WO2020035345A1 - Traitement du cancer colorectal par une association d'un anticorps anti-mica et d'un anticorps anti-nkg2a - Google Patents

Traitement du cancer colorectal par une association d'un anticorps anti-mica et d'un anticorps anti-nkg2a Download PDF

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WO2020035345A1
WO2020035345A1 PCT/EP2019/071045 EP2019071045W WO2020035345A1 WO 2020035345 A1 WO2020035345 A1 WO 2020035345A1 EP 2019071045 W EP2019071045 W EP 2019071045W WO 2020035345 A1 WO2020035345 A1 WO 2020035345A1
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mica
antibody
nkg2a
agent
cells
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PCT/EP2019/071045
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Matthieu ALLEZ
Mathieu Blery
Lionel LE BOURHIS
Tristan COURAU
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Innate Pharma
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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
    • C07K16/2833—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 against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • C07K2317/72—Increased effector function due to an Fc-modification
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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

  • This invention relates to the use of a NKG2A-neutralizing agent and an antibody that binds human MICA to treat cancer.
  • the invention is particularly useful for treating colorectal cancer.
  • NK cell activity is regulated by a complex mechanism that involves both activating and inhibitory signals.
  • Several distinct NK-specific receptors have been identified that play an important role in the NK cell mediated recognition and killing of HLA Class I deficient target cells. Cross-linking of activating receptor proteins leads to NK cell activation resulting in increased intracellular Ca ++ levels, triggering of cytotoxicity, and lymphokine release, and an activation of NK cytotoxicity against many types of target cells.
  • CD94/NKG2A is an inhibitory receptor found on subsets of lymphocytes, including NK cells and CD8 T cells.
  • CD94/NKG2A restricts cytokine release and cytotoxic responses of certain lymphocytes towards cells expressing the CD94/NKG2A-ligand HLA-E.
  • HLA-E has also been found to be secreted in soluble form by certain tumor cells and activated endothelial cells.
  • Antibodies that inhibit CD94/NKG2A signaling may increase the cytokine release and cytolytic activity of lymphocytes towards HLA-E positive target cells, such as responses of CD94/NKG2A-positive NK cells towards HLA-E expressing tumor cells or virally infected cells. Neutralizing anti-NKG2A antibodies may therefore be useful in the treatment of cancer.
  • NKG2D is an activating receptor normally expressed on human T cells (e.g. CD8 + T cells, gd T cells) and NK cells.
  • T cells e.g. CD8 + T cells, gd T cells
  • NK cells On pre-activated CD8 + cells, NKG2D functions as a synergistic co-stimulator of CD28 and TCR signalling via DAP10 association, whereas in NK cells it functions as a direct activator.
  • NKG2D therefore conveys directly activating or costimulatory signals via the paired DAP10 adaptor protein, thereby promoting cancer and infectious disease immunity.
  • MICA major histocompatibility complex class l-related chain A and B polypeptides
  • ULBP UL16-binding protein
  • RAET1 retinoic acid early transcript-1
  • MICA and its close relative MICB which also serves as a ligand for NKG2D, are both polymorphic and the polymorphism has been shown to affect the affinity for NKG2D (Steinle et al. 2001 Immunogenetics 53:279).
  • membrane bound MICA has been reported to downmodulate the expression of NKG2D on NK and/or T cells (Von Lilienfeld-Toal et al. (2010) Cancer Immunol. Immunother. Volume 59, Issue 6, pp 829-839).
  • WO2013/117647 (Innate Pharma) describes use of anti-MICA antibodies that bind with high affinity to the principal MICA alleles found in the human population (and further to MICB), that bind to MICA on the surface of tumor cells, that are able to mediate ADCC towards MICA-expressing tumors.
  • An extensive set of antibodies were disclosed, including some antibodies that bind the a1 a2 platform domain or the a3 domain, and that inhibit the interaction between MICA and NKG2D and consequently can prevent the MICA-mediated down-modulation of NKG2D on NK and/or T cells by membrane-bound MICA (as well by soluble MICA, if present).
  • anti-MICA antibodies that may be able to prevent the MICA-mediated down-modulation of NKG2D on NK and/or T cells, including antibodies that bind the a3 domain (nos. WO2013/117647, WO2013/049527 and WO2015/085210, WO2018/081648 and WO2019/147863).
  • Some antibodies are stated to neutralize soluble MICA (sMICA) (see WO2015/03114), while others are stated to reduce shedding of MICA from the cell surface (see, e.g., WO2018/081648); additionally, certain anti-a3 domain antibodies blocking the interaction of MICA with NKG2D (see, e.g., antibody 14B4 of WO2013/1 17647).
  • Some anti-MICA antibodies bind membrane-bound MICA (e.g. on the surface of tumor cells), while others bind sMICA without binding membrane-bound MICA (see, e.g., WO2014/140904).
  • CRC Colorectal cancer
  • NKG2D-MICA/B engagement has been shown to be a major mechanism of tumor immune surveillance, and proteolytic shedding of surface MICA/B can saturate NKG2D receptors and/or lead to their down-modulation.
  • MICA/B are major targets of cytotoxic cells that infiltrate human tumors (colorectal cancer spheroids) and that NKG2A expression on infiltrating T cells is serving as a mechanism for tumor cells to avoid lysis by the infiltrating immune cells.
  • anti-MICA antibodies induced an upregulation of the inhibitory receptor NKG2A on infiltrating NK cells, which led to study of the expression patterns of NKG2A and its ligand HLA-E during co-cultures.
  • NKG2A expression was increased on infiltrating T cells compared to non-infiltrating cells.
  • Tumor cells did not express HLA-E in control spheroids, but its expression was strongly induced by the presence of immune cells. This suggested that tumor cells in the spheroids could evade the immune response through NKG2A-HLA-E interactions.
  • neutralization of the HLA-E/NKG2A axis could render the MICA-expressing and anti-MICA- treated tumors sensitive to recognition and/or lysis by the host immune system.
  • Neutralization of the HLA-E/NKG2A axis may furthermore provide for improved anti-tumor responses when used with treatments (e.g.
  • anti-MICA antibodies that are capable of restoring or enhancing the functioning of the activating NKG2D pathway in T and/or NK cells (by preventing or reducing the MICA-induced downmodulation of NKG2D).
  • Neutralizing anti- NKG2A-antibodies were then tested and found to be able to enhance the anti-tumor effect of anti-MICA antibodies; the combination was particularly effective and/or synergistic in eliminating cancer cells in co-cultures of immune cells (including autologous cells) with colorectal tumor spheroids produced from human cancer patients.
  • the present invention provides methods of treating and/or preventing a cancer and/or eliciting an anti-tumor immune response in an individual having a MICA- positive cancer (or a MICA and/or MICB-positive cancer), comprising treating said individual with an agent that neutralizes the inhibitory receptor NKG2A.
  • the present invention provides methods of treating and/or preventing a cancer and/or eliciting an anti- tumor immune response in an individual in need thereof, comprising treating said individual with an agent that neutralizes the inhibitory receptor NKG2A and an antibody that binds a MICA polypeptide (optionally wherein the antibody binds MICA and MICB).
