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WO2025230843A2 - Anticorps se liant au kir et leurs fragments se liant à l'antigène - Google Patents

Anticorps se liant au kir et leurs fragments se liant à l'antigène

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Publication number
WO2025230843A2
WO2025230843A2 PCT/US2025/026470 US2025026470W WO2025230843A2 WO 2025230843 A2 WO2025230843 A2 WO 2025230843A2 US 2025026470 W US2025026470 W US 2025026470W WO 2025230843 A2 WO2025230843 A2 WO 2025230843A2
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WIPO (PCT)
Prior art keywords
amino acid
antibody
substituted
seq
kir
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/026470
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English (en)
Other versions
WO2025230843A3 (fr
Inventor
Evan TENG
ChengRui LI
Takatoku Oida
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Biolegend Inc
Original Assignee
Biolegend Inc
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Publication of WO2025230843A2 publication Critical patent/WO2025230843A2/fr
Publication of WO2025230843A3 publication Critical patent/WO2025230843A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the technology relates in part to antibodies or antigen-binding fragments thereof that bind one or more members of the KIR family of proteins (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 detection of KIR.
  • the technology relates to antibodies or antigen binding fragments thereof for use in determining levels of KIR in a sample containing or suspected of containing KIR.
  • the technology relates to antibodies or antigen-binding fragments thereof for use in diagnosing or treating an individual with or suspected of having a disease or disorder associated with KIR.
  • KIR Killer Cell Immunoglobulin-like Receptors
  • the KIR family of proteins are transmembrane glycoproteins and part of the immunoglobulin superfamily.
  • KIR proteins include an extracellular structure and an intracellular structure. Extracellular KIR domains may be characterized as immunoglobulin-like domains. Intracellular domains contain ITAM (activating) and/or ITIM (inhibitory) regions depending on the variant.
  • KIR proteins range in size from about 30 kDa to about 50 kDa.
  • KIR proteins interact with MHC Class 1 molecules (HLA) on a target cell to prevent cell killing and lysis of the target cell by NK cells while other KIR proteins react to HLAs to cause activation or inhibition in NK cells (Pende, D. et al. Front Immunol, (2019), Debska-Zielkowska, J. et al. Cells (2021 )).
  • HLA MHC Class 1 molecules
  • KIR receptors High homology exists among KIR receptors. Accordingly, antibodies specifically recognizing certain members of the KIR family have not been developed. In order to generate antibodies targeting certain members of the KIR family specifically, a collection of monoclonal antibodies was generated against certain KIR receptor family members, including KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and KIR3DL3, for example. The specificity of these antibodies was verified using engineered cell lines and human peripheral blood samples.
  • KIR2DS1 -specific monoclonal antibody described herein can distinguish between the closely related KIR2DL1 versus KIR2DS1 proteins; KIR2DL5-specific monoclonal antibodies described herein can better target broader KIR2DL5 variants compared to current commercially available clones.
  • multiple clones are able to determine shared epitopes of two or more KIRs including KIR2DL2, KIR2DL3, and KIR2DS2.
  • Such monoclonal antibodies may have applications in immunophenotyping, diagnostics, and therapeutics, for example.
  • antibodies or antigen binding fragments thereof that bind one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, 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 that is at least 80 percent identical to 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 that is at least 80 percent identical to 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 that is at least 80 percent identical to a CDRH3 polypeptide provided in Table 3; and (b) an immunoglobulin heavy chain
  • isolated antibodies or antigen binding fragments thereof that bind one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, where the antibody or antigen binding fragment thereof blocks or is capable of blocking KIR ligand binding.
  • KIRs killer cell immunoglobulin-like receptors
  • isolated antibodies or antigen binding fragments thereof that bind one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, where the antibody or antigen binding fragment thereof enhances or is capable of enhancing KIR ligand binding.
  • KIRs killer cell immunoglobulin-like receptors
  • isolated antibodies or antigen binding fragments thereof that bind one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, where the antibody or antigen binding fragment thereof inhibits or is capable of inhibiting natural killer cell activity.
  • KIRs killer cell immunoglobulin-like receptors
  • isolated antibodies or antigen binding fragments thereof that bind one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, where the antibody or antigen binding fragment thereof activates or is capable of activating natural killer cell activity.
  • KIRs killer cell immunoglobulin-like receptors
  • antibodies or antigen binding fragments thereof that bind one or more killer cell immunoglobulin-like receptor (KIR) protein family members or a portion or portions thereof, where the antibody comprises: (i) an immunoglobulin heavy chain comprising a set of heavy chain complementarity determining region (CDR) amino acid sequences, CDRH1 ,
  • an immunoglobulin light chain comprising a set of light chain CDR amino acid sequences, CDRL1 , CDRL2, and CDRL3, wherein the sets of heavy chain and light chain CDRs are each chosen from the same of set A, B, C, D, E, F, G, H, I, J, K, or L:
  • Fig. 1 shows a screening matrix of potential KIR2DL4 staining clones using transfected cell line Ba/F3. Positives are noted as ‘Y’ and negatives are noted as N’. This matrix shows specificity for a particular KIR, in this case KIR2DL4 for S23004D, S22010B, S22010H, and mAb-33.
  • Fig. 2 shows histograms of clones tested for two positive cell lines (KIR2DL4-55 and KIR2DL4-71 transfected Ba/F3) which the clones show positivity for KIR2DL4.
  • Fig. 3 shows titration data from 2 pg to 0.0625 pg for each clone on two KIR2DL4 (2DL4-55 and 2DL4-71 ) cell lines showing high mean fluorescence signals at each concentration. S23004D is highest shown here.
  • Fig. 4 shows KIR2DL4 antibody crossblocking. Specifically, shown here is results of an epitope interaction assay where one antibody is used to block another to create the matrix. No changes from blocking are ‘signal’, loss of signal is ‘block’, and partial decreases are ‘partial’. Some interactions have increases in signal designated ‘higher’. This shows S23004D has a different epitope than the others and/or has much stronger binding.
  • Fig. 5 shows primary peripheral blood lymphocyte staining using CD56 (NK marker) and KIR2DL4 testing antibodies. The positive population in this case is the CD56 high and Y axis positive population, showing positive primary cell population identification.
  • Fig. 6 shows KIR2DL4 expression in a population of IL-2 stimulated PBMCs.
  • stimulation may show a larger population of positives.
  • the comparison stain shows S23004D can identify the population better than other clones.
  • Fig. 7 shows interactions of test clones and reference clones with KIR2DL4 recombinant protein.
  • this figure shows recombinant protein neutralization of the test clones which show reduced staining when blocked by recombinant protein KIR2DL4. This indicates the antibodies are specific to KIR2DL4.
  • Fig. 8 shows certain clones as novel anti-KIR binding agents (single-specific in bold, multi-specific non-bolded).
  • Figs. 9-18 show data generated for anti-KIR2DS1 clones by methodologies described herein.
  • Figs. 19-28 show data generated for anti-KIR3DL2 clones by methodologies described herein.
  • Figs. 29-35 show data generated for anti-KIR2DS4 clones by methodologies described herein.
  • Figs. 36-41 show data generated for anti-KIR3DL1 clones by methodologies described herein.
  • Figs. 42-48 show data generated for anti-KIR2DL5 clones by methodologies described herein.
  • Fig. 49 shows certain clones cover broader polymorphic variants than currently available clones for KIR2DL5.
  • clone S22010E is able to bind to more polymorphic KIR2DL5 variants than commercial clone UP-R1 , better covering for donor-dependent KIR expression differences.
  • Top panel Staining of Ba/F3 cells expressing different KIR2DL5 polymorphic variants with clones S22010E and UP-R1 .
  • Bottom panel SNP variants of KIR2DL5 at positions 141 , 173, and 195.
  • Figs. 50-54 show data generated for anti-KIR2DL5 clones by methodologies described herein.
  • Figs. 55-59 show data generated for anti-KIR3DL3 clones by methodologies described herein.
  • Figs. 60-63 show data generated for anti-KIR2DS5 clones by methodologies described herein.
  • Figs. 64-101 show data generated for anti-KIR2DL1 , anti-KIR2DL2, anti-KIR2DS2, and/or anti- KIR2DL3 clones by methodologies described herein.
  • Fig. 102 shows certain KIR-specific clones can characterize KIR positive NK populations. Data shown is pre-gated on a lymphocyte population of peripheral blood, and samples were stained with anti-CD56 APC and specific clones as indicated. Cells were analyzed by flow cytometry. Figs. 103A-103C show certain clones can affect KIR-ligand interactions. Fig. 103A: For KIR2DS1 , HLA-C*04:01 is a reported ligand for KIR2DS1 . The results indicate KIR2DS1 -binding clones S22019F and S22013A block HLA-C*04:01 tetramer from binding to KIR2DS1. Fig.
  • KIR3DL2 For KIR3DL2, HLA-A*11 :01 is a reported ligand for KIR3DL2. The results indicate KIR3DL2-binding clone S22025B enhances HLA-A*1 1 :01 tetramer binding to KIR3DL2.
  • Fig. 107C For KIR3DL3, HHLA2 is a reported ligand for KIR3DL3. The results indicate KIR3DL3-binding clone S23011 E blocks HHLA2 from binding to KIR3DL3.
  • Figs. 104-121 show data characterizing the aforementioned anti-KIR clones in relation to other commercial antibodies.
  • Fig. 122 shows a representative image of activation in a KIR3DL1 cytotoxicity assay and representative effects of HLA or antibody addition.
  • Fig. 123 shows CD107a activation change results from a KIR3DL1 cytotoxicity assay.
  • Fig. 124 shows an effect of KIR3DL1 -binding antibody addition to normalized CD107a activation ratio of NK cells in NK-K562 co-culture.
  • NK cells in NK-K562 co-culture were divided and analyzed according to KIR3DL1 positivity.
  • CD107a activation ratio was calculated by the ratio of percentage of NK cells with CD107a positivity compared to percentage of NK cells with CD107a negativity. All samples were normalized to isotype control measurements (MOPC-21 ).
  • **** p-value ⁇ 0.0001
  • *** p-value ⁇ 0.001 by Brown-Forsythe and Welch one-way ANOVA.
  • n 7 to 21 samples per group.
  • Fig. 125 shows a representative image of activation in a KIR2DS1 cytotoxicity assay and representative effects of HLA or antibody addition.
  • Fig. 126 shows an effect of KIR2DS1 -specific antibody addition to normalized CD107a activation ratio of NK cells in NK-K562 co-culture.
  • NK cells in NK-K562 co-culture were divided and analyzed according to KIR2DS1 positivity.
  • CD107a activation ratio was calculated by the ratio of percentage of NK cells with CD107a positivity compared to percentage of NK cells with CD107a negativity. All samples were normalized to isotype control measurements (MOPC-21 ).
  • **** p-value ⁇ 0.0001
  • *** p-value ⁇ 0.001 by Brown-Forsythe and Welch two-way ANOVA.
  • n 16 samples per group.
  • Fig. 127 shows a representative image of activation in a KIR2DS4 cytotoxicity assay and representative effects of HLA or antibody addition.
  • Fig. 128 shows an effect of KIR2DS4-specific antibody addition to change in activated NK cells in NK-K562 co-culture.
  • NK cells in NK-K562 co-culture were divided and analyzed according to CD107a positivity. Samples were normalized to isotype control (MOPC-21 ) and statistical comparisons of CD107a+ made to isotype control.
  • *** p-value ⁇ 0.001
  • ** p-value ⁇ 0.01
  • ns not significant by Turkey two-way ANOVA.
  • n 11 to 22 samples per group.
  • Fig. 129 shows an effect of KIR2DS4-specific antibody addition to TNFa and IFNy secretion in NK- K562 co-culture.
  • NK-K562 co-culture supernatant was analyzed for TNFa and IFNy concentration in solution.
  • n 8 to 16 samples per group.
  • Fig. 130 shows an effect of KIR2DS4-specific antibody addition to K562 survival in NK-K562 coculture.
  • Figs. 131 -132 show ligand binding interaction data generated for anti-KIR2DL1 clones by methodologies described herein.
  • Figs. 133-136 show ligand binding interaction data generated for anti-KIR2DL2, anti-KIR2DS2, and anti-KIR2DL3 clones by methodologies described herein.
  • Figs. 137-139 show ligand binding interaction data generated for anti-KIR3DL1 clones by methodologies described herein.
  • Figs. 140-142 show ligand binding interaction data generated for anti-KIR2DS4 clones by methodologies described herein.
  • Fig. 143 shows ligand binding interaction data generated for anti-KIR2DL5 clones by methodologies described herein.
  • Fig. 144 shows certain clones can affect KIR-ligand interactions.
  • KIR2DL5A transfected Ba/F3 cells were incubated with CD155-FC, CD112-FC, or CD1 13-FC. Anti-FC antibody was used to detect the binding in flow cytometry.
  • certain clones were added to cells before ligand incubation to determine if the clones can affect ligand binding.
