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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
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  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
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  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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  • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
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  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/31—Chimeric antigen receptors [CAR]
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  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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• • A—HUMAN NECESSITIES
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  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], 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
  • C07K16/283—Immunoglobulins [IG], 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 against Fc-receptors, e.g. CD16, CD32, CD64
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
  • A61K2039/55527—Interleukins
  • A61K2039/55533—IL-2
• • A—HUMAN NECESSITIES
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  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • 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
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
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  • C07—ORGANIC CHEMISTRY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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  • C07K2317/77—Internalization into the cell
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  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the antibody binds human CD94 expressed on the surface of a cell (e.g., a human natural killer (NK) cell). In some embodiments, the antibody binds cynomolgus CD94 expressed on the surface of a cell (e.g., a cynomolgus NK cell, or a cell such as a human cell that overexpresses cynomolgus CD94). In some embodiments, binding of the antibody to human CD94 does not block binding between human CD94 and human HLA-E. In some embodiments, incubating the antibody with a cell expressing human CD94 on its surface for 24 hours at 37°C results in a decrease in surface antibody staining of less than 50% due to internalization.
• a cell expressing human CD94 on its surface for 24 hours at 37°C results in a decrease in surface antibody staining of less than 50% due to internalization.
• the VH domain comprises an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 19, and the VL domain comprises an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO:20.
• the VH domain comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19 and the VL domain comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20.
• the VH domain comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 19, and the VL domain comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:20.
• the VH domain comprises an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO:21
• the VL domain comprises an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO:22.
• the VH domain comprises the amino acid sequence of SEQ ID NO:21
• the VL domain comprises the amino acid sequence of SEQ ID NO:22.
• the antibody comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein: (a) the VH domain comprises the amino acid sequence of SEQ ID NO:65, and the VL domain comprises the amino acid sequence of SEQ ID NO:66; (b) the VH domain comprises the amino acid sequence of SEQ ID NO:67, and the VL domain comprises the amino acid sequence of SEQ ID NO:68; (c) the VH domain comprises the amino acid sequence of SEQ ID NO:69, and the VL domain comprises the amino acid sequence of SEQ ID NO:70; (d) the VH domain comprises the amino acid sequence of SEQ ID NO:71, and the VL domain comprises the amino acid sequence of SEQ ID NO:72; (e) the VH domain comprises the amino acid sequence of SEQ ID NO:73, and the VL domain comprises the amino acid sequence of SEQ ID NO:74; (I) the VH domain comprises the amino acid sequence of SEQ ID NO:75, and the VL domain comprises the amino acid sequence of SEQ ID NO:
• the number of leukemic cells in the human IgGl and anti-CD94 antibody treated conditions were normalized to leukemic cell number from human IgGl treated wells. Partially non- fucosylated, human IgGl 18H3 depleted human CLPD-NK leukemic cells in a concentration dependent manner.
• ATX-130 antibody was titrated from 50 nM to 0.02 nM. Secondary antihuman antibody labeled with Alexa Fluor 647 was used to detect binding on CD3-CD56 bright NK cells. ATX-130-KIF antibody conjugated with Alexa Fluor 647 was titrated from 50 nM to 0.02 nM. T-LGLL cells were identified using CD3+CD16+ gating, while CLPD-NK cells were identified using CD3-CD16+ gating strategy. Titration curves and EC50 were generated using Graphpad Prism. ATX-130 bound to CD3-CD56 bright NK cells with an affinity of 0.6 nM.
• CD3+CD8+NKG2A+ gating strategy was used to identify cynomolgus CD8+ T cells.
• CD8 T cells were quantified by calculating the percentage of the cell population out of total PBMCs. Identifiers at the top of each bar indicate time point at which PMBCs were isolated and correspond to time points depicted in figure legend.
• an antibody of the disclosure has a K D of any of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, or less than
• an antibody of the disclosure specifically binds to human CD94, wherein the antibody does not bind to the same epitope on human CD94 as anti-CD94 antibody clones DX22, HP-3B1, or 131412. In some embodiments, an antibody of the disclosure specifically binds to human CD94, wherein the antibody binds to the same epitope on human CD94 as anti-CD94 antibody clones HP-3D9, DX22, HP-3B1, 131412, or 12K45. In some embodiments, an antibody of the disclosure specifically binds to human CD94, wherein the antibody binds to the same epitope on human CD94 as anti-CD94 antibody clone HP-3D9. In some embodiments, an antibody of the disclosure specifically binds to human CD94, wherein the antibody binds to the same epitope on human CD94 as anti-CD94 antibody clone 12K45.
• an antibody of the disclosure comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18.
• an antibody of the disclosure comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:57, a CDR-H2 comprising an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 9
• an antibody of the disclosure comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:48, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:64.
• VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:48, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62
• the VL domain comprises a CDR-
• an antibody of the disclosure comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:81, and the VL domain comprises the amino acid sequence of SEQ ID NO:82.
• an antibody of the disclosure comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 116, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 118; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:99, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:56.
• VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 116, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 118
• the VL domain comprises
• an antibody of the disclosure comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 109, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 110, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 111; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 112, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 113, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:64.
• VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 109, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 110, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 111
• the VL domain comprises a
• an antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:71 and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:72.
• an antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of ATX-125 as described herein (see, e.g, Table 2) and/or a VL domain comprising 1,
• an antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of ATX-130 as described herein (see, e.g, Table 2) and/or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of ATX-130 as described herein (see, e.g., Table 2).
• an antibody of the disclosure binds to an epitope on human or cynomolgus monkey CD94 that is the same as the CD94 epitope bound by an anti-CD94 antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR- L3 comprising the amino acid sequence of SEQ ID NO:6.
• VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1
• CDR-H2 comprising the amino acid sequence of SEQ ID NO:
• an antibody of the disclosure binds to an epitope on human or cynomolgus monkey CD94 that is the same as the CD94 epitope bound by an anti-CD94 antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:30, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:31, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:32; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:33, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:35.
• VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:30, a CDR-H2 comprising the amino acid sequence of
• an antibody of the disclosure binds to an epitope on human or cynomolgus monkey CD94 that is the same as the CD94 epitope bound by an anti-CD94 antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:65, and the VL domain comprises the amino acid sequence of SEQ ID NO:66.
• VH heavy chain variable
• VL light chain variable
• an antibody of the disclosure binds to an epitope on human or cynomolgus monkey CD94 that is the same as the CD94 epitope bound by an anti-CD94 antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:69, and the VL domain comprises the amino acid sequence of SEQ ID NO:70.
• VH heavy chain variable
• VL light chain variable
• an antibody of the disclosure binds to an epitope on human or cynomolgus monkey CD94 that is the same as the CD94 epitope bound by an anti-CD94 antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:71, and the VL domain comprises the amino acid sequence of SEQ ID NO:72.
• VH heavy chain variable
• VL light chain variable
• an antibody of the disclosure binds to an epitope on human or cynomolgus monkey CD94 that is the same as the CD94 epitope bound by an anti-CD94 antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:47, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:48, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:62; and wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:63, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:64.
• an antibody does not block binding of HLA-E to the CD94/NKG2A heterodimer if it blocks about 20% or less of HLA-E binding to the CD94/NKG2A heterodimer, e.g., compared to an isotype control antibody.
• ADCC activity is determined using an ex vivo assay using PBMCs, LGL cells, and/or NK cells, e.g., as described in the Examples, and the ADCC activity of an antibody of the disclosure is described as the percent of target cells remaining after the ADCC assay and/or the IC50 or EC50 of the antibody (i.e., the concentration of an antibody of the disclosure at which half the maximum target cell depletion or cell lysis is achieved).
• the IC50 or EC50 of an antibody may be determined using any method known in the art, e.g., using a dosage response curve and GraphPad Prism.
• the antibodies provided herein induce ADCC activity with an IC50 or EC50 measured using an ex vivo assay of between about 1 ng/ml to about 100 ng/ml (e.g., any of about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, or about 100 ng/ml).
• an ex vivo assay
• an antibody of the disclosure exhibits an IC50 or EC50 that is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than the IC50 or EC50 of a control antibody (e.g., a wild type control antibody, or an antibody known in the art or commercially available against the same target).
• a control antibody e.g., a wild type control antibody, or an antibody known in the art or commercially available against the same target.
• the antibodies provided herein include a human immunoglobulin Fc region that has enhanced ADCC activity compared to a wild type Fc region. In some embodiments, the antibodies provided herein bind to a human cellular Fc receptor to a greater extent than an antibody comprising a wild type Fc region.
• an Fc receptor is a receptor that is capable of binding to an Fc region of an antibody. Certain Fc receptors can bind to IgG (i.e., y-receptor); such receptors include subclasses of FcyRI, FcyRII and FcyRIII, as well as their allelic variants and alternative splicing events.
• compositions of a partially fucosy lated antibody has a degree of fucosy lation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%).
• cell lines that may be used to produce non-fucosylated or defucosy lated antibodies or antibodies with reduced fucosy lation are known in the art, e.g., include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Y amane- Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and cells overexpressing (31,4-N- acetylglycosminyltransferase III (GnT-III) and Golgi p-mannosidase II (Manll).
• Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. In some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated.
• an antibody of the disclosure is modified such that less than about 90%, e.g., less than any of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1%, of the carbohydrates of the antibody contain fucose. In some embodiments, an antibody of the disclosure is modified such that less than about 40% of the carbohydrates of the antibody contain fucose. In some embodiments, the antibodies provided herein are non-fucosylated.
