of time, or via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes, to mammals, such as humans. For the prevention or treatment of disease, the appropriate dosage of the reagent or antibody will depend on the type of disease being treated, the severity and progression of the disease, whether the antibody is administered for prevention or treatment, prior therapy, the patient's clinical history and response to the antibody, and the discretion of the healthcare professional. Antibodies can be appropriately administered to patients as a single dose or as part of a series of treatments.

[0137] Depending on the type and severity of the disease, antibodies of approximately 1 µg / kg to approximately 50 mg / kg (e.g., 0.1–20 mg / kg) may be the initial candidate dose for administration to the patient, whether by single or multiple administrations or by continuous infusion. Typical daily or weekly dose ranges may be approximately 1 µg / kg to approximately 20 mg / kg or more, depending on the factors mentioned above. Treatment may be repeated after several days or longer of repeated administration, depending on the condition, until the desired suppression of disease symptoms occurs. However, other dosing regimens may be used. Progression of the therapy can be easily monitored using conventional techniques and assays, including, for example, radiographic imaging. To optimize patient exposure to the therapeutic antibody, assays that utilize the antibody to measure KIR levels in body fluids or tissues can be used.

[0138] In some embodiments, compositions containing the antibodies described herein may be administered as a monotherapy, and in some embodiments, compositions containing antibodies may be administered as part of a combination therapy. Therefore, therapeutic compositions are provided herein in which anti-KIR agents / antibodies are used as adjuvants or in combination with adjuvants. In some cases, the effectiveness of antibodies in preventing or treating disease can be improved by administering antibodies sequentially or in combination with another drug effective for these purposes (e.g., a chemotherapeutic agent for treating cancer or microbial infections). In other cases, antibodies can be used to enhance or sensitize cells to chemotherapy, thereby allowing efficacy at lower doses and with less toxicity. In addition to administering compositions containing antibodies that block KIR activity, some combination therapies include the delivery of a second treatment regimen selected from chemotherapeutic agents, radiotherapy, surgery, and any combination of the foregoing therapies. Such other agents may be present in the administered composition or may be administered alone. Furthermore, the antibodies may suitably be administered sequentially or in combination with other agents or methods, such as chemotherapeutic agents or radiation for treating cancer, infections, etc., or immunosuppressive agents.

[0139] Research and diagnosis This document provides diagnostic reagents comprising the anti-KIR reagents / antibodies described herein. For example, the antibodies provided herein can be used to detect and / or purify KIR from body fluids or tissues. Anti-KIR antibodies can be used, for example, for diagnostic assays of KIR, such as detecting its presence in specific cells, tissues, or body fluids. Such diagnostic methods can be used to diagnose diseases or conditions. This document also provides methods for detecting KIR in a subject or in a sample from a subject and / or measuring KIR levels. For example, the method may include contacting a sample (e.g., a biological sample known or suspected of containing KIR) with an antibody provided herein, and detecting a KIR:antibody complex if the sample contains KIR. In some embodiments, the KIR detection method is performed in vitro. In some embodiments, the KIR detection method is performed in vivo. For in vivo diagnostic assays, the antibody can be used with a radionuclide (e.g., a radionuclide). 111 In、 99 Tc, 14 C 131 l、 125 l、 3 H, 32 P or 35 The S) labeling allows the bound target molecules to be located using immunoscintigraphy.

[0140] This document also provides reagents containing the anti-KIR reagents / antibodies described herein for non-diagnostic and non-therapeutic purposes. For example, this document provides reagents containing the anti-KIR reagents / antibodies described herein for use in research applications. Research applications may include studying KIRs and their roles in immune cell function and / or regulation (e.g., natural killer cell function and / or regulation, T lymphocyte function and / or regulation), cell signal transduction, signal transduction, disease, infection, inflammation, etc. This document also provides methods for detecting KIRs and / or measuring KIR levels in non-biological samples. For example, methods may include contacting a non-biological sample (e.g., a laboratory research sample known or suspected of containing KIRs) with an antibody provided herein, and detecting the KIR:antibody complex if the sample contains KIRs. Laboratory research samples may include non-human animal models, samples from non-human animal models, cell lines, products generated from cell lines, etc.

[0141] The anti-KIR reagents and antibodies provided in this article can be used for any suitable assay, such as flow cytometry, immunohistochemistry, immunofluorescence, mass flow cytometry, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0142] Flow cytometry and mass cytometry assays typically involve using a single primary antibody to specifically identify the presence of target molecules exposed on the surface of a dispersion suspension of single cells. The dispersed cells are typically obtained from biological fluid samples, such as blood, but can also be obtained from solid tissue samples, such as single-cell dispersions prepared from tumor biopsies. The primary antibody may be conjugated directly to a detectable component, such as a fluorophore like phycoerythrin, for flow cytometry, or conjugated to a heavy metal chelate for mass cytometry. Alternatively, the primary antibody may be unlabeled or labeled with a non-detectable tag (e.g., biotin), and the primary antibody is then detected by a secondary antibody that specifically recognizes the primary antibody itself or a detectable tag on the primary antibody. The labeled cells are then analyzed in an instrument capable of single-cell detection (e.g., flow cytometer, mass cytometer, fluorescence microscope, or bright-field microscope) to identify those single cells expressing the target recognized by the primary antibody in a dispersed population or tissue sample. In some cases, fixed and permeabilized cells may be used.