  • CD155 has previously been reported to be a ligand of KIR2DL5; CD112 and CD113 which are similar to CD155, were used as negative controls. The results indicate KIR2DL5A-binding clone S22010G enhances CD155 binding to KIR2DL5A.
  • Figs. 145-147 show ligand binding interaction data generated for anti-KIR2DS1 clones by methodologies described herein.
  • Figs. 148-150 show ligand binding interaction data generated for anti-KIR3DL2 clones by methodologies described herein.
  • Figs. 151 -152 show ligand binding interaction data generated for anti-KIR3DL3 clones by methodologies described herein.
  • killer cell immunoglobulin-like receptor (KIR) binding antibodies e.g., antibodies that bind to KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3
  • KIR killer cell immunoglobulin-like receptor
  • vectors e.g., recombinant expression vectors
  • cells e.g., recombinant cells
  • antibodies and antigen binding fragments thereof can bind to KIR under physiological and/or in vitro conditions.
  • methods of producing and using KIR binding antibodies and antigen-binding fragments thereof such as in methods for detecting KIR in a sample from an individual, including methods for laboratory/ research purposes (e.g., flow cytometry, ELISA, and/or Western blot), and/or for the use and treatment and/or prevention of various diseases or disorders through the delivery of pharmaceutical or other compositions that contain such antibodies or antigen-binding fragments thereof.
  • KIR killer cell immunoglobulin-like receptor
  • agents that bind KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3 or portions thereof are also known as CD158.
  • anti-KIR agents may include anti-KIR antibodies, anti-KIR antibody fragments (e.g., antigen binding fragments), and anti-KIR antibody derivatives.
  • the agent is isolated (e.g., separated from a component of its natural environment (e.g., an animal, a biological sample)).
  • the agent is non-naturally occurring (e.g., produced by human intervention).
  • the agent is a humanized antibody, or an antigen binding fragment thereof.
  • the agent is a derivative of a humanized antibody that binds KIR.
  • the agent binds KIR under laboratory conditions (e.g., binds KIR in vitro, binds KIR in a flow cytometry assay, binds KIR in an ELISA). In some embodiments, the agent binds KIR under physiological conditions (e.g., binds KIR in a cell in a subject). In some embodiments, the agent affects KIR signaling. In some embodiments, the agent blocks or is capable of blocking KIR signaling. In some embodiments, the agent enhances or is capable of enhancing KIR signaling. In some embodiments, the agent affects ligand binding to KIR. In some embodiments, the agent blocks or is capable of blocking ligand binding to KIR.
  • an anti-KIR agent herein may block binding of HLA-C*04:01 to KIR2DS1.
  • an anti- KIR agent herein may block binding of HHLA2 to KIR3DL3.
  • the agent enhances or is capable of enhancing ligand binding to KIR.
  • an anti-KIR agent provided herein may enhance binding of CD155 to KIR2DL5A.
  • an anti-KIR agent provided herein may enhance binding of HLA-A*11 :01 to KIR3DL2.
  • KIR ligand binding may be assessed using any suitable assay, such as a ligand binding assay as demonstrated in Example 2, Figs. 103A-C, and Figs. 131 -152, for example.
  • the agent affects immune cell activity. In some embodiments, the agent affects natural killer cell activity. In some embodiments, the agent inhibits natural killer cell activity. In some embodiments, the agent activates natural killer cell activity. Natural killer cell activity may be assessed using any suitable assay, such as a cytotoxicity assay (e.g., degranulation assay as measured by a release of cytotoxic granules, which is marked by surface expression of CD107a), a cytokine (e.g., TNFa, IFNy) expression and/or secretion assay, and a natural killer target cell survival assay (e.g., K562 cell survival assay) as demonstrated in Example 2 and Figs. 122-130.
  • a cytotoxicity assay e.g., degranulation assay as measured by a release of cytotoxic granules, which is marked by surface expression of CD107a
  • a cytokine e.g., TNFa, IFNy
  • secretion assay e
  • an anti-KIR agent provided herein comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain.
  • an anti-KIR agent herein comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • each immunoglobulin heavy chain variable domain of the anti-KIR agent comprises first, second, and third heavy chain complementarity determining regions (CDRs; CDRH1 , CDRH2, CDRH3)
  • each immunoglobulin light chain variable domain of the anti-KIR agent comprises first, second, and third light chain CDRs (CDRL1 , CDRL2, CDRL3).
  • an anti-KIR agent provided herein comprises a heavy chain complementarity determining region 1 (CDRH1 ) comprising a CDRHI polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to a CDRH1 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH1 comprising a polypeptide that is at least 85 percent identical to a CDRH1 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH1 comprising a polypeptide that is at least 90 percent identical to a CDRH1 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH1 comprising a polypeptide that is at least 95 percent identical to a CDRH1 polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises a CDRH1 comprising a polypeptide that is 100 percent identical to a CDRH1 polypeptide provided in Table 3.
  • an anti-KIR agent provided herein comprises a heavy chain complementarity determining region 2 (CDRH2) comprising a CDRH2 polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to a CDRH2 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH2 comprising a polypeptide that is at least 85 percent identical to a CDRH2 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH2 comprising a polypeptide that is at least 90 percent identical to a CDRH2 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH2 comprising a polypeptide that is at least 95 percent identical to a CDRH2 polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises a CDRH2 comprising a polypeptide that is 100 percent identical to a CDRH2 polypeptide provided in Table 3.
  • an anti-KIR agent provided herein comprises a heavy chain complementarity determining region 3 (CDRH3) comprising a CDRH3 polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to a CDRH3 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH3 comprising a polypeptide that is at least 85 percent identical to a CDRH3 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH3 comprising a polypeptide that is at least 90 percent identical to a CDRH3 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRH3 comprising a polypeptide that is at least 95 percent identical to a CDRH3 polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises a CDRH3 comprising a polypeptide that is at 100 percent identical to a CDRH3 polypeptide provided in Table 3.
  • an anti-KIR agent provided herein comprises a light chain complementarity determining region 1 (CDRL1 ) comprising a CDRL1 polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to a CDRL1 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL1 comprising a polypeptide that is at least 85 percent identical to a CDRL1 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL1 comprising a polypeptide that is at least 90 percent identical to a CDRL1 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL1 comprising a polypeptide that is at least 95 percent identical to a CDRL1 polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises a CDRL1 comprising a polypeptide that is 100 percent identical to a CDRL1 polypeptide provided in Table 3.
  • an anti-KIR agent provided herein comprises a light chain complementarity determining region 2 (CDRL2) comprising a CDRL2 polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to a CDRL2 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL2 comprising a polypeptide that is at least 85 percent identical to a CDRL2 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL2 comprising a polypeptide that is at least 90 percent identical to a CDRL2 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL2 comprising a polypeptide that is at least 95 percent identical to a CDRL2 polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises a CDRL2 comprising a polypeptide that is 100 percent identical to a CDRL2 polypeptide provided in Table 3.
  • an anti-KIR agent provided herein comprises a light chain complementarity determining region 3 (CDRL3) comprising a CDRL3 polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to a CDRL3 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL3 comprising a polypeptide that is at least 85 percent identical to a CDRL3 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL3 comprising a polypeptide that is at least 90 percent identical to a CDRL3 polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises a CDRL3 comprising a polypeptide that is at least 95 percent identical to a CDRL3 polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises a CDRL3 comprising a polypeptide that is at least 100 percent identical to a CDRL3 polypeptide provided in Table 3.
  • an anti-KIR agent provided herein binds to KIR2DS1 or a portion thereof, which may be referred to herein as an anti-KIR2DS1 agent.
  • an anti- KIR2DS1 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti-KIR2DS1 agent comprises a CDRH1 comprising the polypeptide GYX1FTX2X3W where Xi is S or T, X2 is T or R, and X 3 is Y or N.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e. , conservative to S and/or T).
  • the amino acid Xi may be substituted with S, T, C, N, A, or G.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or R). In some embodiments, the amino acid X 2 may be substituted with T, R, S, C, N, A, K, or H.
  • the amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to Y and/or N). In some embodiments, the amino acid X 3 may be substituted with Y, N, F, W, S, T, H, Q, or D.
  • the amino acid X 3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • an anti-KIR2DS1 agent comprises a CDRL2 comprising the polypeptide XiTS where Xi is Y or R.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Y and/or R).
  • the amino acid Xi may be substituted with Y, R, F, W, H, S, T, K, H, or Q.
  • an anti-KIR2DS1 agent comprises a CDRL3 comprising the polypeptide QQGXiX 2 X 3 PX4T where Xi is K or S, X 2 is T or S, X 3 is L or I, and X4 is Y or L.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to K and/or S).
  • the amino acid Xi may be substituted with K, S, R, H, T, C, N, or G.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or S). In some embodiments, the amino acid X 2 may be substituted with T, S, C, N, A, or G. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to L and/or I). In some embodiments, the amino acid X 3 may be substituted with L, I, V, M, A, or F.
  • a conservative amino acid i.e., conservative to T and/or S
  • the amino acid X 2 may be substituted with T, S, C, N, A, or G.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e
  • the amino acid X 3 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • the amino acid X4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to Y and/or L).
  • the amino acid X 4 may be substituted with Y, L, F, W, H, S, T, I, V, M, or A.
  • an anti-KIR agent provided herein binds to KIR2DS4 or a portion thereof, which may be referred to herein as an anti-KIR2DS4 agent.
  • an anti- KIR2DS4 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti-KIR2DS4 agent comprises a CDRH1 comprising the polypeptide GYSFXIGYX 2 where Xi is T or S and X 2 is T or Y.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to T and/or S).
  • the amino acid Xi may be substituted with T, S, C, N, A, or G.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or Y).
  • the amino acid X 2 may be substituted with T, S, C, N, A, F, W, or H.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • an anti-KIR2DS4 agent comprises a CDRH2 comprising the polypeptide IHPYNGX1T where Xi is D or A.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to D and/or A). In some embodiments, the amino acid Xi may be substituted with D, A, E, N, S, G, V, or L. In some embodiments, an anti-KIR2DS4 agent comprises a CDRH2 comprising the polypeptide ISYSXIX 2 T where Xi is G or A and X 2 is S or T. The amino acid Xi may be substituted with any amino acid. In some embodiments, the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to G and/or A).
  • the amino acid Xi may be substituted with G, A, V, S, L, P, or C. In some embodiments, the amino acid Xi may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to S and/or T). In some embodiments, the amino acid X 2 may be substituted with T, S, C, N, A, or G.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • an anti-KIR2DS4 agent comprises a CDRH3 comprising the polypeptide ARXIX 2 GSX 3 X4X5AWFAY where Xi is Q or R, X 2 is Y or A, X 3 is no amino acid or S, X4 is no amino acid or Y, and X 5 is L or K.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Q and/or R).
  • the amino acid Xi may be substituted with Q, R, N, E, H, S, T, or K.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to Y and/or A).
  • the amino acid X 2 may be substituted with Y, A, F, W, H, S, T, G, V, or L.
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to S).
  • the amino acid X 3 may be substituted with S, T, C, N, or G. In some embodiments, the amino acid X 3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 4 may be substituted with any amino acid. In some embodiments, the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to Y). In some embodiments, the amino acid X 4 may be substituted with Y, F, W, H, S, or T.
  • the amino acid X 4 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, the amino acid X 4 may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • the amino acid X 5 may be substituted with any amino acid. In some embodiments, the amino acid Xs may be substituted with a conservative amino acid (i.e., conservative to L and/or K). In some embodiments, the amino acid Xs may be substituted with L, K,
  • an anti-KIR2DS4 agent comprises a CDRH3 comprising the polypeptide XiX 2 SKYX 3 NSYAX 4 DY where Xi is T or A, X 2 is R or Q, X 3 is A or S, and X 4 is V or M.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to T and/or A).
  • the amino acid Xi may be substituted with T, A,
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to R and/or Q). In some embodiments, the amino acid X 2 may be substituted with R, Q, K, H, N, E, S, or T.
  • the amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to A and/or S). In some embodiments, the amino acid X 3 may be substituted with A, S, G, V, L, T, C, or N.
  • the amino acid X 4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to V and/or M).
  • the amino acid X 4 may be substituted with V, M, I, L, A, or F.
  • the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • an anti-KIR2DS4 agent comprises a CDRL2 comprising the polypeptide GX1S where Xi is A or S.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to A and/or S). In some embodiments, the amino acid Xi may be substituted with A, S, G, V, L, T, C, or N. In some embodiments, an anti-KIR2DS4 agent comprises a CDRL3 comprising the polypeptide QNDHX1YPPT where Xi is S or I. The amino acid Xi may be substituted with any amino acid. In some embodiments, the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or I).
  • amino acid Xi may be substituted with S, I, T, C, N, G, L, V, M, A, or F.
  • an anti-KIR agent provided herein binds to KIR3DL1 or a portion thereof, which may be referred to herein as an anti-KIR3DL1 agent.