  • the cancer or tumor is colorectal cancer (CRC).
  • a method for treating or preventing a cancer, optionally a CRC, in an individual comprising: (i) identifying an individual who has a MICA- positive cancer, and (ii) administering to the individual an effective amount of agent that neutralizes the inhibitory receptor NKG2A and an effective amount of an agent (e.g. an antibody or antibody fragment) that binds a MICA polypeptide.
  • an agent e.g. an antibody or antibody fragment
  • a method for treating or preventing a cancer, optionally a CRC, in an individual comprising: (i) identifying an individual who has a MICA- and/or MICB-positive cancer, and (ii) administering to the individual an effective amount of agent that neutralizes the inhibitory receptor NKG2A and an effective amount of an agent (e.g. an antibody or antibody fragment) that binds a MICA and a MICB polypeptide.
  • an agent e.g. an antibody or antibody fragment
  • a MICA-positive cancer is a cancer known to be generally characterized by presence of MICA-expressing cells in the tumor or tumor environment.
  • the MICA-positive cancer is characterized by a tumor determined (e.g. by in vitro detection of MICA in a tumor biopsy) to comprise MICA-expressing cells.
  • the MICA-positive cancer is characterized by tumor tissue comprising malignant cells that express MICA, thus comprising MICA polypeptide.
  • the MICA-positive cancer is characterized by tumor tissue or tumor-adjacent tissue characterized by infiltration of MICA-expressing immune cells (e.g. myeloid derived suppressor cells (MDSCs)).
  • the MICA-positive cancer is characterized by tumor tissue or tumor-adjacent tissue characterized by the presence of soluble MICA polypeptide (and/or by cells that shed MICA giving rise to soluble MICA).
  • a method for treating or preventing a cancer in an individual comprising: (i) identifying an individual who has a tumor comprising MICA- expressing cells, and (ii) administering to the individual an effective amount of agent that neutralizes the inhibitory receptor NKG2A and an effective amount of an agent (e.g. an antibody or antibody fragment) that binds a MICA polypeptide.
  • an agent e.g. an antibody or antibody fragment
  • a method for determining whether an individual having a cancer, optionally a CRC, will derive particular benefit from, be responsive to and/or suitable for treatment with an agent that neutralizes the inhibitory receptor NKG2A and an antibody that binds a MICA polypeptide comprising determining whether said individual has a tumor characterized by presence of MICA polypeptide (e.g.
  • a method of increasing the activity and/or numbers of tumor-infiltrating CD8+ T cells and/or NK cells in an individual who has a CRC or more generally a MICA-expressing tumor comprising administering to the individual a therapeutically active amount of an agent that neutralizes the inhibitory receptor NKG2A, and optionally further a therapeutically active amount of an agent (e.g. an antibody or antibody fragment) that binds a MICA polypeptide.
  • an agent e.g. an antibody or antibody fragment
  • an agent that neutralizes NKG2A optionally an anti- NKG2A antibody or antibody fragment, for use in the treatment of cancer (e.g., an epithelial cancer, a CRC), wherein the agent that neutralizes NKG2A is administered in combination with an agent (e.g. an antibody) that binds MICA.
  • cancer e.g., an epithelial cancer, a CRC
  • an agent e.g. an antibody
  • an agent e.g. an antibody or antibody fragment
  • MICA binds MICA
  • cancer e.g., an epithelial cancer, a CRC
  • the agent that neutralizes NKG2A is administered in combination with an agent that neutralizes NKG2A, optionally an antibody or antibody fragment.
  • agents that bind MICA
  • molecules e.g. an antibody or antibody fragment
  • bind MICA and deplete MICA- expressing tumor cells and/or that reduces MICA-mediated down-modulation of NKG2D at the surface of immune effector cells (e.g. NK cells, CD8 T cells).
  • immune effector cells e.g. NK cells, CD8 T cells.
  • molecules that bind MICA on MICA-expressing tumor cells and inhibit the amount of MICA released or shed from cells e.g. an antibody that reduces MICA shedding from tumors
  • molecules that bind MICA and modulate the interaction between MICA and NKG2D e.g.
  • an agent that depletes MICA-expressing tumor cells is antibody that binds MICA and is capable of mediating ADCC (e.g. an antibody of human lgG1 isotype).
  • an agent that reduces MICA-mediated down-modulation of NKG2D at the surface of immune effector cells is an antibody that (a) inhibits the amount of MICA released or shed from cells and/or (b) modulates the interaction between MICA and NKG2D.
  • an antibody or antibody fragment that binds MICA reduces (e.g. substantially eliminates) binding between MICA and hNKG2D (e.g., the antibody or antibody fragment reduces or blocks the interaction of surface MICA on tumor cells with surface NKG2D on effector cells), optionally wherein the antibody or antibody fragment further is a depleting antibody or antibody fragment capable of mediating ADCC (e.g. an antibody of human lgG1 isotype).
  • an antibody or antibody fragment that binds MICA is capable of binding MICA expressed at the surface of a tumor cell and modulating the interaction between MICA expressed at the surface of a tumor cell and NKG2D polypeptides expressed at the surface of an immune cell (e.g. a T cell, an NK cell), optionally wherein the antibody or antibody fragment further is a depleting antibody or antibody fragment capable of mediating ADCC (e.g. an antibody of human lgG1 isotype).
  • an antibody or antibody fragment that binds MICA is capable of inducing or increasing immune cell (e.g. CD8 T cell, NK cell) infiltration into a tumor (e.g. a MICA-expressing tumor).
  • the antibody is capable of mediating ADCC and/or is capable of binding MICA expressed at the surface of a tumor cell and modulating the interaction between MICA expressed at the surface of a tumor cell and NKG2D polypeptides expressed at the surface of an immune cell.
  • antibody or antibody fragment that binds MICA further specifically binds to a MICB polypeptide and reduces (e.g. substantially eliminates) binding between MICB and human NKG2D.
  • antibody or antibody fragment that binds MICA competes with hNKG2D in binding to MICA (and optionally MICB) and prevents human NKG2D from binding to MICA (and optionally MICB).
  • the antibody that binds MICA is capable of (a) mediating ADCC (e.g. comprises an Fc region of human origin that is bound by human CD16A), and/or (b) reduces MICA-mediated down-modulation of NKG2D at the surface of immune effector cells.
  • ADCC e.g. comprises an Fc region of human origin that is bound by human CD16A
  • antibody 19E19 induces ADCC and inhibits or blocks the interaction of a MICA (and optionally MICB) polypeptide with both human NKG2D chains of an hNKG2D homodimer.
  • the antibody blocks the interaction of both an a1 domain of a MICA polypeptide (and optionally MICB polypeptide) and/or an a2 domain of a MICA polypeptide (and optionally MICB polypeptide), with an NKG2D homodimer (e.g., the antibody inhibits the interaction of a MICA a1 a2 platform with both NKG2D chains of an NKG2D homodimer).
  • the antibody binds the a3 domain of a MICA polypeptide and prevents shedding of MICA form the tumor cell surface.
  • the antibody binds the a3 domain of a MICA polypeptide and non-competitively inhibits the interaction of MICA with NKG2D.