[0143] Sandwich assays involve the use of two antibodies, each capable of binding to different immunogenic moieties or epitopes of the protein being detected. In a sandwich assay, the test sample analyte is bound by a first antibody immobilized on a solid support, followed by the binding of a second antibody, thereby forming an insoluble three-part complex. The second antibody itself can be labeled with the detectable moiety (direct sandwich assay), or it can be measured using an anti-immunoglobulin antibody labeled with the detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme. In cellular ELISA, a target cell population can be attached to a solid support using antibodies that first attach to the support and recognize different cell surface proteins. These first antibodies capture the cells to the support. In some cases, immobilized and permeabilized cells can be used.

[0144] In some embodiments, the reagents / antibodies provided herein are formulated for immunohistochemical analysis. In some embodiments, the immunohistochemical analysis includes the use of a sample. In some embodiments, the immunohistochemical analysis includes the use of blood and / or tissue samples. The sample may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, the FFPE sample is saturated with formalin (i.e., formaldehyde) and then embedded in a paraffin block. In some embodiments, the FFPE sample is stable at room temperature. In some embodiments, all or most of the structures in the FFPE sample are preserved. In some embodiments, intracellular and surface proteins in the FFPE sample are preserved. In some embodiments, mRNA in the FFPE sample is preserved. In some embodiments, mRNA, intracellular proteins, and surface proteins in the FFPE sample are preserved. In some embodiments, surface proteins in the FFPE sample are denatured.

[0145] In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect KIR in formalin-fixed paraffin-embedded samples. In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect KIR on the surface of formalin-fixed paraffin-embedded samples. In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect intracellular KIR in formalin-fixed paraffin-embedded samples. In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect both intracellular KIR in formalin-fixed paraffin-embedded samples and KIR on the surface of formalin-fixed paraffin-embedded samples.

[0146] In some embodiments, the sample is a fresh sample that has been frozen. In some embodiments, the sample is a fresh sample that has been cryogenically frozen. In some embodiments, the sample is rapidly frozen. In some embodiments, the sample is rapidly frozen and stored at -80°C. In some embodiments, all or most of the structures in the rapidly frozen sample are preserved. In some embodiments, intracellular and surface proteins in the rapidly frozen sample are preserved. In some embodiments, mRNA in the rapidly frozen sample is preserved. In some embodiments, mRNA, intracellular proteins, and surface proteins in the rapidly frozen sample are preserved. In some embodiments, surface proteins in the rapidly frozen sample are denatured.

[0147] In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect KIR in frozen samples. In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect KIR on the surface of frozen samples. In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect intracellular KIR in frozen samples. In some embodiments, the anti-KIR antibody or its antigen-binding fragment provided herein can detect both intracellular KIR and KIR on the surface of frozen samples.

[0148] KIR detection This document provides antibodies and methods for detecting KIRs (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3). In some embodiments, antibodies and methods for detecting KIRs in biological samples are provided. In some embodiments, KIRs are detected on cell surfaces. In some embodiments, KIRs are detected intracellularly. In some embodiments, KIR detection is in vitro. In some embodiments, KIR detection is in vivo.

[0149] In some implementations, the biological sample is a solid tissue, a fluid, or cells. Solid tissue samples may include one or more solid tissues from adipose tissue, bladder, bone, brain, mammary gland, cervix, endothelium, gallbladder, kidney, liver, lung, lymph, ovary, prostate, salivary gland, stomach, testis, thyroid gland, urethra, uterus, vagina, and vulva. Fluid samples may include one or more of amniotic fluid, bile, blood, breast milk, mammary fluid, cerebrospinal fluid, lavage fluid, lymph, mucus, plasma, saliva, semen, serum, cerebrospinal fluid, sputum, tears, umbilical cord blood, urine, and vaginal fluid.

[0150] In some embodiments, the sample contains immune cells. In some embodiments, the sample contains one or more immune cells selected from macrophages, neutrophils, B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, myeloid dendritic cells (mDCs), intrinsic lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample contains non-immune cells. For example, the sample may contain one or more of fibroblasts, vascular smooth muscle cells, endothelial cells, brain cells, and hepatocytes. In some embodiments, the sample contains tumor cells.

[0151] In some embodiments, the biological sample is obtained from a healthy subject. In some embodiments, the sample is obtained from a subject with a disease or condition. In some embodiments, the detection of KIR indicates the presence or absence of a disease or condition. In some embodiments, the disease or condition is cancer, an autoimmune disease, an inflammatory disease, a neurological disease, or an infection. In some embodiments, the disease or condition is associated with KIR expression. In some embodiments, the disease or condition is associated with abnormal KIR expression. In some embodiments, the disease or condition is associated with immune cells. In some embodiments, the disease or condition is associated with natural killer cells. In some embodiments, the disease or condition is associated with T lymphocytes.