  • an anti- KIR3DL1 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti-KIR3DL1 agent comprises a CDRH1 comprising the polypeptide GYX1FX2X3YX4 where Xi is S or T, X 2 is T or S, X 3 is D or T or N, and X4 is T, Y, S, A or W.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or T).
  • the amino acid Xi may be substituted with S, T, C, N, A, or G.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or S).
  • the amino acid X 2 may be substituted with S, T, C, N, A, or G.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to D and/or T and/or N). In some embodiments, the amino acid X 3 may be substituted with D, T, N, E, S, C, A, Q, or H.
  • the amino acid X 4 may be substituted with any amino acid. In some embodiments, the amino acid X4 may be substituted with a conservative amino acid (i.e., conservative to T and/or Y and/or S and/or A and/or W). In some embodiments, the amino acid X4 may be substituted with S, T, C, N, A, G, F, W, H, V, L, Y, or M.
  • an anti- KIR3DL1 agent comprises a CDRH2 comprising the polypeptide INPXiX 2 GX 3 T where Xi is S or Y, X 2 is S or N, and X 3 is Y or G.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or Y).
  • the amino acid Xi may be substituted with S, Y, T, C, N, G, F, W, or H.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or N).
  • the amino acid Xi may be substituted with S, N, T, C, N, G, Q, D, or H.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Y and/or G). In some embodiments, the amino acid Xi may be substituted with Y, G, A, S, P, C, F, W, H, or T. In some embodiments, an anti-KIR3DL1 agent comprises a CDRH2 comprising the polypeptide INTXIX 2 GX 3 X 4 where Xi is E or Y, X 2 is T or Y, X 3 is E or D, and X 4 is P or A. The amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to E and/or Y). In some embodiments, the amino acid Xi may be substituted with E, Y, F, W, H, S, T, D, Q, or N.
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or Y). In some embodiments, the amino acid X 2 may be substituted with T, Y, F, W, H, S, C, N, or A.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to E and/or D).
  • the amino acid X 3 may be substituted with E, D, N, S, or Q.
  • the amino acid X 3 may be substituted with a negatively charged amino acid (e.g., E or D).
  • the amino acid X 4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to P and/or A). In some embodiments, the amino acid X 4 may be substituted with P, A, G, L, V, or S. In some embodiments, the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, an anti-KIR3DL1 agent comprises a CDRL1 comprising the polypeptide ENIYSX1 where Xi is N or Y. The amino acid Xi may be substituted with any amino acid.
  • a conservative amino acid i.e., conservative to P and/or A
  • the amino acid X 4 may be substituted with P, A, G, L, V, or S.
  • the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to N and/or Y). In some embodiments, the amino acid Xi may be substituted with N, Y, F, W, H, S, T, Q, or D. In some embodiments, the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, an anti-KIR3DL1 agent comprises a CDRL1 comprising the polypeptide QSLLXISX 2 NQKNY where Xi is F or N and X 2 is S or G. The amino acid Xi may be substituted with any amino acid.
  • a conservative amino acid i.e., conservative to N and/or Y
  • the amino acid Xi may be substituted with N, Y, F, W, H, S, T, Q, or D.
  • the amino acid Xi may be substituted with a polar, un
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to F and/or N). In some embodiments, the amino acid Xi may be substituted with F, N, Q, S, T, D, H, Y, W, L, M, I, or V.
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to S and/or G). In some embodiments, the amino acid X 2 may be substituted with S, G, T, C, N, A, or P.
  • an anti-KIR3DL1 agent comprises a CDRL2 comprising the polypeptide AAX1 where Xi is R or S.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to R and/or S).
  • the amino acid Xi may be substituted with R, S, T, C, N, G, K, H, or Q.
  • an anti- KIR3DL1 agent comprises a CDRL2 comprising the polypeptide XiAS where Xi is W or G. The amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to W and/or G). In some embodiments, the amino acid Xi may be substituted with W, G, A, S, P, C, F, Y, H, L, or M. In some embodiments, the amino acid Xi may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, an anti-KIR3DL1 agent comprises a CDRL3 comprising the polypeptide QHX1X1GTPLT where Xi is F or H and X 2 is W or Y. The amino acid Xi may be substituted with any amino acid.
  • a conservative amino acid i.e., conservative to W and/or G
  • the amino acid Xi may be substituted with W, G, A, S, P, C, F, Y, H, L, or M.
  • the amino acid Xi may be substituted with
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to F and/or H). In some embodiments, the amino acid Xi may be substituted with F, H, Y, W, L, M, I, V, K, R, N, or Q. In some embodiments, the amino acid Xi may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to W and/or Y).
  • an anti- KIR3DL1 agent comprises a CDRL3 comprising the polypeptide XIQSX 2 X 3 X4PYT where Xi is S or Q, X 2 is T or N, X3 is H or E, and X4 is V or D.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or Q). In some embodiments, the amino acid Xi may be substituted with S, Q, T, C, N, G, E, H, or K. In some embodiments, the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or N).
  • the amino acid X 2 may be substituted with T, N, S, C, A, Q, D, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to H and/or E). In some embodiments, the amino acid X 3 may be substituted with H, E, D, Q, N, S, K, R, Y, or W.
  • the amino acid X4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to V and/or D). In some embodiments, the amino acid X 4 may be substituted with V, D, I, L, A, M, E, N, or S.
  • an anti-KIR agent provided herein binds to KIR3DL3 or a portion thereof, which may be referred to herein as an anti-KIR3DL3 agent.
  • an anti- KIR3DL3 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti-KIR3DL3 agent comprises a CDRH2 comprising the polypeptide IHYSGXiT where Xi is T or S.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e. , conservative to T and/or S).
  • the amino acid Xi may be substituted with T, S, C, N, A, or G.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • an anti-KIR3DL3 agent comprises a CDRL3 comprising the polypeptide QQWSSX1PLT where Xi is D or N.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to D and/or N).
  • the amino acid Xi may be substituted with D, N, E, S, Q, T, or H.
  • an anti-KIR agent provided herein binds to KIR2DL2 or a portion thereof and KIR2DS2 or a portion thereof, which may be referred to herein as an anti-KIR2DL2/KIR2DS2 agent.
  • an anti-KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti-KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising the polypeptide GX1SX2TSX3X4 where Xi is D or F, X 2 is I or L, X 3 is G or Y, and X 4 is F or G.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to D and/or F).
  • the amino acid Xi may be substituted with D, F, Y, W, L, M, I, V, E, N, or S.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to I and/or L). In some embodiments, the amino acid X 2 may be substituted with I, L, V, M, A, or F. In some embodiments, the amino acid X 2 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to G and/or Y).
  • the amino acid X 3 may be substituted with G, Y, F, W, H, S, T, A, P, or C.
  • the amino acid X 4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to F and/or G).
  • the amino acid X 4 may be substituted with F, G, A, S, P, C, Y, W, L, M, I, or V.
  • the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • an anti-KIR2DL2/KIR2DS2 agent comprises a CDRH2 comprising the polypeptide IX1X2X3GST where Xi is S or W, X 2 is Y or T, and X 3 is S or G.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or W).
  • the amino acid Xi may be substituted with S, W, F, Y, H, L, M, T, C, N, or G.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to Y and/or T). In some embodiments, the amino acid X 2 may be substituted with Y, T, S, C, N, A, F, W, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to S and/or G).
  • a conservative amino acid i.e., conservative to Y and/or T
  • the amino acid X 2 may be substituted with Y, T, S, C, N, A, F, W, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N,
  • an anti- KIR2DL2/KIR2DS2 agent comprises a CDRL1 comprising the polypeptide QSLLNSXiX 2 X 3 KNY, where Xi is F or R, X 2 is N or T, and X 3 is Q or R.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to F and/or R).
  • the amino acid Xi may be substituted with F, R, Y, W, L, M, I, V, K, H, Q, or N.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to N and/or T).
  • the amino acid X 2 may be substituted with N, T, S, C, A, Q, D, or H.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to Q and/or R). In some embodiments, the amino acid X 3 may be substituted with Q, R, N, E, H, S, T, or K. In some embodiments, an anti-KIR2DL2/KIR2DS2 agent comprises a CDRL2 comprising the polypeptide X1AS where Xi is F or W. The amino acid Xi may be substituted with any amino acid. In some embodiments, the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to F and/or W).
  • the amino acid Xi may be substituted with F, W, Y, H, L, M, or V. In some embodiments, the amino acid Xi may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the amino acid Xi may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • an anti-KIR2DL2/KIR2DS2 agent comprises a CDRL3 comprising the polypeptide XIQX 2 YNX 3 PX 4 T where Xi is Q or K, X 2 is Y or S, X 3 is T or L, and X 4 is V or W.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Q and/or K).
  • the amino acid Xi may be substituted with Q, K, N, E, H, S, T, or R.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to Y and/or S). In some embodiments, the amino acid X 2 may be substituted with Y, S, T, C, N, G, F, W, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to T and/or L).
  • a conservative amino acid i.e., conservative to Y and/or S
  • the amino acid X 2 may be substituted with Y, S, T, C, N, G, F, W, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N,
  • the amino acid X 3 may be substituted with T, L, S, C, N, A, I, V, M, or F.
  • the amino acid X 4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to V and/or W).
  • the amino acid X 4 may be substituted with V, W, I, L, A, M, F, Y, or H.
  • the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • an anti-KIR agent 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 agent.
  • an anti- KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti- KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising the polypeptide GXiTFX 2 X 3 YX 4 where Xi is Y or F, X 2 is T, K or N, X 3 is D or N, and X 4 is A or D.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Y and/or F).
  • the amino acid Xi may be substituted with Y, F, W, H, S, T, L, M, I, or V.
  • the amino acid Xi may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or K and/or N).
  • the amino acid X 2 may be substituted with T, K, S, C, N, A, R, and H.
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to D and/or N).
  • the amino acid X 3 may be substituted with D, N, E, S, Q, T, or H.
  • the amino acid X 4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to A and/or D).
  • the amino acid X 4 may be substituted with A, D, G, V, S, L, E, or N.
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising the polypeptide GFTFX1X2X3X4 where Xi is N or K, X 2 is T or N, X 3 is N or Y, and X 4 is A or D.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to N and/or K).
  • the amino acid Xi may be substituted with N, K, Q, S, T, D, H, or R.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to T and/or N). In some embodiments, the amino acid X 2 may be substituted with T, N, S, C, A, Q, D, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to N and/or Y).
  • the amino acid X 3 may be substituted with N, Y, Q, S, T, D, H, F, or W. In some embodiments, the amino acid X 3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 4 may be substituted with any amino acid. In some embodiments, the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to A and/or D). In some embodiments, the amino acid X 4 may be substituted with A, D, G, V, S, L, E, or N.
  • a conservative amino acid i.e., conservative to A and/or D
  • the amino acid X 4 may be substituted with A, D, G, V, S, L, E, or N.
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising the polypeptide GXITFX 2 X 3 X 4 A where Xi is F or Y, X 2 is N or T, X 3 is T or D, and X 4 is N or Y.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to F and/or Y).
  • the amino acid Xi may be substituted with F, Y, W, H, S, T, L, M, I, or V.
  • the amino acid Xi may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to N and/or T).
  • the amino acid X 2 may be substituted with N, T, Q, S, D, H, C, or A.
  • the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to T and/or D). In some embodiments, the amino acid X 3 may be substituted with T, D, S, C, N, A, or E.
  • the amino acid X 4 may be substituted with any amino acid. In some embodiments, the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to N and/or Y). In some embodiments, the amino acid X 4 may be substituted with N, Y, F, W, H, S, T, Q, or D.
  • the amino acid X 4 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH1 comprising the polypeptide GYXI FTDYX 2 where Xi is S or T and X 2 is N or A.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or T).
  • the amino acid Xi may be substituted with S, T, C, N, A, or G.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to N and/or A).
  • the amino acid X 2 may be substituted with N, A, G, V, S, L, Q, T, D, or H.
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH2 comprising the polypeptide IXIX 2 X 3 NGX4T where Xi is D or S, X 2 is P or T, X 3 is D or Y, and X4 is G or N.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to D and/or S).
  • the amino acid Xi may be substituted with D, S, E, N, T, C, or G.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to P and/or T). In some embodiments, the amino acid X 2 may be substituted with P, T, S, C, N, A, G, L, or V.
  • the amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to D and/or Y). In some embodiments, the amino acid X 3 may be substituted with D, Y, E, N, S, F, W, H, or T.
  • the amino acid X 4 may be substituted with any amino acid.
  • amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to G and/or N). In some embodiments, the amino acid X 4 may be substituted with G, N, A, S, P, C, Q, T, D, or H. In some embodiments, an anti- KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH2 comprising the polypeptide
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or T).
  • the amino acid Xi may be substituted with S, T, C, N, A, or G.
  • the amino acid Xi may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to S and/or Y). In some embodiments, the amino acid X 2 may be substituted with S, Y, T, C, N, G, F, W, or H. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to R and/or I).