  • the agent that neutralizes the activity of a human NKG2A polypeptide is an antibody that reduces the inhibitory activity of NKG2A by blocking binding of its ligand, HLA-E, i.e., the anti-NKG2A antibody interferes with the binding of NKG2A by HLA-E.
  • the antibody having the heavy chain variable regions of any one of SEQ ID NOS: 2- 6 and a light chain variable region of SEQ ID NO: 7 is an example of such an antibody.
  • the anti-NKG2A antibody reduces the inhibitory activity of NKG2A without blocking binding of its ligand, HLA-E, i.e., the anti-NKG2A agent is a non-competitive antagonist and does not interfere with the binding of NKG2A by HLA-E.
  • the antibody having the heavy and light chain variable regions of SEQ ID NOS: 20 and 21 respectively is an example of such an antibody.
  • the anti-NKG2A agent is an antibody which binds with a significantly higher affinity to NKG2A than to one or more activating NKG2 receptors.
  • the agent is an antibody which binds with a significantly higher affinity to NKG2A than to NKG2C.
  • the agent is an antibody which binds with a significantly higher affinity to NKG2A than to NKG2E.
  • the agent is an antibody which binds with a significantly higher affinity to NKG2A than to NKG2H.
  • the anti-NKG2A agent competes with the antibody having the heavy chain variable region of any one of SEQ ID NOS: 2-6 and light chain variable region of SEQ ID NO: 7, and/or the antibody having the heavy and light chain variable regions of SEQ ID NOS: 20 and 21 respectively, in binding to CD94/NKG2A.
  • the agent can be, e.g., a human or humanized anti-NKG2A antibody.
  • the anti-NKG2A antibody is a humanized antibody having the heavy chain variable region of any one of SEQ ID NOS: 2-6 and light chain variable region of SEQ ID NO: 7.
  • FIG. 1 shows Anti-MICA/B antibodies induce immune-mediated anti-tumor effects through increased NK cell infiltration and activation in HT29 spheroids.
  • HT29 spheroids were cocultured or not with CD19-CD14- PBMCs in the presence or not of ADCC-inducing anti- MICA/B antibodies or corresponding control isotype.
  • Figure 2 shows NKG2A-HLA-E pathway is engaged during the cocultures and NKG2A blockade synergizes with anti-MICA/B antibodies.
  • (B) HLA-E expression by tumor cells in the spheroids cocultured or not with CD19-CD14- PBMCs, analyzed by flow cytometry (n 16 independent experiments) and immunohistochemistry (representative pictures of 1 experiment) at 24h.
  • Figure 3 shows change in spheroid volume 48h after culturing primary CRC-derived spheroids with (A) or without (B) autologous TILs, in the presence or not of anti-MICA/B, anti- NKG2A or corresponding control isotypes.
  • a or B panels spheroid volumes are normalized to the culture condition without stimulation.
  • n 5 independent experiments.
  • FIG 4 shows T cell subsets and NKG2D expression by CD8 T cells after MICA/B treatment.
  • HT29 spheroids were cocultured or not with CD19-CD14- PBMCs in the presence of ADCC-enhanced anti-MICA/B antibodies or corresponding control isotype.
  • Statistical significance was analyzed using the Wilcoxon matched-pairs signed rank test ( * p ⁇ 0.05; ** p ⁇ 0.005, *** p ⁇ 0.001 , **** p ⁇ 0.0001).
  • FIG. 5 shows T and NK cells proportions and CD137 expression by CD8 T cells after combination therapy.
  • HT29 spheroids were cocultured or not with CD19-CD14- PBMCs in the presence of either ADCC-enhanced anti-MICA/B antibodies alone or combined with anti-NKG2A blocking antibodies, or with corresponding control isotype antibodies alone or combined.
  • Statistical significance was analyzed using the Wilcoxon matched-pairs signed rank test ( * p ⁇ 0.05; ** p ⁇ 0.005, *** p ⁇ 0.001 , **** p ⁇ 0.0001 ).
  • Figure 6 shows MICA expression on cancer cells as a proportion score across a number of patient samples for several different tumor types, as assessed by IHC in FFPE samples.
  • a or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • NKG2A (OMIM 161555, the entire disclosure of which is herein incorporated by reference) is a member of the NKG2 group of transcripts (Houchins, et al. (1991 ) J. Exp. Med. 173:1017-1020). NKG2A is encoded by 7 exons spanning 25 kb, showing some differential splicing. Together with CD94, NKG2A forms the heterodimeric inhibitory receptor CD94/NKG2A, found on the surface of subsets of NK cells, a/b T cells, g/d T cells, and NKT cells. Similar to inhibitory KIR receptors, it possesses an ITIM in its cytoplasmic domain.
  • NKG2A refers to any variant, derivative, or isoform of the NKG2A gene or encoded protein.
  • Human NKG2A comprises 233 amino acids in 3 domains, with a cytoplasmic domain comprising residues 1-70, a transmembrane region comprising residues 71-93, and an extracellular region comprising residues 94-233, of the following sequence: MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQDFQGNDKTYHC KDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGHCP EEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSS HHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO:
  • NKG2C (OMIM 602891 , the entire disclosure of which is herein incorporated by reference) and NKG2E (OMIM 602892, the entire disclosure of which is herein incorporated by reference) are two other members of the NKG2 group of transcripts (Gilenke, et al. (1998) Immunogenetics 48:163-173).
  • the CD94/NKG2C and CD94/NKG2E receptors are activating receptors found on the surface of subsets of lymphocytes such as NK cells and T-cells.
  • HLA-E (OMIM 143010, the entire disclosure of which is herein incorporated by reference) is a nonclassical MHC molecule that is expressed on the cell surface and regulated by the binding of peptides, e.g. such as fragments derived from the signal sequence of other MHC class I molecules. Soluble versions of HLA-E have also been identified. In addition to its T-cell receptor binding properties, HLA-E binds subsets of natural killer (NK) cells, natural killer T-cells (NKT) and T cells (a/b and g/d), by binding specifically to CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C (see, e.g., Braud et al.
  • NK natural killer
  • NKT natural killer T-cells
  • T cells a/b and g/d
  • HLA-E refers to any variant, derivative, or isoform of the HLA-E gene or encoded protein.
  • “NKG2A-”, or “CD94/NKG2A-”, “positive lymphocyte”, or“restricted lymphocyte”, refers to cells of the lymphoid lineage (e.g. NK-, NKT- and T-cells) expressing CD94/NKG2A on the cell-surface, which can be detected by e.g. flow-cytometry using antibodies that specifically recognize a combined epitope on CD94 and NKG2A or and epitope on NKG2A alone.“NKG2A positive lymphocyte” also includes immortal cell lines of lymphoid origin (e.g. NKL, NK-92).
  • “reduces the inhibitory activity of NKG2A”, “neutralizes NKG2A” or“neutralizes the inhibitory activity of NKG2A” refers to a process in which CD94/NKG2A is inhibited in its capacity to negatively affect intracellular processes leading to lymphocyte responses such as cytokine release and cytotoxic responses. This can be measured for example in a NK- or T-cell based cytotoxicity assay, in which the capacity of a therapeutic compound to stimulate killing of HLA-E positive cells by CD94/NKG2A positive lymphocytes is measured.