[0152] Reagent test kit This document provides kits containing the anti-KIR reagents / antibodies described herein. A kit generally refers to a packaged combination of a predetermined amount of reagents and instructions for use (e.g., instructions for performing a diagnostic assay). In some embodiments, the kit is a diagnostic kit configured to detect KIRs (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3) in a sample (e.g., a biological sample). In some embodiments, the kit is a non-diagnostic kit configured to detect KIRs (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, and / or KIR3DL3) in a non-biological sample (e.g., a research sample). When the anti-KIR reagent is labeled with a fluorophore, the kit may include an isotype negative control antibody to control for nonspecific binding to the anti-KIR reagent. When the anti-KIR reagent is labeled with an enzyme, the kit may include the substrate and cofactor required for the enzyme (e.g., providing a substrate precursor that can detect chromophores or fluorophores). Additionally, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can be widely varied to provide reagent concentrations in solution that significantly optimize the sensitivity of the assay. In particular, reagents may be provided as dry powders, typically lyophilized, including excipients that, upon dissolution, provide a reagent solution with an appropriate concentration.

[0153] Products This document provides articles containing materials that can be used to treat or prevent the aforementioned conditions. The articles may include containers and labels. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from various materials, such as glass or plastic. The container contains a composition that effectively treats the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The active anti-KIR reagent in the composition may be an anti-KIR antibody. A label on or associated with the container indicates that the composition is intended for the treatment or diagnosis of selected conditions. The articles may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or glucose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use.

[0154] Some implementation methods The following are some non-limiting examples of implementations of this technology.

[0155] A1. An isolated antibody or antigen-binding fragment thereof that binds to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof, said antibody or antigen-binding fragment comprising: (a) Variable domains of the immunoglobulin heavy chain, which include: (i) Heavy chain complementarity-determining region 1 (CDRH1), which contains the CDRH1 peptide provided in Table 3 or a peptide that has at least 80% identity with the CDRH1 peptide provided in Table 3. (ii) Heavy chain complementarity-determining region 2 (CDRH2), comprising the CDRH2 peptides provided in Table 3 or peptides having at least 80% identity with the CDRH2 peptides provided in Table 3; and (iii) Heavy chain complementarity-determining region 3 (CDRH3), comprising the CDRH3 peptides provided in Table 3 or peptides having at least 80% identity with the CDRH3 peptides provided in Table 3; and (b) Variable domains of the immunoglobulin light chain, which include: (i) Light chain complementarity-determining region 1 (CDRL1), which contains the CDRL1 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the CDRL1 polypeptide provided in Table 3. (ii) Light chain complementarity-determining region 2 (CDRL2), comprising the CDRL2 peptides provided in Table 3 or peptides having at least 80% identity with the CDRL2 peptides provided in Table 3; and (iii) Light chain complementarity-determining region 3 (CDRL3), which contains the CDRL3 polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the CDRL3 polypeptide provided in Table 3.

[0156] A1.1 The isolated antibody or its antigen-binding fragment according to embodiment A1, wherein one or more KIRs are selected from KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and KIR3DL3.

[0157] A2. The isolated antibody or its antigen-binding fragment according to embodiment A1 or A1.1, wherein the immunoglobulin heavy chain variable domain comprises the immunoglobulin heavy chain variable domain polypeptide provided in Table 3 or a polypeptide having at least 80% identity with the immunoglobulin heavy chain variable domain polypeptide provided in Table 3.

[0158] A3. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A2, wherein the immunoglobulin light chain variable domain comprises an immunoglobulin light chain variable domain polypeptide provided in Table 3 or a polypeptide having at least 80% identity with an immunoglobulin light chain variable domain polypeptide provided in Table 3.

[0159] A4. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A3, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.

[0160] A5. The isolated antibody or its antigen-binding fragment according to any one of embodiments A1-A4, further comprising one or more human frame regions.

[0161] A6. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A5, wherein the antibody or antigen-binding fragment thereof is humanized.

[0162] A7. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A6, wherein the antibody or antigen-binding fragment thereof contains a detectable marker or label, or is coupled to a detectable marker or label.

[0163] A8. The isolated antibody or its antigen-binding fragment as described in embodiment A7, wherein the detectable marker or label comprises a detectable portion.

[0164] A9. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A8, further comprising oligonucleotides.

[0165] A10. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A9, wherein the antibody or antigen-binding fragment thereof is immobilized on a solid support.

[0166] A11. An isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A10, wherein the antibody or antigen-binding fragment thereof is immobilized on a solid support in a non-diffusion manner.

[0167] A12. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments A1-A11, wherein the antibody is a single-chain fragment.

[0168] A13. The isolated antibody or its antigen-binding fragment according to embodiment A12, wherein the single-chain fragment is a single-chain variable fragment (scFv).