  • the amino acid X 3 may be substituted with R, I, K, H, Q, N, L, V, M, A, or F.
  • the amino acid X4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e. , conservative to I and/or T).
  • the amino acid X 4 may be substituted with I, T, L, V, M, A, F, S, C, or N.
  • an anti- KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRH3 comprising the polypeptide ARXIX 2 X 3 X4YX5X 6 WFAY where Xi is G or L, X 2 is D or G, X 3 is Y or N, X 4 is G or N, X 5 is L or G, and X 6 is R or G.
  • the amino acid Xi may be substituted with any amino acid. In some embodiments, the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to G and/or L).
  • the amino acid Xi may be substituted with G, L, I, V, M, A, F, S, P, or C. In some embodiments, the amino acid Xi may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to D and/or G). In some embodiments, the amino acid X 2 may be substituted with D, G, A, S, P, C, E, or N.
  • the amino acid X 3 may be substituted with any amino acid.
  • the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to Y and/or N). In some embodiments, the amino acid X 3 may be substituted with Y, N, F, W, H, S, T, Q, or D. In some embodiments, the amino acid X 3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 4 may be substituted with any amino acid. In some embodiments, the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to G and/or N).
  • the amino acid X 4 may be substituted with G, N, Q, S, T, D, H, A, P, or C. In some embodiments, the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • the amino acid X 5 may be substituted with any amino acid. In some embodiments, the amino acid X 5 may be substituted with a conservative amino acid (i.e., conservative to L and/or G). In some embodiments, the amino acid X 5 may be substituted with L, G, A, S, P, C, I, V, M, or F.
  • the amino acid X5 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • the amino acid X 6 may be substituted with any amino acid.
  • the amino acid X 6 may be substituted with a conservative amino acid (i.e., conservative to R and/or G).
  • the amino acid X 6 may be substituted with R, G, K, H, Q, N, A, S, P, or C.
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRLI comprising the polypeptide QX1 INX1Y where Xi is D or N and X 2 is S or K.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to D and/or N).
  • the amino acid Xi may be substituted with D, N, Q, S, T, H, or E.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to S and/or K).
  • the amino acid X 2 may be substituted with S, K, T, C, N, G, R, or H.
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRL2 comprising the polypeptide KX1S where Xi is V or E.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to V and/or E).
  • the amino acid Xi may be substituted with V, E, I, L, A, M, D, Q, N, or S.
  • an anti- KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRL2 comprising the polypeptide XiAN where Xi is R or N.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to R and/or N).
  • the amino acid Xi may be substituted with R, N, Q, S, T, D, H, or K.
  • an anti-KIR2DL3/KIR2DL2/KIR2DS2 agent comprises a CDRL3 comprising the polypeptide X1QX2X3EX4PYT where Xi is Q or L, X 2 is N or Y, X3 is N or D, and X4 is D or F.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Q and/or L).
  • the amino acid Xi may be substituted with Q, L, I, V, M, A, F, N, E, H, S, T, or K.
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to N and/or Y). In some embodiments, the amino acid X 2 may be substituted with N, Y, F, W, H, S, T, Q, or D. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to N and/or D).
  • the amino acid X 3 may be substituted with N, D, Q, S, T, H, or E.
  • the amino acid X4 may be substituted with any amino acid.
  • the amino acid X4 may be substituted with a conservative amino acid (i.e., conservative to D and/or F).
  • the amino acid X4 inay be substituted with D, F, E, N, S, Y, W, L, M, I, or V.
  • an anti-KIR agent provided herein binds to 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 an anti- KIR2DL1/KIR2DL2/KIR2DL3/KIR2DS1/KIR2DS2 agent.
  • an anti- KIR2DL1/KIR2DL2/KIR2DL3/KIR2DS1/KIR2DS2 agent comprises a CDRH1 comprising a polypeptide provided in Table 3, a CDRH2 comprising a polypeptide provided in Table 3, a CDRH3 comprising a polypeptide provided in Table 3, a CDRL1 comprising a polypeptide provided in Table 3, a CDRL2 comprising a polypeptide provided in Table 3, and a CDRL3 comprising a polypeptide provided in Table 3, or one or more variants of each thereof.
  • an anti-KIR2DL1/KIR2DL2/KIR2DL3/KIR2DS1/KIR2DS2 agent comprises a CDRH3 comprising the polypeptide ARXIX 2 X3X4X 5 YYGSSX6X7YX 8 MDY where Xi is no AA or R, X 2 is no AA or F, X 3 is S or R, X4 is S or P, X5 is L or N, X 6 is Y or F, X7 is D or no AA, and X 8 is V or A.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to R). In some embodiments, the amino acid Xi may be substituted with R, K, H, Q, N. In some embodiments, the amino acid Xi may be substituted with a positively charged amino acid (e.g., K, R, H).
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to F). In some embodiments, the amino acid X 2 may be substituted with F, Y, W, L, M, I, or V.
  • the amino acid X 2 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V). In some embodiments, the amino acid X 2 may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • the amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to S and/or R). In some embodiments, the amino acid X 3 may be substituted with S, R, T, C, N, G, K, H, or Q.
  • the amino acid X 4 may be substituted with any amino acid.
  • the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to S and/or P). In some embodiments, the amino acid X 4 may be substituted with S, P, T, C, N, G, A, L, or V.
  • the amino acid X5 may be substituted with any amino acid. In some embodiments, the amino acid Xs may be substituted with a conservative amino acid (i.e., conservative to L and/or N). In some embodiments, the amino acid X 5 may be substituted with L, N, I, V, M, A, F, Q, S, T, D, or H.
  • the amino acid X 6 may be substituted with any amino acid.
  • the amino acid X 6 may be substituted with a conservative amino acid (i.e., conservative to Y and/or F). In some embodiments, the amino acid X 6 may be substituted with Y, F, W, H, S, T, L, M, I, or V. In some embodiments, the amino acid X 6 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). The amino acid X 7 may be substituted with any amino acid. In some embodiments, the amino acid X 7 may be substituted with a conservative amino acid (i.e., conservative to D). In some embodiments, the amino acid X 7 may be substituted with D, E, N, or S.
  • a conservative amino acid i.e., conservative to Y and/or F.
  • the amino acid X 6 may be substituted with Y, F, W, H, S, T, L, M, I, or V. In some embodiments, the amino acid X 6 may be substituted with an aromatic amino acid (e
  • the amino acid X 7 may be substituted with a negatively charged amino acid (e.g., D, E).
  • the amino acid X 8 may be substituted with any amino acid.
  • the amino acid X 8 may be substituted with a conservative amino acid (i.e., conservative to V and/or A).
  • the amino acid X 8 may be substituted with V, A, G, S, L, I, or M.
  • the amino acid X 8 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • an anti- KIR2DL1/KIR2DL2/KIR2DL3/KIR2DS1/KIR2DS2 agent comprises a CDRL1 comprising the polypeptide QXIVSX 2 D where Xi is S or D and X 2 is N or T.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to S and/or D).
  • the amino acid Xi may be substituted with S, D, T, C, N, G, or E.
  • the amino acid X 2 may be substituted with any amino acid.
  • the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to N and/or T). In some embodiments, the amino acid X 2 may be substituted with N, T, Q, S, D, H, C, or A. In some embodiments, the amino acid X 2 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C). In some embodiments, an anti- KIR2DL1/KIR2DL2/KIR2DL3/KIR2DS1/KIR2DS2 agent comprises a CDRL2 comprising the polypeptide XiAS where Xi is Y or W. The amino acid Xi may be substituted with any amino acid.
  • a conservative amino acid i.e., conservative to N and/or T
  • the amino acid X 2 may be substituted with N, T, Q, S, D, H, C, or A.
  • the amino acid X 2 may be substituted with a polar, uncharged
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to Y and/or W). In some embodiments, the amino acid Xi may be substituted with Y, W, F, H, S, T, L, or M. In some embodiments, the amino acid Xi may be substituted with an aromatic amino acid (e.g., F, Y, W, H).
  • an anti- KIR2DL1/KIR2DL2/KIR2DL3/KIR2DS1/KIR2DS2 agent comprises a CDRL3 comprising the polypeptide QQXIX 2 SX 3 PX 4 T where Xi is D or H, X 2 is Y or F, X 3 is S or T, and X 4 is P or W.
  • the amino acid Xi may be substituted with any amino acid.
  • the amino acid Xi may be substituted with a conservative amino acid (i.e., conservative to D and/or H).
  • the amino acid Xi may be substituted with D, H, K, R, N, Q, Y, W, E, or S.
  • the amino acid X 2 may be substituted with any amino acid. In some embodiments, the amino acid X 2 may be substituted with a conservative amino acid (i.e., conservative to Y and/or F). In some embodiments, the amino acid X 2 may be substituted with Y, F, W, H, S, T, L, M, I, or V. In some embodiments, the amino acid X 2 may be substituted with an aromatic amino acid (e.g., F, Y, W, H). The amino acid X 3 may be substituted with any amino acid. In some embodiments, the amino acid X 3 may be substituted with a conservative amino acid (i.e., conservative to S and/or T).
  • the amino acid X 3 may be substituted with S, T, C, N, A, or G. In some embodiments, the amino acid X 3 may be substituted with a polar, uncharged amino acid (e.g., S, T, N, Q, Y, C).
  • the amino acid X 4 may be substituted with any amino acid. In some embodiments, the amino acid X 4 may be substituted with a conservative amino acid (i.e., conservative to P and/or W). In some embodiments, the amino acid X 4 may be substituted with P, W, F, Y, H, L, M, A, G, or V. In some embodiments, the amino acid X 4 may be substituted with a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, V).
  • an anti-KIR agent provided herein comprises an immunoglobulin heavy chain variable domain comprising an immunoglobulin heavy chain variable domain polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to an immunoglobulin heavy chain variable domain polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises an immunoglobulin heavy chain variable domain comprising an immunoglobulin heavy chain variable domain polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises an immunoglobulin heavy chain variable domain comprising a polypeptide that is at least 90 percent identical to an immunoglobulin heavy chain variable domain polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises an immunoglobulin heavy chain variable domain comprising a polypeptide that is at least 95 percent identical to an immunoglobulin heavy chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises an immunoglobulin heavy chain variable domain comprising a polypeptide that is 100 percent identical to an immunoglobulin heavy chain variable domain polypeptide provided in Table 3.
  • an anti-KIR agent provided herein comprises an immunoglobulin light chain variable domain comprising an immunoglobulin light chain variable domain polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to an immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises an immunoglobulin light chain variable domain comprising a polypeptide that is at least 85 percent identical to an immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises an immunoglobulin light chain variable domain comprising a polypeptide that is at least 90 percent identical to an immunoglobulin light chain variable domain polypeptide provided in Table 3.
  • the anti-KIR agent provided herein comprises an immunoglobulin light chain variable domain comprising a polypeptide that is at least 95 percent identical to an immunoglobulin light chain variable domain polypeptide provided in Table 3. In some embodiments, the anti-KIR agent provided herein comprises an immunoglobulin light chain variable domain comprising a polypeptide that is at least 100 percent identical to an immunoglobulin light chain variable domain polypeptide provided in Table 3.
  • Percent amino acid sequence identity with respect to a reference VH, VL, and CDR polypeptide sequences herein generally refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • an anti-KIR agent 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).
  • an anti-KIR agent 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).
  • Sets of CDRs may comprise any combination of CDR amino acid sequences (i.e., CDRH1 , CDRH2, CDRH3; and CDRL1 , CDRL2, CDRL3) provided herein.
  • an immunoglobulin heavy chain variable domain comprises a set of CDRH1 , CDRH2, and CDRH3 amino acid sequences
  • an immunoglobulin light chain variable domain comprises a set of CDRL1 , CDRL2, and CDRL3 amino acid sequences chosen from the sets provided in Table 3 herein.
  • each immunoglobulin heavy chain variable domain may comprise a set of CDRH1 , CDRH2, and CDRH3 amino acid sequences
  • each immunoglobulin light chain variable domain may comprise a set of CDRL1 , CDRL2, and CDRL3 amino acid sequences chosen from the sets provided in Table 3.
  • all CDRs are from the same set.
  • each immunoglobulin heavy chain variable domain may comprise a set of CDRH1 , CDRH2, and CDRH3 amino acid sequences from a single clone in Table 3
  • each immunoglobulin light chain variable domain may comprise a set of CDRL1 , CDRL2, and CDRL3 amino acid sequences from the same clone in Table 3.
  • CDRs are from the different sets.
  • each immunoglobulin heavy chain variable domain may comprise a set of CDRH1 , CDRH2, and CDRH3 amino acid sequences from a first clone in Table 3
  • each immunoglobulin light chain variable domain may comprise a set of CDRL1 , CDRL2, and CDRL3 amino acid sequences from a second clone Table 3.