• an antibody has a high degree of internalization if it results in an MFI decrease of greater than 50%, calculated by computing the difference in MFI over a 24 hour period (e.g., between 0.5 and 24 hours) in cells incubated with antibody at 37°C, and multiplying by 100, as measured by an ex vivo assay, e.g., using PBMCs and/or NK cells, as described in the Examples.
• an antibody has a high degree of internalization if incubating the antibody with a cell expressing human CD94 on its surface for 24 hours at 37°C results in a decrease in surface antibody staining of greater than 50% due to internalization, assessed using methods known in the art and/or as described above.
• an antibody has a low degree of internalization if it results in an MFI decrease of less than 50% (e.g., any of about 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, or 0%), calculated by computing the difference in MFI over a 24 hour period (e.g., between 0.5 and 24 hours) in cells incubated with antibody at 37°C, and multiplying by 100, as measured by an ex vivo assay, e.g., using PBMCs and/or NK cells, as described in the Examples.
• an ex vivo assay e.g., using PBMCs and/or NK cells, as described in the Examples.
• the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.
• recombinant targets e.g., CD94
• PBL Peripheral blood lymphocytes isolated from the blood of normal (i.e., healthy) donors are incubated with antibodies that have a human Fc region with and without fucose and/or with and without Fc region mutations.
• the level of killing of NK cells and/or T cells that express CD94 in the PBLs is measured using any method known in the art, such as flow cytometry (e.g., as described in the Examples).
• an antibody of the disclosure has stability (e.g., lack of degradation products, e.g., as measured by SDS-PAGE) during storage, e.g., for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, or more, at any of about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, or about 8°C.
• stability e.g., lack of degradation products, e.g., as measured by SDS-PAGE
• NK cells and/or T cells that express CD94 may play a role are: LGL leukemia, Rheumatoid arthritis, Felty’s syndrome, CLPD-NK, aggressive NK leukemia, IBM, IBD, or an NK/T cell lymphoma, such as extranodal NK/T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy -associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma/delta TCL, or subcutaneous pannic
• the disease or disorder is selected from chronic lymphoproliferative disorder of NK cells (CLPD-NK), LGL leukemia, Felty’s syndrome, rheumatoid arthritis, aggressive NK leukemia, inclusion body myositis, inflammatory bowel disease, or an NK/T cell lymphoma, such as extranodal NK/T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy- associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epidermotropic CD8+ cutaneous TCL, primary cutaneous gamma/delta TCL, subcutaneous panniculitis TCL, or microscopic colitis.
• CLPD-NK chronic lymphoproliferative disorder of NK cells
• administration of the antibody results in a reduction in the number of NK cells and/or T cells that express CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood NK cells, CD8+, CD4+, or CD8+/CD4+ T cells, and/or LGL cells in the subject, e.g., that express CD94. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood LGL cells, e.g., that express CD94, in the subject. In some embodiments, administration of the antibody results in a reduction in the number of peripheral blood NK cells, e.g., that express CD94, in the subject.
• the reduction in the number of peripheral blood NK cells and/or T cells that express CD94 in the subject is reversible. In some embodiments, the reduction in the number of peripheral blood NK cells and/or T cells that express CD94 in the subject is reversible within any of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or more, after administration of the antibody to the subject.
• an antibody of the disclosure binds to CD94.
• an antibody of the disclosure depletes and/or reduces the level of NK cells and/or T cells that express CD94.
• an antibody of the disclosure has clear benefits for a patient (e.g., a human patient) having a disease or disorder, such as CLPD-NK, LGL leukemia, rheumatoid arthritis, Felty’s syndrome, aggressive NK leukemia, IBM, IBD, or an NK/T cell lymphoma, such as extranodal NK/T cell lymphoma, hepatosplenic T cell lymphoma (TCL), enteropathy-associated TCL, cutaneous TCL, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), peripheral TCL (not otherwise specified), angioimmunoblastic TCL, adult TCL, monomorphic epitheliotropic intestinal TCL, epithelial a a fibroblasts, a
• the methods described herein may be used, e.g., to reduce the number of abnormal or pathologic NK cells and/or T cells (e.g., CD4+ T cells, CD8+ T cells, CD4+ and CD8+ T cells) that express CD94 via mechanisms such as ADCC that employ NK cells, essentially using the pathologic cells to eliminate each other.
• NK cells and/or T cells e.g., CD4+ T cells, CD8+ T cells, CD4+ and CD8+ T cells
• LGL leukemia is a chronic lymphoproliferative disorder that exhibits a chronic elevation in large granular lymphocytes (LGLs) in the peripheral blood and is called T- cell LGL leukemia.