[0169] A14. The isolated antibody or its antigen-binding fragment according to any one of embodiments A1-A13, for non-diagnostic and / or non-therapeutic uses.

[0170] B1. A kit comprising an isolated antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A14 and instructions for use.

[0171] C1. A diagnostic reagent comprising an isolated antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A13.

[0172] D1. A diagnostic kit configured to detect KIR or a portion thereof in a biological sample, wherein the kit comprises an isolated antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A13 or a diagnostic reagent according to embodiment C1.

[0173] E1. A therapeutic composition comprising an isolated antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A13 and a pharmaceutically acceptable excipient.

[0174] E2. The therapeutic composition according to embodiment E1, wherein the antibody or its antigen-binding fragment is used as an adjuvant or in combination with an adjuvant.

[0175] F1. An isolated polynucleotide comprising a nucleic acid sequence encoding an immunoglobulin heavy chain variable domain according to any one of embodiments A1-A13.

[0176] F2. A recombinant expression vector comprising isolated polynucleotides according to embodiment F1.

[0177] F3. A host cell transfected with the recombinant expression vector according to embodiment F2.

[0178] F4. An isolated polynucleotide comprising a nucleic acid sequence encoding a variable domain of an immunoglobulin light chain according to any one of embodiments A1-A13.

[0179] F5. A recombinant expression vector comprising isolated polynucleotides according to embodiment F4.

[0180] F6. A host cell transfected with the recombinant expression vector according to embodiment F5.

[0181] F7. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a polynucleotide, the polynucleotide comprising a nucleic acid sequence encoding an immunoglobulin heavy chain variable domain according to any one of embodiments A1-A13; and the second expression cassette comprises a polynucleotide, the polynucleotide comprising a nucleic acid sequence encoding an immunoglobulin light chain variable domain according to any one of embodiments A1-A13.

[0182] F8. The recombinant expression vector according to embodiment F7, wherein the first expression cassette and the second expression cassette each contain a promoter.

[0183] F9. A host cell transfected with a recombinant expression vector according to embodiment F7 or F8.

[0184] G1. A method for detecting one or more killer cell immunoglobulin-like receptors (KIRs), the method comprising: a) Contact the sample with the antibody or its antigen-binding fragment according to any one of embodiments A1-A13, and b) If the sample contains one or more KIRs, detect the KIR:anti-KIR complex.

[0185] G2. The method according to embodiment G1, wherein one or more KIRs are selected from KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 and KIR3DL3.

[0186] G3. The method according to implementation scheme G1 or G2, wherein the method is performed in vitro.

[0187] G3. The method according to any one of embodiments G1-G3, wherein the sample is a biological sample.

[0188] G4. The method according to any one of embodiments G1-G3, wherein the sample is a non-biological sample.

[0189] H1. A first anti-KIR reagent that binds to one or more killer cell immunoglobulin-like receptors (KIRs), wherein the first reagent competitively binds to or is capable of competitively binding to a second anti-KIR reagent, wherein the second reagent is an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A13.

[0190] H2. A first anti-KIR reagent that binds to one or more killer cell immunoglobulin-like receptors (KIRs), wherein the first reagent binds to or is capable of binding to the same epitope as a second anti-KIR reagent, wherein the second reagent is an antibody or an antigen-binding fragment thereof according to any one of embodiments A1-A13.

[0191] H3. An isolated antibody or antigen-binding fragment thereof that binds to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof, wherein the antibody or antigen-binding fragment thereof blocks or is capable of blocking KIR ligand binding.

[0192] H4. An isolated antibody or antigen-binding fragment thereof that binds to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof, wherein the antibody or antigen-binding fragment thereof enhances or is capable of enhancing KIR ligand binding.

[0193] H5. An isolated antibody or antigen-binding fragment thereof that binds to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof, wherein the antibody or antigen-binding fragment thereof inhibits or is capable of inhibiting natural killer cell activity.

[0194] H6. An isolated antibody or antigen-binding fragment thereof that binds to one or more killer cell immunoglobulin-like receptors (KIRs) or portions thereof, wherein said antibody or antigen-binding fragment thereof activates or is capable of activating natural killer cell activity.

[0195] Example The embodiments described below illustrate certain implementation methods and do not limit the technology.

[0196] Example 1: Generation of anti-KIR antibody expression hybridoma This embodiment describes the generation and characterization of hybridomas that secrete monoclonal antibodies that react with various KIR family members.

[0197] In short, mice with myeloma cell lines expressing the human KIR immunogen shown in Table 2 were immunized with recombinant KIR protein. Myeloma cells were fused with spleens to form hybridomas using a standard protocol, and lymph node cells were drained and harvested. Successful fusions were selected for HAT medium and cloned to approximately 1 cell / well in microtiter plates, followed by flow cytometry testing of the culture supernatant against KIR-expressing cell transfectants. Wells were selected by evaluating staining profiles, then passaged into larger containers for subcloning.

[0198] Certain clones were selected for further analysis, as shown in Table 2 below. The amino acid and nucleic acid sequences of some clones are provided in Table 3 below. Example 2: Function of anti-KIR antibody method The following methods were used to evaluate the function of anti-KIR antibodies. Results were shown... Figure 1-152 As described below.