  • one immunoglobulin heavy chain variable domain may comprise a set of CDRH1 , CDRH2, and CDRH3 amino acid sequences from a first clone in Table 3 and the other immunoglobulin heavy chain variable domain may comprise a set of CDRH1 , CDRH2, and CDRH3 amino acid sequences from a second clone Table 3; and one immunoglobulin light chain variable domain may comprise a set of CDRL1 , CDRL2, and CDRL3 amino acid sequences from a first clone in Table 3 and the other immunoglobulin light chain variable domain may comprise a set of CDRL1 , CDRL2, and CDRL3 amino acid sequences from a second clone in Table 3. Competitor agents
  • anti-KIR agents that competitively bind, or are capable of competitively binding, with one or more anti-KIR agents described herein.
  • anti-KIR agents that compete, or are capable of competing, with one or more anti-KIR agents described herein for binding to KIR (e.g., KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3).
  • Such agents that compete, or are capable of competing, with anti-KIR agents described herein may be referred to as competitor agents.
  • an agent i.e.
  • competitor agent may be considered to compete for binding to KIR when the competitor binds to the same general region of KIR as an anti- KIR agent described herein.
  • an agent i.e., competitor agent
  • an agent may be considered to compete for binding to KIR when the competitor binds to the exact same region of KIR as an anti-KIR agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)).
  • an agent i.e., competitor agent
  • an agent i.e., competitor agent
  • an agent may be considered capable of competing for binding to KIR when the competitor binds to the exact same region of KIR as an anti- KIR agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)) under suitable assay conditions.
  • an agent i.e., competitor agent
  • KIR KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3
  • an agent i.e., competitor agent
  • KIR KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3 when the competitor blocks the binding of one or more anti-KIR agents described herein to KIR.
  • Whether a competitor blocks the binding of one or more anti-KIR agents described herein to KIR may be determined using a suitable competition assay or blocking assay, such as, for example, a blocking assay as described in Example 4 herein.
  • a competitor agent may block binding of one or more anti-KIR agents described herein to KIR in a competition or blocking assay by 50% or more, and conversely, one or more anti-KIR agents described herein may block binding of the competitor agent to KIR in a competition or blocking assay by about 50% or more.
  • an agent i.e., competitor agent
  • one or more anti-KIR agents described herein may block binding of the competitor agent to KIR in a competition or blocking assay by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • an agent i.e., competitor agent
  • KIR KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3
  • an agent i.e., competitor agent
  • KIR3DL1 , KIR3DL2, and/or KIR3DL3DL3DL3DL3DL3DL3DL3DL3DL2 and/or KIR3DL3 KIR2DL1 , KIR2DL2, and/or KIR3DL3DL3 when the competitor binds to KIR with a similar affinity as one or more anti-KIR agents described herein.
  • an agent i.e., competitor agent
  • an agent is considered to compete for binding to KIR when the competitor binds to KIR with an affinity that is at least about 50% of the affinity of one or more anti-KIR agents described herein.
  • an agent i.e., competitor agent
  • a competitor agent may comprise any feature described herein for anti-KIR agents.
  • anti-KIR agents that bind to, or are capable of binding to, the same epitope as one or more anti-KIR agents described herein.
  • anti-KIR agents that compete with one or more anti-KIR agents described herein for binding to the same epitope on KIR (e.g., an epitope on KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3).
  • Such agents that bind the same epitope may be referred to as epitope competitors.
  • an epitope competitor may bind to the exact same region of KIR as an anti-KIR agent described herein (e.g., exact same peptide (linear epitope) or exact same surface amino acids (conformational epitope)).
  • an epitope competitor blocks the binding of one or more anti-KIR agents described herein to KIR.
  • An epitope competitor may block binding of one or more anti-KIR agents described herein to KIR in a competition assay by about 50% or more, and conversely, one or more anti-KIR agents described herein may block binding of the epitope competitor to KIR in a competition assay by 50% or more.
  • an epitope competitor binds to KIR with a similar affinity as one or more anti-KIR agents described herein. In some embodiments, an epitope competitor binds to KIR with an affinity that is at least about 50% of the affinity of one or more anti- KIR agents described herein. For example, an epitope competitor may bind to KIR with an affinity that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-KIR agents described herein. An epitope competitor may comprise any feature described herein for anti-KIR agents. Antibodies
  • an anti-KIR agent is a KIR binding antibody or antigen binding fragment thereof (e.g., a KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3 binding antibody or antigen binding fragment thereof).
  • An antibody as described herein generally includes of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • Immunoglobulin genes generally include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An antibody herein may be classified as IgG (e.g., lgG1 , lgG2, lgG3, lgG4), IgM, IgA, IgD, or IgE.
  • An antibody may be monoclonal.
  • a monoclonal antibody generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical (as assessed at the level of Ig heavy and/or light chain amino acid sequence) and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present technology may be made 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.
  • An antibody structural unit may be in the form of a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies typically include one or more variable regions or variable domains, which refer to portions of light and heavy chains of an antibody that include amino acid sequences of complementarity determining regions (CDRs, e.g., CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3) and framework regions (FRs).
  • CDRs complementarity determining regions
  • the variable domain for heavy chains and light chains is commonly designated VH and VL, respectively.
  • the variable domain is included on Fab, F(ab’)2, Fv and scFv antigen binding fragments, for example, and is involved in specific antigen recognition.
  • a complementarity determining region (CDR) refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1 , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • Antibodies typically have 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 the four heavy chain framework regions and CDRH1 -3 represent the three hypervariable regions of an antibody variable heavy domain.
  • FRH1 -4 are derived from a consensus sequence (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or are derived from an individual human antibody framework region or from a combination of different framework region sequences.
  • 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 the four framework regions and CDRL1 -3 represent the three hypervariable regions of an antibody variable light domain.
  • FRL1-4 are derived from a consensus sequence (for example the most common amino acids of a class, subclass or subgroup of heavy or light chains of human immunoglobulins) or are derived from an individual human antibody framework region or from a combination of different framework region sequences.
  • an antibody is monovalent (an antibody with one antigen binding site). In some embodiments, an antibody is bivalent (an antibody with two antigen binding sites). In some embodiments, an antibody is in a multivalent form, e.g., a trivalent (an antibody with three antigen binding sites) or tetravalent form (an antibody with four antigen binding sites). In some embodiments, an antibody is monospecific (binds to one antigen or epitope). In some embodiments, an antibody is multi-specific (binds to more than one antigen or epitope; e.g., bispecific, trispecific, and the like).
  • Anti-KIR antibodies herein may include VH-VL dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light domain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • a single chain Fv antibody typically is a covalently linked VH-VL which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker. While the VH and VL are connected to each other as a single polypeptide chain, the VH and VL domains typically associate non- covalently.
  • VH and VL domains together typically include six complementarity determining regions (CDR) (three in each from the heavy and light chain) that contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody.
  • CDR complementarity determining regions
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen has the ability to recognize and bind antigen.
  • a dsFv is an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.
  • Anti-KIR antibodies herein may include antigen binding fragments produced by the modification of whole antibodies. For example, pepsin digests an antibody C-terminal to the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer.
  • the Fab' monomer is essentially a Fab with part of the hinge region.
  • antigen binding fragments are defined in terms of the digestion of an intact antibody, antigen binding fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • an antibody may refer to antigen binding fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies.
  • Anti-KIR antibodies herein may include antibody fragments or antigen binding fragments thereof that retain binding specificity.
  • An antibody fragment or antigen binging fragment thereof may include a portion of an intact antibody (e.g., an antigen binding region of the intact antibody and/or a variable region of the intact antibody).
  • Antibody fragments or antigen binding fragments thereof include but are not limited to Fv fragments, disulf ide-linked Fvs (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.
  • a fragment of an antibody containing a variable domain (VH) and one constant region domain (CHI) of an antibody heavy chain Fd' fragments (i.e., a fragment of an antibody containing one heavy chain portion of a F(ab')2 fragment), diabodies (i.e., dimeric scFvs), linear antibodies, and single-chain antibody molecules (e.g., single-chain Fvs (scFv) or single-chain Fabs (scFab)).
  • an anti-KIR antibody or antigen binding fragment thereof is a chimeric antibody.
  • Chimeric antibodies may include immunoglobulin molecules in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, and the like.
  • Chimeric antibodies may also include immunoglobulin molecules in which the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigen specificity; or with corresponding sequences from another species or from another antibody class or subclass.
  • an anti-KIR antibody or antigen binding fragment thereof comprises one or more human framework regions.
  • an anti-KIR antibody or antigen binding fragment thereof is humanized.
  • Humanized antibodies generally refer to immunoglobulin molecules in which CDRs from a donor antibody (e.g., mouse, rabbit) are grafted onto human framework sequences. Humanized antibodies also may include residues of donor origin in the framework sequences. A humanized antibody also may include at least a portion of a human immunoglobulin constant region. Humanized antibodies also may include residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed using methods known in the art.
  • an anti-KIR antibody or antigen binding fragment thereof comprises one or more variations (e.g., amino acid substitutions, deletions, and/or insertions),
  • An example method for identification of certain residues or regions of an antibody that are preferred locations for amino acid substitutions is alanine scanning mutagenesis.
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with an antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • Amino acid sequence insertions may include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include an N-terminal methionyl residue or the antibody fused to an epitope tag.
  • Other insertional variants include the fusion of an enzyme or a polypeptide that increases the serum halflife of the antibody to the N- or C-terminus of the antibody.
  • Sites for amino acid substitutions may include sites in the hypervariable regions and may include sites in the framework regions.
  • Amino acid substitutions may include conservative substitutions or non-conservative substitutions. Examples of substitutions are listed below.
  • Substantial modifications in the biological properties of an antibody may be accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues can be divided into groups based on common side-chain properties listed below.
  • Non-conservative substitutions generally entail exchanging a member of one of these classes for another class.
  • cysteine residue not involved in maintaining the proper conformation of an antibody also may be substituted, e.g., to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (e.g., where the antibody is an antibody fragment such as an Fv fragment).
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity).
  • alanine-scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody.
  • one or more carbohydrate moieties found in the antibody may be deleted, and/or one or more glycosylation sites that are not present in the antibody may be added.
  • Glycosylation of antibodies is typically either N-linked and/or or O-linked.
  • N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue.
  • 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 enzymatic attachment of a carbohydrate moiety to an asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • Addition of glycosylation sites to an antibody may be accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N- linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of an original antibody (for O-linked glycosylation sites).
  • an antibody or antigen binding fragment thereof comprises a fragment crystallizable region (Fc region), also referred to as an Fc polypeptide.
  • Fc region also referred to as an Fc polypeptide.
  • An Fc polypeptide is part of each of the two heavy chains in the antibody and can interact with certain cell surface receptors and certain components of the complement system.
  • An Fc polypeptide typically includes the CH2 domain and the CH3 domain, which are immunoglobulin constant region domain polypeptides.
  • the Fc polypeptide in an antibody described herein can be a wild-type Fc polypeptide, e.g., a human lgG1 Fc polypeptide.
  • an antibody described herein can comprise a wild-type Fc polypeptide having the following sequence:
  • an antibody or antigen binding fragment thereof comprises a variant of the wild-type Fc polypeptide that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity to the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 1) and at least one amino acid substitution relative to the sequence of a wild-type Fc polypeptide (e.g., SEQ ID NO: 1 ).
  • an Fc polypeptide 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 comprising modified Fc polypeptides typically have altered phenotypes relative to antibodies comprising wild-type Fc polypeptides.
  • antibodies comprising modified Fc polypeptides can have altered serum half-life, altered stability, altered susceptibility to cellular enzymes, and/or altered effector function (e.g., as assayed in an NK-dependent or macrophagedependent assay).
  • an Fc polypeptide in an antibody or antigen binding fragment thereof can include amino acid substitutions that modulate effector function.
  • an Fc polypeptide in an antibody described herein can include amino acid substitutions that reduce or eliminate effector function.
  • Illustrative Fc polypeptide amino acid substitutions that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 4 and 5 (position numbering relative to the sequence of SEQ ID NO: 1).
  • one or both Fc polypeptides in an antibody described herein can comprise L4A and L5A substitutions.
  • Additional Fc polypeptide amino acid substitutions that modulate an effector function include, e.g., substitution at position 99 (position numbering relative to the sequence of SEQ ID NO: 1 ).
  • one or both Fc polypeptides in an antibody described herein can comprise a P99G substitution.
  • one or both Fc polypeptides in an antibody described herein can have L4A, L5A, and P99G substitutions.
  • an Fc polypeptide includes one or more modifications that alter (relative to a wild-type Fc polypeptide) the Ratio of Affinities of the modified Fc polypeptide to an activating FcvR (such as FcvRIlA or FcvRIIIA) relative to an inhibiting FcvR (such as FcvRHB):
  • Ratio of Affinities WT to variant change in affinity to FevRactivating WT to variant change in affinity to FcvR in hibiting
  • an antibody herein may have particular use in providing a therapeutic or prophylactic treatment of a disease, disorder, or infection, or the amelioration of a symptom thereof, where an enhanced efficacy of effector cell function mediated by FcvR is desired, e.g., cancer or infectious disease.