  • an NKG2A-neutralizing antibody preparation causes at least a 10% augmentation in the cytotoxicity of a CD94/NKG2A-restricted lymphocyte, optionally at least a 40% or 50% augmentation in said lymphocyte cytotoxicity, optionally at least a 70% augmentation in said lymphocyte cytotoxicity”, optionally at least a 70% augmentation of NK cytotoxicity, and referring to the cytotoxicity assays described herewith. If an anti-NKG2A antibody reduces or blocks CD94/NKG2A interactions with HLA- E, it may increase the cytotoxicity of CD94/NKG2A-restricted lymphocytes.
  • NK cells that express CD94/NKG2A
  • target cells that express HLA-E.
  • NK cells do not efficiently kill targets that express HLA-E because CD94/NKG2A recognizes HLA-E, leading to initiation and propagation of inhibitory signaling that prevents lymphocyte-mediated cytolysis.
  • Such an in vitro cytotoxicity assay can be carried out by standard methods that are well known in the art, as described for example in Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993).
  • Chromium release and/or other parameters to assess the ability of the antibody to stimulate lymphocytes to kill target cells are also disclosed in Sivori et al., J. Exp. Med. 1997;186:1 129-1 136; Vitale et al., J. Exp. Med. 1998; 187:2065-2072; Pessino et al. J. Exp. Med. 1998;188:953-960; Neri et al. Clin. Diag. Lab. Immun. 2001 ;8:1131-1135; Pende et al. J. Exp. Med. 1999;190:1505-1516, the entire disclosures of each of which are herein incorporated by reference.
  • the target cells are labeled with 51 Cr prior to addition of NK cells, and then the killing is estimated as proportional to the release of 51 Cr from the cells to the medium, as a result of killing.
  • the addition of an antibody that prevents CD94/NKG2A from binding to HLA-E results in prevention of the initiation and propagation of inhibitory signaling via CD94/NKG2A. Therefore, addition of such agents results in increases in lymphocyte-mediated killing of the target cells. This step thereby identifies agents that prevent CD94/NKG2A-induced negative signaling by, e.g., blocking ligand binding.
  • CD94/NKG2A- expressing NK effector-cells can kill HLA-E-negative LCL 721.221 target cells, but less well HLA-E-expressing LCL 721.221-Cw3 control cells.
  • YTS effector-cells that lack CD94/NKG2A kill both cell-lines efficiently.
  • NK effector cells kill less efficiently HLA-E + LCL 721.221-Cw3 cells due to HLA-E-induced inhibitory signaling via CD94/NKG2A.
  • NK cells When NK cells are pre-incubated with blocking anti-CD94/NKG2A antibodies described herewith in such a 51 Cr-release cytotoxicity assay, HLA-E-expressing LCL 721.221-Cw3 cells are more efficiently killed, in an antibody-concentration-dependent fashion.
  • the inhibitory activity (i.e. cytotoxicity enhancing potential) of an anti-NKG2A antibody can also be assessed in any of a number of other ways, e.g., by its effect on intracellular free calcium as described, e.g., in Sivori et al., J. Exp. Med. 1997;186:1129-1 136, the disclosure of which is herein incorporated by reference.
  • Activation of NK cell cytotoxicity can be assessed for example by measuring an increase in cytokine production (e.g. IFN-g production) or cytotoxicity markers (e.g. CD107 or CD137 mobilization).
  • IFN-y production from PBMC is assessed by cell surface and intracytoplasmic staining and analysis by flow cytometry after 4 days in culture. Briefly, Brefeldin A (Sigma Aldrich) is added at a final concentration of 5 pg/ml for the last 4 hours of culture.
  • GM-CSF and IFN-y production from polyclonal activated NK cells are measured in supernatants using ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, MN, IFN-y: OptEIA set, Pharmingen).
  • MICA (PERB11.1 ) refers to MHC class I polypeptide-related sequence A (see, e.g., UniProtKB/Swiss-Prot Q29983), its gene and cDNA and its gene product, or naturally occurring variants thereof. Nomenclature of MICA genes and proteins, together with reference to accession number of sequence for different alleles are described in Frigoul A. and Lefranc, M-P. Recent Res. Devel. Human Genet., 3(2005): 95-145 ISBN: 81-7736-244- 5, the disclosure of which is incorporated herein by reference.
  • MICA genes and protein sequence, including polymorphisms at the protein and DNA level, are also available from http://www.ebi.ac.uk/ipd/imgt/hla/align.html maintained by Cancer Research UK and the European Bioinformatics Institute (EBI).
  • MICA amino acid sequences of MICA were first described in Bahram et al (1994) Proc. Nat. Acad. Sci. 91 : 6259-6263 and Bahram et al. (1996) Immunogenetics 44:80-81 , the disclosures of which are incorporated herein by reference.
  • the MICA gene is polymorphic, displaying an unusual distribution of a number of variant amino acids in their extracellular cd , a2, and a3 domains.
  • Petersdorf et al. (1999) examined its alleles among 275 individuals with common and rare HLA genotypes.
  • the full MICA sequence further comprises a leader sequence of 23 amino acids, as well as a transmembrane domain and a cytoplasmic domain.
  • the amino acid sequence of MICA * 001 is shown in SEQ ID NO : 22, corresponding to Genbank accession no. AAB41060.
  • the amino acid sequence of human MICA allele MICA * 004 is shown in SEQ ID NO 23, corresponding to Genbank accession no. AAB41063.
  • the amino acid sequence of human MICA allele MICA * 007 is shown in SEQ ID NO : 24, corresponding to Genbank accession no. AAB41066.
  • the amino acid sequence of human MICA allele MICA * 008 is shown in SEQ ID NO : 25, corresponding to Genbank accession no. AAB41067.
  • MICB also known as PERB1 1.2 refers to MHC class I polypeptide-related sequence B (See, e.g., UniProtKB/Swiss-Prot Q29980).
  • the amino acid sequence of an exemplary human MICB polypeptide is shown Genbank accession no. CAI18747 (SEQ ID NO: 27).
  • the MICA gene encodes a protein that belongs to the MHC SF (superfamily) and to the IgSF.
  • This protein is a transmembrane MHC-l-alpha-like (l-alpha-like) chain, which comprises three extracellular domains, two distal G-like domains, G-alpha1-like (also referred to as“D1” or“a1”) and G-alpha2-like (also referred to as“D2” or“a2”), and a C-like- domain (also referred to as“D3” or“a3”) proximal to the cell membrane, and three regions, a connecting-region, a transmembrane-region and a cytoplasmic-region (labels according to the IMGT Scientific Chart of the IMGT (international ImMunoGeneTics information system®), http://imgt.org and LeFranc et al.
  • the MICA mature protein including leader, ECD, TM and CY domains is made up of 360 to 366 amino acids, the difference arising from a microsatellite polymorphism in the transmembrane region.
  • the a1 , a2 and a3 can be defined according to any suitable numbering system (e.g., the IMGT numbering system).
  • the amino acid sequences of the a1 , a2 and a3 domains are shown in SEQ ID NO: 28, 29 and 30, respectively.