[0199] Cell culture Ba / F3 mouse interleukin-3 dependent progenitor B cell lines were cultured in RPMI containing 10% fetal bovine serum (FBS) + 1% Pen / Strep + 2 ng / mL recombinant mouse IL-3. K562 cells were cultured in RPMI + 10% FBS + 1% Pen / Strep at 37°C. For NK cell isolation, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood via LYMPHOPURE phase separation. NK cells were then purified from the isolated PBMCs using the MOJOSORT Human NK Cell Isolation Kit (Biolegend 480054). NK cells were then incubated in RPMI + 10% FBS + 1% Pen / Strep + 50 ng / mL recombinant human IL-2 + 10 ng / mL recombinant human IL-15 at 37°C for 16 hours prior to harvest on the day of the experiment.

[0200] Transfection of cell lines Ba / F3 mouse interleukin-3 dependent progenitor B cell lines were transfected with a construct encoding the extracellular domain of KIR. The accession numbers for KIR molecules are as follows: NM_014218.3, NM_014219.2, NM_015868.3, AF034771.1, NM001080770, NM_002255.6, NM_014512.1, NM_012312.4, NM_012313.1, NM_012314.6, NM_014513.2, NM_020535.3, NM_001018081.1, NM_013289.3, NM_001322168.1, NM_006737, NM_153443.5, NM_001083539.2. Cells were maintained in RPMI containing 10% fetal bovine serum (FBS) and recombinant mouse interleukin-3.

[0201] Flow cytometry staining Cells were stained according to the flow cytometry staining protocol described on the BioLegend website. In short, the fluorophore-conjugated antibody was added to the cell solution at the stated concentration for each test and incubated at room temperature for 15 minutes. Cells were then washed twice with Fluorescence Activated Cell Sorting (FACS) buffer and evaluated using flow cytometry. When using unconjugated antibodies, the antibody was added to the cell solution as previously described. Cells were then washed twice with FACS buffer, and a second species- or allotype-specific antibody conjugated to the fluorophore was added to the cell solution. Cells were then washed twice with FACS buffer, and additional fluorophore-conjugated antibody was added for co-staining and incubated at room temperature for 15 minutes. Cells were then washed twice with FACS buffer and analyzed using flow cytometry.

[0202] Ligand binding assay Ligands specific to specific KIR proteins were added to cells and incubated at room temperature for 15 minutes. For tagged recombinant protein ligands, binding was detected by anti-tagged antibody. The cell suspension was then washed and analyzed by flow cytometry. When determining the effect of the antibody on ligand-KIR interactions, cells were first incubated with KIR-specific antibodies at room temperature for 15 minutes, and then the ligands were added to the cell solution.

[0203] NK cell toxicity assay On the day of harvest, K562 cells were counted and washed with culture medium. Then, K562 cells were stained with TAG-IT VIOLET dye (BioLegend 425101) for 12 minutes. The TAG-IT VIOLET dye reaction was then quenched with cold culture medium and allowed to stand for 15 minutes. The cells were centrifuged and washed with culture medium.

[0204] On the day of harvest, NK cells were counted and washed with RPMI + 10% FBS + 1% Pen / Strep, then resuspended in culture medium. NK cells were incubated with 10 µg of the designated antibody or HLA protein at 37ºC for 15 min to allow sufficient antibody interaction with NK cells.

[0205] Subsequently, the Tag-it Violet-stained K562 cells were mixed with NK cells at a 1:1 ratio and incubated at 37ºC for 1 hour. The NK-K562 cell co-culture was centrifuged and the supernatant was used for in-solution analyte analysis. The NK-K562 cell co-culture was then stained using a standard staining procedure.

[0206] Quantitative assay of cytokines Cytokine quantification of NK-K562 culture supernatant was performed using the Human CD8 / NK V02 Panel LEGENDPLEX Assay Kit (BioLegend 741187). Detailed procedures are available on the BioLegend website and should be performed according to the manufacturer's instructions. In short, the co-culture supernatant was stained with the kit's capture beads for 2 hours, then centrifuged and washed three times with LEGENDPLEX wash buffer. The capture beads were then stained with biotinylated analyte detection antibodies for 1 hour, followed by the addition of PE-streptavidin to the detection antibody and incubation at room temperature for 30 minutes. The analyte-stained beads were then centrifuged and washed three times with LEGENDPLEX wash buffer. The analyte beads were then run on a flow cytometer to collect analyte quantification data. Data were analyzed using LEGENDPLEX software.

[0207] Functional results KIR-specific antibodies may disrupt or enhance MHC class I or ligand interactions. Each member of the KIR protein family binds to multiple MHC class I proteins or certain other ligand proteins. To determine whether KIR protein-ligand interactions can be disrupted by each specific KIR-protein interaction, a developed anti-KIR antibody was added to a Ba / F3 cell line transfected with a single KIR protein, followed by the addition of a known ligand for that single KIR protein. The presence of the ligand was then measured by the conjugate fluorophore or with the anti-ligand antibody. The effect of each tested KIR protein and the developed anti-KIR antibody on their corresponding KIR ligand interactions is detailed below.