  • a modified Fc region has a Ratio of Affinities less than 1
  • an antibody herein may have particular use in providing a therapeutic or prophylactic treatment of a disease or disorder, or the amelioration of a symptom thereof, where a decreased efficacy of effector cell function mediated by FcvR is desired, e.g., autoimmune or inflammatory disorders.
  • the following are examples of single, double, triple, quadruple, and quintuple amino acid substitutions in an Fc polypeptide that provide a Ratio of Affinities greater than 1 or less than 1 .
  • An antibody may bind to one or more antigens.
  • An antigen generally refers to a molecule, compound, or complex that is recognized by an antibody, i.e., can be specifically bound by the antibody.
  • the term antigen may be used interchangeably with immunogen, antibody target, target analyte, and the like.
  • An antigen may refer to any molecule that can be specifically recognized by an antibody, e.g., a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.).
  • the term antigen does not necessarily indicate that the molecule is immunogenic in every context, but generally indicates that it can be targeted by an antibody.
  • Antibodies typically bind to an epitope on an antigen.
  • An epitope is the localized site on an antigen that is recognized and bound by an antibody.
  • Epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids.
  • the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid.
  • the epitope is a three-dimensional moiety.
  • the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).
  • a discontinuous epitope typically includes 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.
  • an epitope includes 8-10 amino acids in a unique spatial conformation.
  • the terms specific for, specifically binds, and like terms generally refer to a molecule (e.g., antibody or antigen binding fragment) that binds to a target with at least 2-fold greater affinity than non-target compounds, e.g., at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity.
  • a molecule e.g., antibody or antigen binding fragment
  • binds to a target with at least 2-fold greater affinity than non-target compounds e.g., at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity.
  • an antibody that specifically binds a target will typically bind the target with at least a 2-fold greater affinity than a non-target. Specificity can be determined using standard methods, e.g., solid-phase ELISA
  • the term binds with respect to an antibody target typically indicates that an antibody binds a majority of the antibody targets in a pure population (assuming appropriate molar ratios).
  • an antibody that binds a given antibody target typically binds to at least about 2/3 of the antibody targets in a solution (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the antibody targets in a solution).
  • an agent/antibody comprises one or more modifications.
  • immunoconjugates comprising an antibody described herein may be conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug.
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), a radioactive isotope (for example, a radioconjugate), or a cytotoxic drug.
  • ADCs antibody-drug conjugates
  • Conjugates can be made using any suitable bifunctional protein coupling agent such as N-succinimidyl-3-(2- pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4- dinitrobenzene).
  • SPDP N-succinimidyl-3-(2- pyridyld
  • an agent/antibody disclosed herein may be formulated as an immunoliposome.
  • Liposomes containing an antibody may be prepared by a suitable method known in the art. Liposomes with enhanced circulation time may be generated. For example, liposomes can be generated by a reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes may be extruded through filters of defined pore size to yield liposomes with a desired diameter. Fab' fragments of an antibody provided herein may be conjugated to the liposomes via a disulfide interchange reaction, for example. Other active ingredients may be contained within the liposome.
  • enzymes or other polypeptides may be covalently bound to an agent/antibody disclosed herein by a suitable technique such as the use of the heterobifunctional cross-linking reagents.
  • fusion proteins comprising at least the antigen binding region of an antibody provided herein linked to at least a functionally active portion of an enzyme can be constructed using recombinant DNA techniques.
  • This may be achieved, for example, by incorporation of a salvage receptor binding epitope into the antigen binding fragment (e.g., by mutation of the appropriate region in the antigen binding fragment or by incorporating the epitope into a peptide tag that is then fused to the antigen binding fragment at either end or in the middle, e.g., by DNA or peptide synthesis.
  • a salvage receptor binding epitope into the antigen binding fragment (e.g., by mutation of the appropriate region in the antigen binding fragment or by incorporating the epitope into a peptide tag that is then fused to the antigen binding fragment at either end or in the middle, e.g., by DNA or peptide synthesis.
  • an antibody or antigen binding fragment thereof comprises, is conjugated to, or is hybridized to one or more oligonucleotides.
  • the oligonucleotide contains a barcode sequence (e.g., a sample barcode sequence).
  • the oligonucleotide further contains a binding site for a primer and/or an anchor.
  • a detectable marker or label e.g., a radioisotope or fluorophore
  • the oligonucleotide is a polymeric sequence.
  • oligonucleotide and polynucleotide are used interchangeably to refer to a single-stranded multimer of nucleotides from about 2 to about 500 nucleotides in length.
  • Oligonucleotides may be synthetic, made enzymatically (e.g., via polymerization), or using a “split-pool” method.
  • Oligonucleotides may include ribonucleotide monomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides).
  • Oligonucleotides may include a combination of both deoxyribonucleotide monomers and ribonucleotide monomers in the oligonucleotide (e.g., random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). Oligonucleotides may include one or more functional moieties that are attached (e.g., covalently or non-covalently) to another structure.
  • An anchor generally refers to a polymer, e.g., a polynucleotide or oligonucleotide, which is designed to hybridize to a complementary oligonucleotide sequence.
  • an anchor is designed for the purpose of generating a double stranded construct oligonucleotide sequence.
  • an anchor is positioned at the 3’ end of the construct oligonucleotide sequence.
  • an anchor is positioned at the 5’ end of a construct oligonucleotide sequence.
  • Each anchor may be specific for its intended complementary sequence.
  • a binding site for a primer is a functional component of the oligonucleotide which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for amplification of the oligonucleotide.
  • a binding site for a primer can be formed of polymers of DNA, RNA, PNA, modified bases or combinations of these bases, or polyamides, etc.
  • a binding site for a primer is about 10 of such monomeric components, e.g., nucleotide bases, in length.
  • a binding site for a primer is at least about 5 to 100 monomeric components, e.g., nucleotides, in length.
  • a binding site for a primer can be a generic sequence suitable as an annealing site for a variety of amplification technologies.
  • Amplification technologies 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 (for example, T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase) and RNA- polymerase based amplification systems (such as T7-, T3-, and SP6-RNA-polymerase amplification), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA),
  • a modification is introduced into an antibody (e.g., within the polypeptide chain or at either the N- or C-terminal), e.g., to extend in vivo half-life, such as PEGylation or incorporation of long-chain polyethylene glycol polymers (PEG).
  • PEG polyethylene glycol polymers
  • Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of polypeptides, for example, to prevent rapid filtration into the urine.
  • a lysine residue in a sequence is conjugated to PEG directly or through a linker.
  • linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain.
  • An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site-specifically attached to a PEG chain containing, for example, a maleimide function.
  • Methods for incorporating PEG or long chain polymers of PEG are known in the art.
  • Covalent modifications of an antibody are also included within the scope of this technology. For example, modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of an antibody. Other types of covalent modifications of an antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues.
  • An example covalent modification of an antibody involves linking the antibody to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes.
  • an anti-KIR agent/antibody comprises a detectable marker or label.
  • an anti-KIR agent/antibody is conjugated to a detectable marker or label.
  • an anti-KIR agent/antibody may be labeled with a detectable moiety. Any suitable marker, label, or moiety may be associated with or conjugated to an anti-KIR agent/antibody herein.
  • an anti-KIR agent/antibody is labeled with one or more radioisotopes such as, for example, 35 S, 14 C, 125 l, 3 H, and 131 1.
  • an anti-KIR agent/antibody is labeled with one or more fluorescent labels such as, for example, rare earth chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, Texas Red and Brilliant VioletTM. Fluorescent labels may be conjugated to an antibody using methods known in the art. Fluorescence can be quantified using a flow cytometer, imaging microscope, or fluorimeter, for example.
  • fluorescent labels such as, for example, rare earth chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, Texas Red and Brilliant VioletTM.
  • Fluorescent labels may be conjugated to an antibody using methods known in the art. Fluorescence can be quantified using a flow cytometer, imaging microscope, or fluorimeter, for example.
  • an anti-KIR agent/antibody is labeled with one or more enzyme-substrate labels.
  • An enzyme can catalyze a chemical alteration of a chromogenic substrate that can be measured using various techniques. For example, an enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, an enzyme may alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying a change in fluorescence are known in the art. For example, a chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemilluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • a label is indirectly conjugated with the agent/antibody.
  • an antibody can be conjugated with biotin and any of the labels described above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody may be conjugated with a small hapten (e.g., digoxin) and a label described above can be conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten e.g., digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • an anti-KIR agent/antibody need not be labeled, and the presence thereof can be detected, e.g., using a labeled antibody which binds to an anti-KIR antibody.
  • an anti-KIR agent/antibody herein is immobilized on a solid support or substrate.
  • an anti-KIR agent/antibody herein is non-diffusively immobilized on a solid support (e.g., the anti-KIR agent/antibody does not detach from the solid support).
  • a solid support or substrate can be any physically separable solid to which an anti-KIR agent/antibody can be directly or indirectly attached including, but not limited to, surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles.
  • Solid supports also can include, for example, chips, columns, optical fibers, wipes, filters (e.g., flat surface filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled-pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductive materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethanes, TEFLONTM, polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF), and the like), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon, silica gel, and modified silicon, Sephadex®, Sepharose®, carbon, metals (
  • the solid support or substrate may be coated using passive or chemically-derivatized coatings with any number of materials, including polymers, such as dextrans, acrylamides, gelatins or agarose. Beads and/or particles may be free or in connection with one another (e.g., sintered).
  • a solid support or substrate can be a collection of particles.
  • the particles can comprise silica, and the silica may comprise silica dioxide.
  • the silica can be porous, and in certain embodiments the silica can be non-porous.
  • the particles further comprise an agent that confers a paramagnetic property to the particles.
  • the agent comprises a metal
  • the agent is a metal oxide, (e.g., iron or iron oxides, where the iron oxide contains a mixture of Fe 2+ and Fe 3+ ).
  • An anti-KIR agent/antibody may be linked to a solid support by covalent bonds or by non-covalent interactions and may be linked to a solid support directly or indirectly (e.g., via an intermediary agent such as a spacer molecule or biotin).
  • nucleic acids encoding an anti-KIR agent (e.g., anti-KIR antibody), vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the agent or antibody.
  • an anti-KIR agent e.g., anti-KIR antibody
  • vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the agent or antibody.
  • nucleic acids e.g., isolated nucleic acids
  • a nucleic acid encodes an immunoglobulin heavy chain variable domain of an anti-KIR agent provided herein.
  • a nucleic acid encodes an immunoglobulin light chain variable domain of an anti-KIR agent provided herein.
  • a nucleic acid encodes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an anti-KIR agent provided herein.
  • a nucleic acid comprises a nucleotide sequence provided in Table 3.
  • a nucleic acid comprises a nucleotide sequence that encodes an amino acid sequence provided in Table 3.
  • a nucleic acid may comprise a nucleotide sequence that encodes a CDR amino acid sequence of any one the CDRs provided in Table 3.
  • a nucleic acid may comprise a nucleotide sequence that encodes an immunoglobulin heavy chain variable domain amino acid sequence of any one of the immunoglobulin heavy chain variable domains provided in Table 3.
  • a nucleic acid may comprise a nucleotide sequence that encodes an immunoglobulin light chain variable domain amino acid sequence of any one of the immunoglobulin light chain variable domains provided in Table 3.
  • a nucleic acid encoding the anti-KIR agent or antibody may be isolated and inserted into a replicable vector for further cloning and/or expression.
  • an anti-KIR agent or antibody may be produced by homologous recombination.
  • DNA encoding an anti-KIR agent or antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Any suitable vector may be used.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Suitable host cells for cloning and/or expressing DNA in vectors include prokaryote cells, yeast cells, and higher eukaryote cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning and/or expression hosts for anti-KIR agent/antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for expression of anti-KIR agents/antibodies may be derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silk moth) have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • Suitable host cells for expression of anti-KIR agents/antibodies also may include vertebrate cells (e.g., mammalian cells). Vertebrate cells may be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby 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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary
  • Host cells may be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, and/or amplifying the genes encoding the desired sequences.
  • Host cells used to produce an agent/antibody herein may 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 the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENT MYCINTM), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Certain culture conditions, such as temperature, pH, and the like, may include conditions previously used with the host cell selected for expression.
  • growth factors such as insulin, transferrin, or epidermal growth factor
  • salts such as sodium chloride, calcium, magnesium, and phosphate
  • buffers such as HEPES
  • nucleotides such as adenosine and thymidine
  • antibiotics such as GENT MYC
  • an agent/antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.
  • the agent/antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. Protein A can be used to purify antibodies that are based on human heavy chains.
  • protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein G may be for mouse isotypes and for human g3.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • compositions comprising an anti-KIR agent/antibody described herein, and a pharmaceutically acceptable excipient.
  • antibodies and related compositions which may be useful for blocking KIR ligand binding, for example.
  • antibodies and related compositions which may be useful for enhancing KIR ligand binding.