  • the a1 domain comprises residue positions 1 to 88 of the MICA polypeptide of SEQ ID NO : 22; the a2 domain comprises residue positions 89 to 181 of the MICA polypeptide of SEQ ID NO :22; and the a3 domain comprises residue positions 182 to 274 of the MICA polypeptide of SEQ ID NO : 22.
  • the a1 and a2 domains each comprise A, B, C and D strands, AB, BC and CD turns, and a helix.
  • the a3 domain comprises A, B, C, D, E, F and G strands, a BC loop, a CD strand, a DE-turn and an FG loop.
  • the MICA protein is highly glycosylated with eight potential glycosylation sites, two in a1 , one in a2 and five in the a3 domain, including O-glycans (N- acetyllactosamine linked to serine or threonine) and/or N-glycans. While MICA is expressed constitutively in certain cells, low levels of MICA expression do not usually give rise to host immune cell attack. However, MICA is upregulated on rapidly proliferating cells such as tumor cells.
  • MICA is the most highly expressed of all NKG2D ligands, and it has been found across a wide range of tumor types (e.g., carcinomas in general, bladder cancer, melanoma, lung cancer, hepatocellular cancer, glioblastoma, prostate cancer, hematological malignancies in general, acute myeloid leukemia, acute lymphatic leukemia, chronic myeloid leukemia and chronic lymphatic leukemia. Recently, Tsuboi et al.
  • O-glycan branching enzyme core2 3-1 ,6-N-acetylglucosaminyltransferase (C2GnT) is active in MICA-expressing tumor cells and that MICA from tumor cells contains core2 O-glycan (an O-glycan comprising an N-acetylglucosamine branch connected to N- acetylgalactosamine).
  • hNKG2D refers to a human killer cell activating receptor gene, its cDNA (e.g., GenBank Accession No. NM_007360), and its gene product (GenBank Accession No. NP_031386), or naturally occurring variants thereof.
  • hNKG2D can form heterodimers or higher order complexes with proteins such as DAP10 (GenBank Accession No.
  • hNKG2D Any activity attributed herein to hNKG2D, e.g., cell activation, antibody recognition, etc., can also be attributed to hNKG2D in the form of a heterodimer such as hNKG2D-DAP10, or higher order complexes with these two (and/or other) components.
  • shedding when referring to MICA, refers to release of a soluble extracellular domain (ECD) fragment of MICA from the cell surface of a cell which expresses MICA.
  • ECD extracellular domain
  • Such shedding may be caused by proteolytic cleavage (e.g. through the action of matrix metalloproteinases (MMPs), e.g. ADAM10 and/or ADAM17) of cell surface MICA resulting in release of an ECD fragment from the cell surface, or the soluble ECD or fragment thereof may be encoded by an alternate transcript.
  • MMPs matrix metalloproteinases
  • MICA-expressing cells means a process, method, or compound that can kill, eliminate, lyse or induce such killing, elimination or lysis, so as to negatively affect the number of MICA-expressing cells present in a sample or in a subject.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • therapeutic agent refers to an agent that has biological activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NK natural killer
  • treatment and “treating” and the like generally mean obtaining a desired pharmacological and physiological effect.
  • the effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it such as a preventive early asymptomatic intervention; (b) inhibiting the disease, e.g., arresting its development; or relieving the disease, e.g., causing regression of the disease and/or its symptoms or conditions such as improvement or remediation of damage, for example in a subject who has been diagnosed as having the disease.
  • treatment may cause (e.g.
  • treatment may cause or provide (e.g. may be characterized as a method of causing or providing) stable disease, a partial response or a complete response in a subject, e.g. according to standard criteria, optionally RECIST criteria.
  • biopsy as used herein is defined as removal of a tissue for the purpose of examination, such as to establish diagnosis.
  • types of biopsies include by application of suction, such as through a needle attached to a syringe; by instrumental removal of a fragment of tissue; by removal with appropriate instruments through an endoscope; by surgical excision, such as of the whole lesion; and the like.
  • the term "antigen binding domain” refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope.
  • said domain can comprise a hypervariable region, optionally a VH and/or VL domain of an antibody chain, optionally at least a VH domain.
  • the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain.
  • the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.
  • antibody refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as lgG1 , lgG2, lgG3, lgG4, and the like.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa).
  • each chain defines a variable region of about 100 to 1 10 or more amino acids that is primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed “alpha,”“delta,”“epsilon,”“gamma” and “mu,” respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • IgG are the exemplary classes of antibodies employed herein because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • the antibody is a monoclonal antibody.
  • Particular examples of antibodies are humanized, chimeric, human, or otherwise-human-suitable antibodies.“Antibodies” also includes any fragment or derivative of any of the herein described antibodies.
  • hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity-determining region” or "CDR" (e.g., residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1 ), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al.
  • CDR complementarity-determining region
  • residues from a "hypervariable loop" e.g., residues 26-32 (L1 ), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917), or a similar system for determining essential amino acids responsible for antigen binding.
  • a "hypervariable loop” e.g., residues 26-32 (L1 ), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917
  • typing of amino acid residues in this region is performed by the method de- scribed in Kabat et al., supra.
  • phrases such as“Kabat position”, "variable domain residue numbering as in Kabat” and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains.
  • the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at re- gions of homology of the sequence of the antibody with a "standard" Kabat numbered se- quence.
  • frame or "FR” residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs.
  • Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1 , FR2, FR3 and FR4).
  • binding partner e.g. NKG2A for an anti-NKG2A agent or antibody, MICA (and optionally further MICB) for an anti-MICA antibody, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells.
  • Competitive binding assays and other methods for determining specific binding are well known in the art. For example binding can be detected via radiolabels, physical methods such as mass spectrometry, or direct or indirect fluorescent labels detected using, e.g., cytofluorometric analysis (e.g. FACScan). Binding above the amount seen with a control, non-specific agent indicates that the agent binds to the target.
  • An agent that specifically binds NKG2A may bind NKG2A alone or NKG2A as a dimer with CD94.
  • an antibody When an antibody is said to“compete with” a particular monoclonal antibody, it means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant molecules (e.g., NKG2A) or surface expressed molecules (e.g., NKG2A).
  • recombinant molecules e.g., NKG2A
  • surface expressed molecules e.g., NKG2A
  • a test antibody reduces the binding of an antibody having a heavy chain variable region of any of SEQ ID NOS: 2-6 and a light chain variable region of SEQ ID NO: 7 to a NKG2A polypeptide or NKG2A-expressing cell in a binding assay, the antibody is said to “compete” respectively with such antibody.
  • affinity means the strength of the binding of an antibody to an epitope.
  • the affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • Kd dissociation constant
  • a“determinant” designates a site of interaction or binding on a polypeptide.
  • the term“epitope” refers to an antigenic determinant, and is the area or region on an antigen to which an antibody binds.
  • a protein epitope may comprise amino acid residues directly involved in the binding as well as amino acid residues which are effectively blocked by the specific antigen binding antibody or peptide, i.e., amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural area on a complex antigen molecule that can combine with e.g., an antibody or a receptor. Epitopes can be linear or conformational/structural.