[0208] KIR3DL3 (See example,) Figures 151-152 The interaction between HHLA2 and KIR3DL3 transfected Ba / F3 cells can be completely disrupted by mAb S23011A, B, C, D, E, F and S22010B. mAb S22010G did not show an interaction, but mAb S22010H showed an increased interaction between HHLA2 and KIR3DL3.

[0209] KIR3DL2 (See example,) Figures 148-150 - Although HLA-A with bound P20 peptide The interaction between 11:01APC-coupled tetramer and KIR3DL2-transfected Ba / F3 cells was initially very low, but the interaction between KIR3DL2 and its ligand increased when mAb S22025B was added.

[0210] KIR2DS1 (See example,) Figures 145-147- In HLA-C with a proprietary UVX peptide that binds The interaction between the 04:01 monomer and the KIR2DS1 transfected Ba / F3 was eliminated by the addition of mAbs S22019B, S22019F, S22013A and S22021B.

[0211] KIR2DL5 (See example,) Figures 143-144 Although the initial interaction between recombinant CD155-biotin and KIR2DL5A-transfected Ba / F3 cells was extremely low, the interaction between KIR2DL5 and its ligand increased upon the addition of mAb S22010G. This KIR-ligand enhancement was found to be specific to the interaction between KIR2DL5 and CD155, while this was not the case with ligands containing similar amino acid sequences such as CD112 or CD113.

[0212] KIR2DS4 (See example,) Figures 140-142 - In HLA-C with bound P2-AW peptide (IIDKSGAWV) 05:01 The interaction between the APC-coupled tetramer and KIR2DS4 is completely disrupted by mAb S22012B, C, E, F, and G. mAb S22012A and S22012H also slightly disrupt this KIR-ligand interaction.

[0213] KIR3DL1 (See example,) Figures 137-139 - In HLA-B with bound "self-peptide" (LSSPVTKSF) 57:01 The interactions between the tetramers coupled with APC were completely disrupted by mAb S22024A, B, C, D, E, F, G, H and S22026A, B and S23011E, F, G.

[0214] KIR2DL1 (See example,) Figures 131-132 - In HLA-C with bound CMV-modified peptide (QYDDAVYKL) 04:01 The interaction between the tetramers coupled with APC was completely disrupted by mAb S22039A, B, C, D and S22013A.

[0215] KIR2DL2 (See example,) Figures 133-134 - In HLA-C with a proprietary UVX peptide that binds The interaction between the monomer and KIR2DL2 transfected Ba / F3 cells was completely disrupted by mAbs S22036B, S22036D, and S22037E, F, and G. mAbs S22020A and S22034A, B, C, and D also partially reduced KIR-ligand interactions.

[0216] KIR2DS2 (See example,) Figure 133 , 135 - In HLA-C with a proprietary UVX peptide that binds The interaction between the monomer and the KIR2DS2 transfected Ba / F3 was completely disrupted by mAbs S22032A, S22032C, S22034A, B, C, D, S22036B, S22036D and S22038E.

[0217] KIR2DL3 (See example,) Figure 133 , 136 - In HLA-C with a proprietary UVX peptide that binds The interaction between the monomer and KIR2DL3-transfected Ba / F3 cells was completely disrupted by mAbs S22036B, S22036D, S22037E, S22037F, and S22037G. mAbs S22034A, B, C, and D also partially reduced KIR-ligand interactions.

[0218] Degranulation of KIR3DL1+ NK cells was reduced in the presence of KIR3DL1-specific antibodies. To determine the effect of KIR3DL1-specific antibodies on primary NK cells, IL-2 and IL-15-activated primary NK cells were cultured with the K562 cell line in the presence of anti-KIR3DL1 antibody and allotype control, and changes in CD107a expression were measured by flow cytometry. Most NK cells without KIR3DL1+ expression did not show significant changes between allotype control and anti-KIR3DL1 antibody samples, except for slight increases observed in mAbs S22026A and S23011B. For samples with the developed anti-KIR3DL1 antibody, CD107a expression was decreased (28.9–51.8%) compared to KIR3DL1- NK cells when comparing KIR3DL1+ NK cells, with the largest decreases observed in mAbs S22024A, S22024B, S22024E, and S22024H. Figure 124 In normalized KIR3DL1+ NK cells, minimal antibody-mediated decrease in CD107a was induced by mAbS23011A, S23011B, and S23011C.

[0219] Degranulation of KIR2DS1+ NK cells is increased due to the presence of KIR2DS1-specific antibodies. Similarly, to determine the effect of KIR2DS1-specific antibodies on primary NK cells, anti-KIR2DS1 antibodies were added to IL-2 and IL-15-activated primary NK cells and the K562 cell line, and changes in CD107a expression were measured by flow cytometry. In KIR2DS1- NK cells, the addition of mAb S22019B resulted in a slight but significant decrease in CD107a expression compared to the isotype, while mAb S22019F showed no significant difference compared to the isotype. In KIR2DS1+ NK cells, based on the normalized CD107a activation ratio, the addition of mAb S22019B and S22019F showed a significant increase in CD107a expression (38% and 36%, respectively). Figure 126 ).