  • antibodies and related compositions which may be useful for inhibiting natural killer cell activity.
  • antibodies and related compositions which may be useful for activating natural killer cell activity.
  • Serum KIR levels may be associated with prognosis in patients with certain diseases or disorders. Accordingly, anti-KIR antibodies may be useful for detecting KIR levels in certain patients.
  • an anti-KIR antibody or antigen binding fragment thereof may be formulated in a pharmaceutical composition that is useful for a variety of purposes, including the treatment of diseases or disorders.
  • Pharmaceutical compositions comprising one or more antibodies may be administered using a pharmaceutical device to a patient in need thereof, and according to one embodiment of the technology, kits are provided that include such devices. Such devices and kits may be designed for routine administration, including self-administration, of the pharmaceutical compositions herein.
  • compositions comprising an anti-KIR agent/antibody described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.
  • Therapeutic formulations of an anti-KIR antibody may be prepared for storage by mixing the agent or antibody having the desired degree of purity with physiologically and/or pharmaceutically acceptable carriers, excipients, or stabilizers, in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • Formulations for in vivo administration generally are sterile. This may be accomplished for instance by filtration through sterile filtration membranes, for example.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot® (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • polymers such as such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • antibodies provided herein may be administered to a mammal, e.g., a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the appropriate dosage of agent or antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventative or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the health professional.
  • the antibody may be suitably administered to the patient at one time or over a series of treatments.
  • about 1 pg/kg to about 50 mg/kg (e.g., 0.1 -20 mg/kg) of antibody may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily or weekly dosage might range from about 1 pg/kg to about 20 mg/kg or more, depending on the factors mentioned above.
  • the treatment may be repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful.
  • the progress of this therapy is easily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using the antibody to determine KIR levels in bodily fluids or tissues may be used in order to optimize patient exposure to the therapeutic antibody.
  • a composition comprising an antibody herein can be administered as a monotherapy, and in some embodiments, the composition comprising the antibody can be administered as part of a combination therapy.
  • the anti-KIR agent/antibody is used as an adjuvant or in conjunction with an adjuvant.
  • the effectiveness of the antibody in preventing or treating diseases may be improved by administering the antibody serially or in combination with another drug that is effective for those purposes, such as a chemotherapeutic drug for treatment of cancer or a microbial infection.
  • the antibody may serve to enhance or sensitize cells to chemotherapeutic treatment, thus permitting efficacy at lower doses and with lower toxicity.
  • Certain combination therapies include, in addition to administration of the composition comprising an antibody that blocks KIR activity, delivering a second therapeutic regimen selected from the group consisting of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
  • a second therapeutic regimen selected from the group consisting of a chemotherapeutic agent, radiation therapy, surgery, and a combination of any of the foregoing.
  • Such other agents may be present in the composition being administered or may be administered separately.
  • the antibody may be suitably administered serially or in combination with the other agent or modality, e.g., chemotherapeutic drug or radiation for treatment of cancer, infection, and the like, or an immunosuppressive drug.
  • diagnostic reagents comprising anti-KIR agent/antibody described herein.
  • antibodies provided herein may be used to detect and/or purify KIR from bodily fluid(s) or tissues.
  • Anti-KIR antibodies for example, may be useful in diagnostic assays for KIR, e.g., detecting its presence in specific cells, tissues, or bodily fluids. Such diagnostic methods may be useful in diagnosis of a disease or disorder.
  • methods for detecting KIR and/or measuring KIR levels in a subject or in a sample from a subject are also provided herein.
  • a method may comprise contacting a sample (e.g., a biological sample known or suspected to contain KIR) with an antibody provided herein, and, if the sample contains KIR, detecting KIR:antibody complexes.
  • a KIR detection method is performed in vitro.
  • a KIR detection method is performed in vivo.
  • the antibody may be labeled with a radionuclide (such as 111 In, "Tc, 14 C, 131 1, 125 l, 3 H, 32 P, or 35 S) so that the bound target molecule can be localized using immunoscintillography.
  • reagents comprising anti-KIR agent/antibody described herein for nondiagnostic use are also provided herein.
  • reagents comprising anti-KIR agent/antibody described herein for non-therapeutic use are also provided herein.
  • reagents comprising anti-KIR agent/antibody described herein for non-diagnostic and non-therapeutic use are also provided herein.
  • a method may comprise contacting a non-biological sample (e.g., a laboratory research sample known or suspected to contain KIR) with an antibody provided herein, and, if the sample contains KIR, detecting KIR:antibody complexes.
  • Laboratory research samples may include non-human animal models, samples from non-human animal models, cell lines, products produced by cell lines, and the like.
  • Anti-KIR agents and antibodies provided herein may be employed in any suitable detection assay, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • detection assay such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • Flow cytometry and mass cytometry assays generally involve the use of a single primary antibody to specifically identify the presence of the target molecule expressed on the surface of a dispersed suspension of individual cells.
  • the dispersed cells are typically obtained from a biological fluid sample, e.g., blood, but may also be obtained from a dispersion of single cells prepared from a solid tissue sample such as a tumor biopsy.
  • the primary antibody may be directly conjugated with a detectable moiety, e.g., a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass cytometry.
  • the primary antibody may be unlabeled or labeled with an undetectable tag such as biotin, and the primary antibody is then detected by a detectably labeled secondary antibody that specifically recognizes the primary antibody itself or the 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 brightfield light microscope, to identify those individual cells in the dispersed population or tissue sample that express the target recognized by the primary antibody.
  • an instrument capable of single cell detection e.g., flow cytometer, mass cytometer, fluorescence microscope or brightfield light microscope, to identify those individual cells in the dispersed population or tissue sample that express the target recognized by the primary antibody.
  • fixed and permeabilized cells may be used.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein that is detected.
  • the test sample analyte is bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the target cell population may be attached to the solid support using antibodies first attached to the support and that recognize different cell surface proteins. These first antibodies capture the cells to the support.
  • fixed and permeabilized cells may be used.
  • an agent/antibody provided herein is formulated for immunohistochemical analysis.
  • immunohistochemical analysis includes the use of samples.
  • immunohistochemical analysis includes the use of blood and/or tissue samples.
  • a sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin.
  • a sample is a formalin-fixed paraffin- embedded (FFPE) sample.
  • an FFPE sample is saturated with formalin (i.e.
  • an FFPE sample is stable at room temperature. In some embodiments, all or most of the structures in an FFPE sample are preserved. In some embodiments, the intracellular and surface proteins in an FFPE sample are preserved. In some embodiments, mRNA in an FFPE sample is preserved. In some embodiments, mRNA, intracellular proteins, and surface proteins in an FFPE sample are preserved. In some embodiments, surface proteins in an FFPE sample are denatured.
  • an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting KIR in a formalin-fixed paraffin-embedded sample. In some embodiments, an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting KIR on the surface of a formalin-fixed paraffin-embedded sample. In some embodiments, an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting intracellular KIR in a formalin-fixed paraffin-embedded sample. In some embodiments, an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting intracellular KIR and KIR on the surface of a formalin-fixed paraffin-embedded sample.
  • a sample is a fresh sample that has been frozen. In some embodiments, a sample is a fresh sample that has been cryogenically frozen. In some embodiments, a sample is flash frozen. In some embodiments, a sample is flash frozen and stored at 80°C. In some embodiments, all or most of the structures in a flash frozen sample are preserved. In some embodiments, intracellular and surface proteins in a flash frozen sample are preserved. In some embodiments, mRNA in a flash frozen sample is preserved. In some embodiments, mRNA, intracellular proteins, and surface proteins in a flash frozen sample are preserved. In some embodiments, surface proteins in a flash frozen sample are denatured.
  • an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting KIR in a frozen sample. In some embodiments, an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting KIR on the surface of a frozen sample. In some embodiments, an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting intracellular KIR in a frozen sample. In some embodiments, an anti-KIR antibody or antigen binding fragment thereof provided herein is capable of detecting intracellular KIR and KIR on the surface of a frozen sample.
  • antibodies and methods for detecting KIR are provided herein.
  • antibodies and methods are provided for detecting KIR in a biological sample.
  • KIR is detected on the surface of a cell.
  • KIR is detected intracellularly.
  • detection of KIR is in vitro.
  • detection of KIR is in vivo.
  • a biological sample is a solid tissue, fluid, or cell.
  • Solid tissue samples may comprise solid tissue from one or more of adipose tissue, bladder, bone, brain, breast, cervix, endothelium, gallbladder, kidney, liver, lung, lymph, ovary, prostate, salivary gland, stomach, testis, thyroid, urethra, uterus, vagina, and vulva.
  • Fluid samples may comprise one or more of amniotic fluid, bile, blood, breast milk, breast fluid, cerebrospinal fluid, lavage fluid, lymphatic fluid, mucous, plasma, saliva, semen, serum, spinal fluid, sputum, tears, umbilical cord blood, urine, and vaginal fluid.
  • a sample comprises immune cells.
  • a sample comprises one or more immune cell chosen from macrophages, neutrophils, B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, myeloid dendritic cells (mDCs), innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs).
  • a sample comprises non-immune cells.
  • a sample may comprise one or more of fibroblasts, vascular smooth muscle cells, endothelial cells, brain cells, and liver cells.
  • a sample comprises tumor cells.
  • the biological sample is from a healthy subject. In some embodiments, the sample is from a subject with a disease or condition. In some embodiments, the detection of KIR indicates the presence or absence of a disease or disorder. In some embodiments, the disease or disorder is a cancer, an autoimmune disorder, an inflammatory disorder, a neurologic disorder, or an infection. In some embodiments, the disease or disorder is associated with KIR expression. In some embodiments, the disease or disorder is associated with aberrant KIR expression. In some embodiments, the disease or disorder is associated with immune cells. In some embodiments, the disease or disorder is associated with natural killer cells. In some embodiments, the disease or disorder is associated with T lymphocytes. Kits
  • kits comprising an anti-KIR agent/antibody described herein.
  • a kit generally refers to a packaged combination of reagents in predetermined amounts with instructions for use (e.g., instructions for performing a diagnostic assay).
  • the kit is a diagnostic kit configured to detect KIR (e.g., KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and/or KIR3DL3) in a sample (e.g., a biological sample).
  • the kit is a non-diagnostic kit configured to detect KIR (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).
  • a non-biological sample e.g., a research sample.
  • the kit may include an identical isotype negative control irrelevant antibody to control for non-specific binding of the anti-KIR agent.
  • the kit may include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides the detectable chromophore or fluorophore
  • other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration.
  • An article of manufacture may comprise a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active anti-KIR agent in the composition may be an anti-KIR antibody.
  • the label on, or associated with, the container indicates that the composition is used for treating, or diagnosing, the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer’s solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • A1 An isolated antibody or antigen binding fragment thereof that binds one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, comprising:
  • an immunoglobulin heavy chain variable domain comprising:
  • CDRH1 heavy chain complementarity determining region 1
  • CDRH2 heavy chain complementarity determining region 2
  • CDRH3 heavy chain complementarity determining region 3
  • an immunoglobulin light chain variable domain comprising:
  • CDRL1 light chain complementarity determining region 1
  • CDRL2 light chain complementarity determining region 2
  • CDRL3 light chain complementarity determining region 3
  • A1 .1 The isolated antibody or antigen binding fragment thereof of embodiment A1 , wherein the one or more KIRs are chosen from KIR2DL1 , KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1 , KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1 , KIR3DL2, and KIR3DL3.
  • immunoglobulin heavy chain variable domain comprises an immunoglobulin heavy chain variable domain polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to an immunoglobulin heavy chain variable domain polypeptide provided in Table 3.
  • immunoglobulin light chain variable domain comprises an immunoglobulin light chain variable domain polypeptide provided in Table 3, or a polypeptide that is at least 80 percent identical to an immunoglobulin light chain variable domain polypeptide provided in Table 3.
  • A4 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A3, comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains.
  • A5 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A4, further comprising one or more human framework regions.
  • A6 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A5, wherein the antibody or antigen binding fragment thereof is humanized.
  • A7 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A6, wherein the antibody or antigen binding fragment thereof comprises or is conjugated to a detectable marker or label.
  • A9 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A8, further comprising an oligonucleotide.
  • A10 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A9, wherein the antibody or antigen binding fragment thereof is immobilized on a solid support.
  • A11 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A10, wherein the antibody or antigen binding fragment thereof is non-diffusively immobilized on a solid support.
  • A12 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A11 , that is a single-chain fragment.
  • A14 The isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A13, for non-diagnostic use and/or non-therapeutic use.
  • a kit comprising the isolated antibody or antigen binding fragment thereof of any one of embodiments A1-A14, and instructions for use.
  • a diagnostic reagent comprising the isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A13.
  • D1 A diagnostic kit configured to detect KIR or a portion thereof in a biological sample, wherein the kit comprises the isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A13 or the diagnostic reagent of embodiment C1 .
  • a therapeutic composition comprising the isolated antibody or antigen binding fragment thereof of any one of embodiments A1 -A13, and a pharmaceutically acceptable excipient.