  • linear epitope is defined as an epitope composed of amino acid residues that are contiguous on the linear sequence of amino acids (primary structure).
  • formational or structural epitope is defined as an epitope composed of amino acid residues that are not all contiguous and thus represent separated parts of the linear sequence of amino acids that are brought into proximity to one another by folding of the molecule (secondary, tertiary and/or quaternary structures).
  • a conformational epitope is dependent on the 3-dimensional structure.
  • the term‘conformational’ is therefore often used interchangeably with‘structural’.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • therapeutic agent refers to an agent that has biological activity.
  • a“humanized” or“human” antibody refers to an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin.
  • Such antibodies are designed to maintain the binding specificity of a non-human antibody from which the binding regions are derived, but to avoid an immune reaction against a non-human antibody.
  • Such antibodies can be obtained from transgenic mice or other animals that have been “engineered” to produce specific human antibodies in response to antigenic challenge (see, e.g., Green et at. (1994) Nature Genet 7:13; Lonberg et at. (1994) Nature 368:856; Taylor et al.
  • a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated in their entirety by reference).
  • Fc domain refers to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human g (gamma) heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, d, e and m for human antibodies), or a naturally occurring allotype thereof.
  • aa amino acid
  • gamma human g
  • a d, e and m for human antibodies
  • the commonly accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991 ) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).
  • isolated refers to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.
  • polypeptide “peptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • antibody that“binds” a polypeptide or epitope designates an antibody that binds said determinant with specificity and/or affinity.
  • identity refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.
  • NCBI National Center for Biotechnology Information
  • the NKG2A neutralizing agent binds an extra-cellular portion of human CD94/NKG2A receptor or its ligand HLA-E and reduces the inhibitory activity of human CD94/NKG2A receptor expressed on the surface of a CD94/NKG2A positive lymphocyte.
  • the agent competes with HLA-E in binding to CD94/NKG2A, i.e. the agent blocks the interaction between CD94/NKG2A and its ligand HLA-E.
  • the agent binds NKG2A but does not compete with HLA-E in binding to CD94/NKG2A; i.e. the agent is capable of binding CD94/NKG2A simultaneously with HLA-E.
  • the agent e.g. an antibody or antibody fragment
  • the agent comprises an antigen binding domain that binds to NKG2A.
  • the antibody may bind a combined epitope on CD94 and NKG2A or and epitope on NKG2A alone.
  • the agent e.g. an antibody or antibody fragment
  • the NKG2A neutralizing agent comprises an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody.
  • the agent comprises a constant domain derived from a human lgG1 , lgG2, lgG3 or lgG4 antibody.
  • the agent is a fragment of an antibody selected from IgA, an IgD, an IgG, an IgE and an IgM antibody.
  • the agent is an antibody fragment selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camel Ig), a V H H fragment, a single domain FV, and a single-chain antibody fragment.
  • the agent is a synthetic or semisynthetic antibody-derived molecule selected from a scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR, and a multispecific antibody.
  • the anti-NKG2A antibodies do not demonstrate substantial specific binding (e.g., via their Fc domains) to human Fey receptors, e.g. CD16.
  • the anti-NKG2A antibodies lack substantial specific binding or have low or decreased specific binding to one or more, or all of, human CD16, CD32A, CD32B or CD64.
  • Exemplary antibodies may comprise constant regions of various heavy chains that are known not to bind or to have low binding to Fey receptors.
  • One such example is a human lgG4 constant region.
  • the lgG4 antibody comprises a modification to prevent the formation of half antibodies (fab arm exchange) in vivo, e.g., the antibody comprises an lgG4 heavy chain comprising a serine to proline mutation in residue 241 , corresponding to position 228 according to the EU-index (Kabat et al.,“Sequences of proteins of immunological interest”, 5 th ed., NIH, Bethesda, ML, 1991 ).
  • modified lgG4 antibodies will remain intact in vivo and maintain a bivalent (high affinity) binding to NKG2A, as opposed to native lgG4 that will undergo fab arm exchange in vivo such that they bind to NKG2A in monovalent manner which can alter binding affinity.
  • antibody fragments that do not comprise constant regions such as Fab or F(ab’)2 fragments, can be used to avoid Fc receptor binding.
  • Fc receptor binding can be assessed according to methods known in the art, including for example testing binding of an antibody to Fc receptor protein in a BIACORE assay.
  • any human antibody type e.g.
  • lgG1 , lgG2, lgG3 or lgG4 can be used in which the Fc portion is modified to minimize or eliminate binding to Fc receptors (see, e.g., W003101485, the disclosure of which is herein incorporated by reference).
  • Assays such as, e.g., cell based assays, to assess Fc receptor binding are well known in the art, and are described in, e.g., WO03101485.
  • the present invention thus concerns antibodies or other agents binding to NKG2A.
  • the antibody binds to NKG2A with a KD at least 100-fold lower than to human NKG2C. In one aspect, the antibody binds to NKG2A with a KD at least 100-fold lower than to human NKG2E.
  • the agent reduces CD94/NKG2A-mediated inhibition of a CD94/NKG2A-expressing lymphocyte by interfering with CD94/NKG2A signalling by, e.g., interfering with the binding of HLA-E by NKG2A, preventing or inducing conformational changes in the CD94/NKG2A receptor, and/or affecting dimerization and/or clustering of the CD94/NKG2A receptor.
  • the agent binds to an extracellular portion of NKG2A with a KD at least 100 fold lower than to NKG2C. In a further preferred aspect, the agent binds to an extracellular portion of NKG2A with a KD at least 150, 200, 300, 400, or 10,000 fold lower than to NKG2C. In another aspect of the invention, the agent binds to an extracellular portion of NKG2A with a KD at least 100 fold lower than to NKG2C, NKG2E and/or NKG2H molecules.
  • the agent binds to an extracellular portion of NKG2A with a KD at least 150, 200, 300, 400, or 10,000 fold lower than to NKG2C, NKG2C and/or NKG2H molecules.
  • a KD at least 150, 200, 300, 400, or 10,000 fold lower than to NKG2C, NKG2C and/or NKG2H molecules.
  • This can be measured, for instance, in BiaCore experiments, in which the capacity of agents to bind the extracellular portion of immobilized CD94/NKG2A (e.g. purified from CD94/NKG2 expressing cells, or produced in a bio-system) is measured and compared to the binding of agents to similarly produced CD94/NKG2C and/or other CD94/NKG2 variants in the same assay.
  • the binding of agents to cells that either naturally express, or over-express e.g.
  • CD94/NKG2A can be measured and compared to binding of cells expressing CD94/NKG2C and/or other CD94/NKG2 variants.
  • Anti-NKG2A antibodies may optionally bind NKG2B, which is an NKG2A splice variant forming an inhibitory receptor together with CD94.
  • affinity can be measured using the methods disclosed in U.S. Patent No 8,206,709, for example by assessing binding to covalently immobilized NKG2A-CD94-Fc fusion protein by Biacore as shown in Example 8 of U.S. Patent No 8,206,709, the disclosure of which is incorporated herein by reference.