• Aggressive NK-cell leukemia is an aggressive disease with systemic proliferation of NK cells and a rapidly declining clinical course. Aggressive NK leukemia may also be referred to as aggressive NK-cell lymphoma. Symptoms of aggressive NK-cell leukemia include constitutional symptoms (e.g., malaise, weight loss, fatigue), hepatosplenomegaly, lymphadenopathy, coagulopathies, hemophagocytic syndrome, multi-organ failure, infections such as Epstein-Barr virus, allergic reactions (e.g., allergic reactions to insect bites, such as mosquito bites) that may result in necrosis and systemic symptoms such as fever, swollen lymph nodes, abdominal pain, diarrhea, and anaphylaxis.
• constitutional symptoms e.g., malaise, weight loss, fatigue
• hepatosplenomegaly e.g., lymphadenopathy
• coagulopathies e.g., hepatosplenomegaly
• lymphadenopathy e.g.
• IBM Inclusion Body Myositis
• sporadic inclusion body myositis is an inflammatory muscle disease characterized by autoimmune and degenerative processes that result in progressive weakness and wasting of distal and/or proximal muscles.
• IBM is characterized by invasion of immune cells into muscle tissues. In some cases, patients with IBM have elevated creatine kinase levels in the blood. Symptoms of IBM include progressive muscle weakness, muscle wasting/atrophy, frequent tripping and falling, difficulty manipulating fingers, foot drop, restricted mobility, impaired balance, muscle pain, dysphagia, and fatigue.
• Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: 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
• the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. IV. Kits and Articles of Manufacture
• the container holds a composition which is by itself or combined with another composition effective for the methods provided herein, e.g., treatment of the diseases or disorders described above, reducing the number of peripheral blood NK cells and/or T cells that express CD94 in a subject, or inducing ADCC activity in a subject, 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).
• At least one active agent in the composition is an antibody of the disclosure.
• mice Prior to cell fusion, mice were administered with one additional boost of CD94-His antigen. The mice were then sacrificed and their spleens harvested. Spleen cells and SP2/0-Agl4 myeloma cells were mixed, and fusion was then induced by incubation at 37 °C in the presence of polyethylene glycol (PEG) or electroporation. The cells were then harvested and plated into 96-well plates with limited dilution to achieve one cell per well. The cells were subsequently treated with hypoxanthine, aminopterin and thymidine (HAT) medium and selected for over 2 weeks in culture.
• PEG polyethylene glycol
• HAT hypoxanthine, aminopterin and thymidine
• Hybridoma supernatant were screened using ELISA and flow cytometry to identify candidates specific towards CD94.
• ELfSA CD94-His antigen was immobilized on plates and 100 pl of each supernatant was incubated with antigen.
• a fluorescently labeled secondary antibody was used to detect antibodies captured on the ELISA plate, and positive hits were validated by analysis of antibody binding on human primary NK cells using flow cytometry. Cynomolgus CD94 cross-reactivity was assessed by antibody binding to cyno-CD94-expressing BaF3 cells using flow cytometry.
• PBMCs Peripheral blood mononuclear cells
• GE Healthcare Pittsburgh, IL
• Bambanker cell freezing media Bulldog-Bio, Portsmouth, NH
• buffy coats were diluted in phosphate buffered saline (PBS) in a 1:1 ratio, followed by layering of the diluted buffy coat and centrifugation at 760g in ficoll.
• PBS phosphate buffered saline
• PBLs Peripheral blood leukocytes
• FIG. 13 shows titration curves generated for anti-CD94 antibody clone ATX-130.
• anti-CD94 antibody clone ATX-130 showed an affinity of 0.3 nM on BaF3 cells overexpressing CD94 homodimer (top panel).
• anti-CD94 antibody clone ATX-130 showed an affinity of 0.8 nM and 1.8 nM on BaF3 cells overexpressing either CD94/NKG2A heterodimer or CD94/NKG2C, respectively (lower panels).
• the validated anti-CD94 antibody was further evaluated for its ability (or lack thereof) to block binding by HLA-E, as described in Example 1.
• HLA-E blocking by anti-CD94 antibody ATX-130-KIF and an isotype control were evaluated by flow cytometry. As shown in FIG. 16, saturating concentrations of ATX-130-KIF did not block HLA-E binding.
• An antibody internalization assay was performed to evaluate internalization of CD94 receptors when bound by anti-CD94 antibody, as described in Example 1. As shown in FIG. 17, CD94 receptor did not become internalized for up to 24 hours at 37°C when bound to unconjugated ATX-130.
• This example describes the characterization of antibodies specific to human CD94 using in vivo mouse studies.
• anti-CD94 antibodies ATX-122, -123, -124, -125, -126, -127, -128, -129, and -130 were characterized in HLA-E tetramer blocking assays to determine whether they block HLA-E binding. Healthy donor PBMCs were incubated with anti-CD94 antibodies. PE labeled HLA-E tetramer was then incubated with cells and antibody mixture and detected by flow cytometry.

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