[0220] The response of isolated NK cells to the K562 cell line was regulated by the presence of a KIR2DS4-specific antibody. The effect of the presence of KIR2DS4-specific antibodies on the activation of NK cell responses was also tested using K562 co-cultures. When CD107a expression was measured in all NK cells within the samples, all samples supplemented with the developed anti-KIR2DS4 antibodies (S22012A, B, C, E, F, G, H) showed a significant increase of 20.2%–20.5% in CD107a expression compared to normalized isotypes. Figure 128 Conversely, the population of CD107a-negative NK cells also decreased. Furthermore, when analyzing in-solution cytokine secretion in response to the K562 cell line and anti-KIR2DS4 antibody, the addition of KIR2DS4 showed an increase in in-solution TNFα concentrations for most developed anti-KIR2DS4 antibodies, while IFNγ also showed a trend towards increased in-solution concentrations compared to the isotype. Figure 129 When measuring changes in K562 cell viability, the addition of the developed anti-KIR2DS4 antibody showed an increase in viable K562 cells (26.5% - 32.5%) compared to the isotype control. Figure 130 ).

[0221] An overview of the functional results of certain clones (see also: Figure 8 and 102 ) S22012A (KIR2DS4): • High flow cytometry performance and specificity - see Figures 29-35 • Higher performance than direct mAb competitors in flow cytometry - see Figure 105 , 113 • Epitopes different from those of competing agents - see [link to product] Figure 114 • Partial functional ligand blocking effect, but not as significant as other clones in this target group - see also Figures 140-142 • Well-defined functional effects on NK cells, including CD107a expression, cytokine expression, and K562 changes - see [link to relevant documentation] Figures 127-130 S22024B (KIR3DL1): • High flow cytometry performance and specificity - see Figure 36-41 • Unique epitopes distinguishing it from competing agents - see [link] Figure 121 • Significant functional effects on ligand binding interactions - see also Figures 137-139 • Well-defined functional data on changes in CD107a expression on NK cells - see [link to relevant documentation] Figures 122-124 S22025B (KIR3DL2): • High flow cytometry performance and specificity - see Figure 19-28 • Epitopes distinct from those in similar clones - see also Figure 24 • Significant functional effects on ligand binding interactions - see also Figures 148-150 S22019F (KIR2DS1): • High flow cytometry performance and specificity - see Figure 9-18 • Significant functional effects on ligand binding interactions - see also Figures 145-147 • Well-defined functional data on changes in CD107a expression on NK cells - see [link to relevant documentation] Figures 125-126 S22013A (KIR2DS1 / KIR2DL1): • High flow cytometry performance and specificity - see Figure 10 , 11 14, 17-18, 80, 83-15 • Higher performance than direct mAb competitors in flow cytometry - see Figure 104 , 112 • Significant functional effects on ligand binding interactions of the two KIR targets - see [link to relevant documentation] Figures 131-132 and 145-147 S22032A (KIR2DL2 / KIR2DS2): • High flow cytometry performance and specificity - see Figures 64-78 86-87, 92-101 • There is no direct mAb competitor for the staining pattern (2DL2 / 2DS2 specificity). • Epitopes distinct from those in similar clones - see also Figures 76-77 • Significant functional effects on ligand-binding interactions of KIR2DS2 - see [link] Figure 133 , 135 S22037G (KIR2DL2 / KIR2DL3): • High flow cytometry performance and specificity - see Figures 64-78 88-89, 92-101 • There is no direct mAb competitor for the staining pattern (2LD2 / 2DL3 specificity). • Epitopes distinct from those in similar clones - see also Figure 76 , 78 • Significant functional effects on ligand binding interactions of the two KIR targets - see [link to relevant documentation] Figure 133 , 134 136 S22038E (KIR2DL2 / KIR2DS2 / KIR2DL3): • High flow cytometry performance and specificity - see Figures 64-78 90-91, 92-101 • Higher performance than direct mAb competitors for staining patterns - see Figures 133-136 S22039A (KIR2DL1): • High flow cytometry performance and specificity - see Figure 64-85 • Epitopes different from those of direct competitors - see [link to relevant documentation] Figure 84 • Significant functional effects on ligand binding interactions - see also Figures 131-132 S22010E (KIR2DL5): • High flow cytometry performance and specificity - see Figures 42-54 • Higher performance than direct mAb competitors - see Figure 49 • Epitopes different from those of direct competitors - see [link to relevant documentation] Figure 50 • Can distinguish more protein variants than direct competitors - see Figures 46-49 S23011E (KIR3DL3): • High flow cytometry performance and specificity - see Figures 55-59 • No direct mouse mAb competitor • Significant functional effects on ligand binding interactions - see also Figures 151-152 S23004D (KIR2DL4): • High flow cytometry performance and specificity - see Figure 1-7 • Higher performance than direct mAb competitors - see Figure 106 , 114 • Epitopes different from those of direct competitors - see [link to relevant documentation] Figure 117 The entire contents of each patent, patent application, publication, and document cited herein are incorporated by reference. The citation of any of these patents, patent applications, publications, and documents does not constitute an admission that any of the foregoing documents is relevant prior art, nor does it constitute any admission of the content or dates of such publications or documents. Their citations do not instruct a search for relevant disclosures. All statements regarding the dates or content of the cited documents are based on available information and do not constitute an admission of their accuracy or correctness.