  • the anti-NKG2A antibody can be a humanized antibody, for example comprising a VH human acceptor framework from a human acceptor sequence selected from, e.g., VH1_18, VH5_a, VH5_51 , VH1_f, and VH1_46, and a JH6 J-segment, or other human germline VH framework sequences known in the art.
  • the VL region human acceptor sequence may be, e.g., VKI_02/JK4.
  • the antibody is a humanized antibody based on antibody Z270.
  • Different humanized Z270 heavy chain variable regions are shown in SEQ ID NOS: 2-6, with optionally further comprising a C-terminal serine (S) residue.
  • the HumZ270VH6 variable region of SEQ ID NO: 2 is based on a human VH5_51 gene; the HumZ270VH1 variable region of SEQ ID NO: 3 is based on a human VH1_18 gene; the humZ270VH5 variable region of SEQ ID NO: 4 is based on a human VH5_a gene; the humZ270VH7 variable region of SEQ ID NO: 5 is based on a human VH1_f gene; and the humZ270VH8 variable region of SEQ ID NO: 6 is based on a human VH1_46 gene; all with a human JH6 J-segment.
  • each of these antibodies retains high affinity binding to NKG2A, with low likelihood of a host immune response against the antibody as the 6 C-terminal amino acid residues of the Kabat H-CDR2 of each of the humanized constructs are identical to the human acceptor framework.
  • the following sequence identities between humZ270VH1 and humZ270VH5, -6, -7, and -8 were obtained: 78,2% (VH1 vs. VH5), 79,0% (VH1 vs. VH6), 88,7% (VH1 vs. VH7), and 96,0% (VH1 vs. VH8).
  • the agent comprises (i) a heavy chain variable region of SEQ ID NOS: 2-6, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical thereto, and (ii) the light chain variable region of SEQ ID NO: 7, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical thereto.
  • the agent comprises (i) a heavy chain comprising the amino acid sequence of any of SEQ ID NOS: 8-12, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical thereto, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical thereto.
  • the antibody having a heavy chain variable region of any of SEQ ID NOS: 2-6 and a light chain variable region of SEQ ID NO: 7 neutralizes the inhibitory activity of NKG2A, but does not substantially bind the activating receptors NKG2C, NKG2E or NKG2H. This antibody furthermore competes with HLA-E for binding to NKG2A on the surface of a cell.
  • the agent comprises H-CDR1 , H-CDR2 and/or H-CDR3 sequences derived from the heavy chain variable region having the amino acid sequence of any of SEQ ID NOS: 2-6.
  • the agent comprises L-CDR1 , L-CDR2 and/or L-CDR3 sequences derived from the light chain variable region having the amino acid sequence of SEQ ID NO: 7.
  • Heavy chain CDRs according to Kabat numbering scheme:
  • H-CDR1 SYWMN (SEQ ID NO: 14)
  • H-CDR2 RIDPYDSETHYAQKLQG (SEQ ID NO: 15)
  • H-CDR3 GGYDFDVGTLYWFFDV (SEQ ID NO: 16)
  • L-CDR1 RASENIYSYLA (SEQ ID NO: 17)
  • L-CDR2 NAKTLAE (SEQ ID NO: 18)
  • the anti-NKG2A antibody is an antibody comprising a H-CDR1 corresponding to residues 31-35 of SEQ ID NOS: 2-6 (or of SEQ ID NOS: 8-12), a H-CDR2 corresponding to residues 50-60 (optionally 50-66 when including amino acids of human origin) of SEQ ID NOS: 2-6 (or of SEQ ID NOS: 8-12), and a H-CDR3 corresponding to residues 99-114 (95-102 according to Kabat) of SEQ ID NOS: 2-6 (or of SEQ ID NOS: 8-12).
  • a CDR may comprise one, two, three, four, or more amino acid substitutions.
  • the anti-NKG2A antibody is an antibody comprising a L-CDR1 corresponding to residues 24-34 of SEQ ID NOS: 7 or 13, a L-CDR2 corresponding to residues 50-56 of SEQ ID NOS: 7 or 13, and an L-CDR3 corresponding to residues 89-97 of SEQ ID NOS: 7 or 13.
  • a CDR may comprise one, two, three, four, or more amino acid substitutions.
  • the anti-NKG2A antibody is an antibody comprising a H-CDR1 corresponding to residues 31-35 of SEQ ID NOS: 2-6, a H-CDR2 corresponding to residues 50-60 (optionally 50-66) of SEQ ID NOS: 2-6, and a H-CDR3 corresponding to residues 99- 1 14 (95-102 according to Kabat) of SEQ ID NOS: 2-6, a L-CDR1 corresponding to residues 24-34 of SEQ ID NO: 7, a L-CDR2 corresponding to residues 50-56 of SEQ ID NO: 7, and an L-CDR3 corresponding to residues 89-97 of SEQ ID NO: 7.
  • the anti-NKG2A antibody is an antibody comprising the heavy chain H- CDR1 , H-CDR2 and H-CDR3 domains having the amino acid sequences of SEQ ID NOS: 14-16, and the light chain L-CDR1 , L-CDR2 and L-CDR3 domains having the amino acid sequences of SEQ ID NOS: 17-19, respectively.
  • the agent is monalizumab, an anti-NKG2A antibody having the heavy chain variable region amino acid sequence of SEQ ID NO: 3 and the light chain variable region amino acid sequence of SEQ ID NO: 7.
  • the agent is monalizumab, an anti-NKG2A antibody having the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 13.
  • the agent comprises H-CDR1 , H-CDR2 and/or H-CDR3 sequences derived from the VH having the amino acid sequence of SEQ ID NO: 20, e.g. according to Kabat numbering (see CDRs underlined in SEQ ID NO: 20, below).
  • the agent comprises L-CDR1 , L-CDR2 and/or L-CDR3 sequences derived from the VL having the amino acid sequence of SEQ ID NO: 21 , e.g. according to Kabat numbering (see CDRs underlined in SEQ ID NO: 21 , below).
  • the agent comprises H-CDR1 , H-CDR2 and/or H-CDR3 sequences derived from the VH having the amino acid sequence of SEQ ID NO: 20, and L-CDR1 , L-CDR2 and/or L-CDR3 sequences derived from the VL having the amino acid sequence of SEQ ID NO: 21.
  • the antibody having the heavy chain variable region of SEQ ID NO: 20 and a light chain variable region of SEQ ID NO: 21 neutralizes the inhibitory activity of NKG2A, and also binds the activating receptors NKG2C, NKG2E and NKG2H. This antibody does not compete with HLA-E for binding to NKG2A on the surface of a cell (i.e. it is a non-competitive antagonist of NKG2A).
  • the agent comprises amino acid residues 31-35, 50-60, 62, 64, 66, and 99-108 of the variable-heavy (V H ) domain (SEQ ID NO: 20 and amino acid residues 24-33, 49-55, and 88-96 of the variable-light (V L ) domain (SEQ ID NO: 21 ), optionally with one, two, three, four, or more amino acid substitutions.
  • the agent is a humanized antibody, for example an agent comprising heavy and light chain variable regions as disclosed in PCT publication no. W02009/092805, the disclosure of which is incorporated herein by reference.