  • F1 An isolated polynucleotide comprising a nucleic acid sequence that encodes the immunoglobulin heavy chain variable domain of any one of embodiments A1 -A13.
  • a recombinant expression vector comprising the isolated polynucleotide of embodiment F1 .
  • F4 An isolated polynucleotide comprising a nucleic acid sequence that encodes the immunoglobulin light chain variable domain of any one of embodiments A1-A13.
  • a recombinant expression vector comprising the isolated polynucleotide of embodiment F4.
  • F6 A host cell transfected with the recombinant expression vector of embodiment F5.
  • a recombinant expression vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a polynucleotide comprising a nucleic acid sequence that encodes the immunoglobulin heavy chain variable domain of any one of embodiments A1-A13, and the second expression cassette comprises a polynucleotide comprising a nucleic acid sequence that encodes the immunoglobulin light chain variable domain of any one of embodiments A1-A13.
  • a method of detecting one or more killer cell immunoglobulin-like receptors comprising a) contacting a sample with the antibody or antigen binding fragment thereof of any one of embodiments A1 -A13, and b) if the sample contains one or more KIRs, detecting KI R:anti-KI R complexes.
  • KIRs killer cell immunoglobulin-like receptors
  • a first anti-KIR agent that binds one or more killer cell immunoglobulin-like receptors (KIRs), wherein the first agent competitively binds, or is capable of competitively binding, with a second anti-KIR agent, wherein the second agent is the antibody or antigen binding fragment thereof of any one of embodiments A1-A13.
  • KIRs killer cell immunoglobulin-like receptors
  • a first anti-KIR agent that binds one or more killer cell immunoglobulin-like receptors (KIRs), wherein the first agent binds to, or is capable of binding to, the same epitope as a second anti-KIR agent, wherein the second agent is the antibody or antigen binding fragment thereof of any one of embodiments A1-A13.
  • KIRs killer cell immunoglobulin-like receptors
  • KIRs killer cell immunoglobulin-like receptors
  • An isolated antibody or antigen binding fragment thereof that binds one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, wherein the antibody or antigen binding fragment thereof enhances or is capable of enhancing KIR ligand binding.
  • KIRs killer cell immunoglobulin-like receptors
  • KIRs killer cell immunoglobulin-like receptors
  • H6 An isolated antibody or antigen binding fragment thereof that binds one or more killer cell immunoglobulin-like receptors (KIRs) or a portion thereof, wherein the antibody or antigen binding fragment thereof activates or is capable of activating natural killer cell activity.
  • KIRs killer cell immunoglobulin-like receptors
  • Example 1 Generation anti-KIR antibody expressing hybridomas
  • This Example describes the generation and characterization of hybridomas that secrete monoclonal antibodies that react with various KIR family members.
  • mice were immunized with recombinant KIR proteins, with mouse myeloma cell lines expressing human KIR immunogens as shown in Table 2 below.
  • Hybridomas were formed using standard protocols to fuse myeloma cells with spleens, and lymph node cells were drained and harvested. Successful fusions were selected into HAT medium, and cloned into approximately one cell per well in microtiter plates, after which culture supernatants were tested against KIR- expressing cell transfectants by flow cytometry. Wells were selected by assessment of staining profiles and then sub-cultured into larger vessels and sub-cloned.
  • PBMCs peripheral blood mononuclear cells
  • NK cells were then incubated in RPMI + 10% FBS + 1% Pen/Strep + 50 ng/mL human recombinant IL2 + 10 ng/mL human recombinant IL15 at 37 e C for 16 hours before harvesting the day of experimentation.
  • Ba/F3 murine interleukin-3 dependent pro-B cell line was transfected using constructs coding for the extracellular domains of KIRs.
  • the accession numbers for the 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 with 10% Fetal Bovine Serum (FBS) and recombinant mouse interleukin-3.
  • FBS Fetal Bovine Serum
  • FACS fluorescence-activated cell sorting
  • the ligands specific to particular KIR proteins were added to cells and incubated at room temperature for 15 minutes.
  • the binding was detected by the anti-tag antibody.
  • Cell suspension was then washed and analyzed on a flow cytometer.
  • cells were first incubated with KIR-specific antibody for 15 minutes at room temperature before the addition of the ligand to the cell solution.
  • K562 cells were counted and washed with culture media on the day of harvest. K562 cells were then stained using TAG-IT VIOLET dye (BioLegend 425101 ) for 12 minutes. TAG-IT VIOLET dye reaction was then quenched using cold culture media and allowed to rest for 15 minutes. Cells were centrifuged and washed using culture media. NK cells were counted and washed with RPMI + 10% FBS + 1% Pen/Strep on the day of harvest before being resuspend in culture media. NK cells were the incubated with 10 pg of designated antibody or HLA protein for 15 minutes at 37 e C to allow for adequate antibody to NK interaction.
  • TAG-IT VIOLET dye BioLegend 425101
  • Tag-it Violet stained K562 cells were then mixed at a 1 :1 ratio to NK cells and incubated at 37 e C for 1 hour.
  • NK-K562 cell co-culture was spun down and supernatant was removed for in-solution analyte analysis.
  • NK-K562 cell co-culture was then stained using standard staining procedure.
  • Cytokine quantification of NK-K562 culture supernatant was analyzed using Human CD8/NK V02 Panel LEGENDPLEX Assay kit (BioLegend 741 187). A detailed procedure can be found on the BioLegend website and should be followed per manufacturer’s instructions. Briefly, co-culture media supernatant was stained using kit capture beads for 2 hours before being spun down and washed three times using LEGENDPLEX wash buffer. Capture beads were then stained using biotinylated analyte detection antibodies for 1 hour before adding PE-streptavidin to the detection antibodies and incubated for 30 minutes at room temperature. Analyte-stained beads were then spun down and washed three times with LEGENDPLEX wash buffer. Analyte beads were then run on a flow cytometer to collect analyte quantification data. Data was analyzed using LEGENDPLEX Software.
  • Each member within the KIR protein family binds to a multitude of MHC class I proteins or certain other ligand proteins.
  • developed anti-KIR antibodies were added to singular KIR protein transfected Ba/F3 cell lines followed by singular KIR protein's known ligand. The ligand presence was then measured through a conjugate fluorophore or with anti-ligand antibody.
  • Each tested KIR protein and the developed anti-KIR antibody’s effect on their KIR’s respective ligand interaction is detailed below.
  • KIR3DL3 see e.g., Figs. 151-152) -
  • the interaction between HHLA2 and KIR3DL3 transfected Ba/F3 was able to be completely disrupted by mAb S23011 A,B,C,D,E,F and S22010B.
  • the mAb S22010G appeared to have no interaction but mAb S22010H appeared to increase the HHLA2’s interaction with KIR3DL3.
  • KIR3DL2 see e.g., Figs.
  • KIR2DS1 see e.g., Figs. 145-147) -
  • the interaction between HLA-C*04:01 monomers with bound proprietary UVX peptide and KIR2DS1 transfected Ba/F3 was abrogated with the addition of mAb S22019B, S22019F, S22013A, and S22021 B.
  • KIR2DL5 see e.g., Figs. 143-144) - While the interaction between recombinant CD155-Biotin and KIR2DL5A transfected Ba/F3 was initially minimal, the interaction of KIR2DL5 and its ligand was increased when mAb S22010G was added. This KIR-ligand enhancement was found to be specific for the interaction of KIR2DL5 and CD155 and not with ligand containing a similar amino acid sequence such as CD1 12 or CD1 13.
  • KIR2DS4 see e.g., Figs. 140-142) - The Interaction between HLA-C*05:01 APC-conjugated tetramer with bound P2-AW peptide (IIDKSGAWV) and KIR2DS4 was completely disrupted by mAb S22012B,C,E,F,G. The mAb S22012A and S22012H also slightly disrupted this KIR-ligand interaction as well.
  • KIR3DL1 see e.g., Figs. 137-139) -
  • the interaction between HLA-B*57:01 APC-conjugated tetramer with bound ‘self-peptide’ (LSSPVTKSF) was completely disrupted by mAb S22024A,B,C,D,E,F,G,H, and S22026A,B and S2301 1 E,F,G.
  • KIR2DL1 see e.g., Figs. 131-132
  • the interaction between HLA-C*04:01 APC-conjugated tetramer with bound CMV-modified peptide was completely disrupted by mAb S22039A,B,C,D and S22013A.
  • KIR2DL2 see e.g., Figs. 133-134) -
  • the mAbs S22020A and S22034A,B,C,D also partially decreased KIR-ligand interaction.
  • KIR2DS2 (see e.g., Figs. 133, 135) - The interaction been HLA-C*07:02 monomer with bound proprietary UVX peptide and KIR2DS2 transfected Ba/F3 was completely disrupted by mAb S22032A, S22032C, S22034A,B,C,D, S22036B, S22036D, and S22038E.
  • KIR2DL3 (see e.g., Figs. 133, 136) - The interaction been HLA-C*07:02 monomer with bound proprietary UVX peptide and KIR2DL3 transfected Ba/F3 was completely disrupted by mAb S22036B, S22036D, S22037E, S22037F, and S22037G.
  • the mAbs S22034A,B,C,D also partially decreased KIR-ligand interaction. Degranulation of KIR3DL1+ NK cells decreased from KIR3DL 1 specific antibody presence
  • IL-2 and IL-15 activated primary NK cells with K562 cell line were cultured in the presence of anti- KIR3DL1 antibodies and isotype control, and the change in CD107a expression was measured through flow cytometry.
  • Most NK cells without KIR3DL1 + expression did not show significant change between isotype control and anti-KIR3DL1 antibodies samples, with the exception of mAb S22026A and S23011 B showing a slight increase.
  • CD107a expression decreased (28.9 - 51 .8%) in comparison to KIR3DL1 - NK cells for samples with developed anti-KIR3DL1 antibodies, with the highest decrease coming from mAb S22024A, S22024B, S22024E, and S22024H (Fig. 124).
  • the lowest antibody mediated CD107a decrease in normalized KIR3DL1 + NK cells was induced by mAb S23011A, S2301 1 B, and S23011 C.
  • KIR2DS1 specific antibodies were added to IL-2 and IL-15 activated primary NK cells with K562 cell line and changes in CD107a expression were measured through flow cytometry.
  • KIR2DS1- NK cells the addition of mAb S22019B caused a slight but significant decrease in CD107a expression compared to isotype, while mAb S22019F did not show any significant difference compared to isotype.
  • KIR2DS1 + NK cells the addition of mAb S22019B and S22019F showed significant increases in CD107a expression (38, 36%, respectively) via the normalized CD107a activation ratio (Fig. 126).

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Abstract

La technologie concerne en partie des anticorps ou des fragments de ceux-ci se liant à des antigènes qui se lient à un ou plusieurs membres de la famille des protéines KIR (par exemple, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2 et/ou KIR3DL3) ou des parties de ceux-ci, ainsi que des méthodes, des systèmes et des kits pour la détection de KIR. Dans certains aspects, la technologie concerne également des anticorps ou des fragments de ceux-ci se liant à l'antigène destinés à être utilisés dans la détermination de niveaux de KIR dans un échantillon contenant ou suspecté de contenir un KIR. Selon certains aspects, la technologie concerne des anticorps ou des fragments se liant à l'antigène de ceux-ci destinés à être utilisés dans le diagnostic ou le traitement d'un individu atteint ou suspecté d'être atteint d'une maladie ou d'un trouble associé au KIR.
PCT/US2025/026470 2024-05-01 2025-04-25 Anticorps se liant au kir et leurs fragments se liant à l'antigène Pending WO2025230843A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0183070A2 (fr) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation de levures du genre Pichia
EP0244234A2 (fr) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation de trichoderma
EP0402226A1 (fr) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Vecteurs de transformation de la levure yarrowia

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Publication number Priority date Publication date Assignee Title
DK2287195T3 (da) * 2004-07-01 2019-08-19 Innate Pharma Pan-kir2dl nk-receptor-antistoffer og anvendelse heraf i diagnostik og terapi
PL1836225T3 (pl) * 2005-01-06 2012-05-31 Novo Nordisk As Czynniki wiążące kir i sposoby ich stosowania
EP2897979B1 (fr) * 2012-09-19 2019-02-13 Innate Pharma Agents de liaison à kir3dl2
JP6654781B2 (ja) * 2014-08-29 2020-02-26 国立大学法人北海道大学 Ｋｉｒ２ｄｓ１に対するモノクローナル抗体

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Publication number Priority date Publication date Assignee Title
EP0183070A2 (fr) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation de levures du genre Pichia
EP0244234A2 (fr) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation de trichoderma
EP0402226A1 (fr) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Vecteurs de transformation de la levure yarrowia

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* Cited by examiner, † Cited by third party
Title
DEBSKA-ZIELKOWSKA, J ET AL., CELLS, 2021
PENDE, D ET AL., FRONT IMMUNOL, 2019

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