[0222] The present invention has been described with reference to specific embodiments. The terminology and expressions used herein to describe the present invention are descriptive and not necessarily limiting. Certain modifications to the disclosed embodiments may be considered within the scope of the present invention. Certain aspects of the disclosed embodiments may be suitably implemented in the presence or absence of certain elements not expressly disclosed herein.

[0223] The terms “comprising,” “substantially consisting of,” and “composed of” can each be replaced by any two other terms. The term “a” or “an” can refer to one or more elements it modifies (e.g., “reagent” can mean one or more reagents) unless describing one or more elements is obvious from the context. The term “about,” as used herein, refers to a value within 10% of the base parameter (i.e., plus or minus 10%; for example, a weight of “about 100 grams” can include a weight between 90 grams and 110 grams). The term “about” is used at the beginning of a list of values ​​to modify each stated value (e.g., “about 1, 2, and 3” means “about 1, about 2, and about 3”). When describing a list of values, the list includes all its intermediate values ​​and all fractional values ​​(e.g., the list of values ​​“80%, 85%, or 90%” includes the intermediate value 86% and the fractional value 86.4%). When a list of values ​​is followed by the term "or more," the term "or more" applies to each of the values ​​listed (for example, the list "80%, 90%, 95%, or more," or "80%, 90%, 95%, or more," or "80%, 90%, or 95% or more" means "80% or more, 90% or more, or 95% or more"). When describing a list of values, the list includes all ranges between any two values ​​listed (for example, the list "80%, 90%, or 95%" includes the ranges "80%-90%," "80%-95%," and "90%-95%").

[0224] Some embodiments of this technology are set forth in the appended claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to one or more members of the killer cell immunoglobulin-like receptor (KIR) protein family or portions thereof, said antibody comprising: (i) an immunoglobulin heavy chain comprising a set of heavy chain complementarity-determining region (CDR) amino acid sequences CDRH1, CDRH2, and CDRH3; and (ii) an immunoglobulin light chain comprising a set of light chain CDR amino acid sequences CDRL1, CDRL2, and CDRL3, wherein the heavy chain and light chain CDR sets are each selected from the same group A, B, C, D, E, F, G, H, I, J, K, or L: 。 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 2 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 2, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 4 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

4.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 62 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 62, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 64 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

64.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 82 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 82, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 84 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

84.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 242 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 242, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 244 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

244.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 402 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 402, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 404 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

404.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 422 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 422, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 424 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

424.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 502 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 502, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 504 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

504.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 602 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 602, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 604 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

604.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 742 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 742, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 744 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

744.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 942 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 942, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 944 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

944.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 962 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 962, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 964 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

964.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 982 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO: 982, and wherein the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 984 or a sequence having at least 80% amino acid sequence identity with SEQ ID NO:

984.

14. A diagnostic antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising the antibody or antigen-binding fragment according to any one of claims 1-13.

15. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-13.

16. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-13 and a pharmaceutically acceptable excipient.

17. An isolated nucleic acid comprising a nucleotide sequence encoding a variable domain of an immunoglobulin heavy chain that encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1-13.

18. An isolated nucleic acid comprising a nucleotide sequence encoding a variable domain of an immunoglobulin light chain that encodes an antibody or an antigen-binding fragment thereof according to any one of claims 1-13.

19. A recombinant expression vector comprising the isolated nucleic acid according to claim 17 and / or the isolated nucleic acid according to claim 18.

20. A host cell comprising the nucleic acid according to claim 17 and / or the nucleic acid according to claim 18, or the recombinant expression vector according to claim 19.

21. A recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding a variable domain of an immunoglobulin heavy chain according to any one of claims 1-13, and the second expression cassette comprises a nucleic acid molecule comprising a nucleotide sequence encoding a variable domain of an immunoglobulin light chain of an antibody or an antigen-binding fragment thereof according to any one of claims 1-13.

22. A host cell comprising the recombinant expression vector according to claim 21.

23. A method for detecting one or more members of the KIR protein family, the method comprising: Under conditions that allow the antibody or its antigen-binding fragment according to any one of claims 1-13 to bind to one or more KIR protein family members in the sample, the sample is contacted with the antibody or its antigen-binding fragment, wherein the binding results in the formation of one or more KIR protein family member / antibody or its antigen-binding fragment complexes.

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