  • the agent is a fully human antibody which has been raised against the CD94/NKG2A epitope to which any of the aforementioned antibodies bind.
  • any fragment of NKG2A preferably but not exclusively human NKG2A, or any combination of NKG2A fragments, can be used as immunogens to raise antibodies, and the antibodies can recognize epitopes at any location within the NKG2A polypeptide, so long as they can do so on NKG2A expressing NK cells as described herein.
  • the epitope is the epitope specifically recognized by an antibody having a heavy chain variable region of SEQ ID NOS: 2-6 and a light chain variable region of SEQ ID NO: 7.
  • the agent is an antibody that is a function-conservative variant of humZ270 or of an antibody having a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 13.
  • “Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein simi- larity is based on the MEGALIGN algorithm.
  • A“function-conservative variant” also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.
  • the agent competes with humZ270 antibody disclosed in U.S. Patent No 8,206,709 (the disclosure of which is incorporated herein by reference) in binding to the extra-cellular portion of human CD94/NKG2A receptor.
  • Competitive binding can be measured, for instance, in BiaCore experiments, in which the capacity of agents is measured, for binding the extracellular portion of immobilized CD94/NKG2A receptor (e.g. purified from CD94/NKG2 expressing cells, or produced in a bio-system) saturated with humZ270.
  • the binding of agents to cells is measured that either naturally express, or over-express (e.g.
  • CD94/NKG2A receptor which have been pre-incubated with saturating doses of Z270.
  • competitive binding can be measured using the methods disclosed in U.S. Patent No 8,206,709, for example by assessing binding to Ba/F3-CD94-NKG2A cells by flow cytometry as shown in Example 15 of U.S. Patent No 8,206,709, the disclosure of which is incorporate herein by reference.
  • the agent e.g. an antibody or antibody fragment
  • the agent comprises an antigen binding domain that binds to a human MICA polypeptide (and optionally further a human MICB polypeptide).
  • the agent comprises an antibody or antibody fragment that is capable of specifically binding a human MICA polypeptide (and optionally further a human MICB polypeptide) and depleting MICA-expressing tumor cells (and optionally further MICB-expressing tumor cells).
  • anti-MICA antibodies include an antibody or antibody fragment capable of binding MICA on MICA-expressing tumor cells and inhibiting the amount of MICA released from cells, as well as an antibody or antibody fragment capable of binding MICA and modulating the interaction between MICA and NKG2D polypeptides.
  • an antibody or antibody fragment is capable of binding MICA expressed at the surface of a tumor cell and modulating the interaction between MICA expressed at the surface of a tumor cell and NKG2D polypeptides expressed at the surface of an immune cell (e.g. a T cell, an NK cell).
  • an antibody or antibody fragment is capable of inducing or increasing immune cell (e.g. T cell) infiltration into a tumor (e.g. a MICA-expressing tumor).
  • the antibody that binds and depletes MICA is capable of mediating ADCC (e.g. an antibody of human lgG1 isotype).
  • an antibody that binds MICA can optionally be specified as lacking ability to mediate ADCC (e.g. an antibody lacking an Fc domain or having an Fc domain of human IgG isotype modified to abolish ADCC and/or binding to human CD16 polypeptides).
  • anti-MICA antibody binds the MICA * 001 , * 004, * 007 and * 008 alleles of the human MICA polypeptide (e.g. a membrane bound MICA polypeptide as expressed at the surface of a cell).
  • antibody further binds the human MICB polypeptide.
  • Such antibodies will be capable of binding to MICA proteins in substantially the entire human population and they can therefore be used in detection and/or treatment steps without a need for prior determination of the MICA allele(s) expressed by an individual.
  • an antibody or antibody fragment that binds MICA reduces (e.g. substantially eliminates) binding between MICA and human NKG2D polypeptides (e.g., the antibody or antibody fragment reduces or blocks the interaction of surface MICA on tumor cells with surface NKG2D on effector cells), optionally wherein the antibody or antibody fragment further is a depleting antibody or antibody fragment capable of mediating ADCC (e.g. an antibody of human lgG1 isotype).
  • antibody or antibody fragment that binds MICA further specifically binds to a MICB polypeptide and reduces (e.g. substantially eliminates) binding between MICB and human NKG2D.
  • antibody or antibody fragment that binds MICA competes with human NKG2D in binding to MICA (and optionally MICB) and prevents NKG2D from binding to MICA (and optionally MICB).
  • the antibody that binds MICA inhibits or blocks the interaction of a MICA (and optionally MICB) polypeptide with both NKG2D chains of an NKG2D homodimer.
  • the antibody blocks the interaction of both an a1 domain of a MICA polypeptide (and optionally MICB polypeptide) and/or an a2 domain of a MICA polypeptide (and optionally MICB polypeptide), with an NKG2D homodimer (e.g., the antibody inhibits the interaction of a MICA a1 a2 platform with both NKG2D chains of an NKG2D homodimer).
  • an antigen binding protein or antibody competes for binding to the MICA polypeptide with any one or more of antibodies 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1 (described, e.g. in Tables 1 and 2).
  • an antigen binding protein or antibody recognizes, binds to, competes for binding to, or has immunospecificity for substantially or essentially the same, or the same, epitope or“epitopic site” on a MICA polypeptide as antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1.
  • the antigen binding protein or antibody binds substantially the same epitope on MICA as antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1 .
  • the epitope recognized, or bound to, by the antigen binding protein or antibodies at least partially overlaps, or includes at least one residue in the segment corresponding to residues 1-88, residues 89-181 , or residues 182-274 of a MICA polypeptide comprising an amino acid sequence of any of SEQ ID NOS: 22-26.
  • all key residues of the epitope are in a segment corresponding to residues 1 -88, residues 89-181 , or residues 182-274 of a MICA polypeptide comprising an amino acid sequence of any of SEQ ID NOS: 22-26.
  • an antibody binds an epitope spanning the junction of (a) the a1 and/or a2 domain and (b) the a3 domain, wherein all key residues of the epitope is in a segment corresponding to residues 1 -181 (e.g., residues 1 -88 (optionally 1 -85) or 89-181 (optionally 86-181 )) of a MICA polypeptide comprising an amino acid sequence of any of SEQ ID NOS: 22-26.
  • residues 1 -181 e.g., residues 1 -88 (optionally 1 -85) or 89-181 (optionally 86-181 )
  • the antigen binding protein or antibodies bind an epitope comprising 1 , 2, 3, 4, 5, 6, 7 or more residues in the segment corresponding to residues 1-88 (optionally 1 -85) or residues 89-181 (optionally 86-181 ) of a MICA polypeptide comprising an amino acid sequence of any of SEQ ID NOS: 22-26.
  • residues bound by the antibody are present on the surface of the MICA polypeptide, e.g. in a MICA polypeptide expressed on the surface of a cell.
  • the epitope of an antigen binding protein or antibody of the invention may be entirely within the a1 and/or a2 domains of MICA.
  • the antibodies can be characterized as not substantially binding to the a3 domain region required for MICA shedding.
  • the epitope of an antigen binding protein or antibody of the invention may be within the a3 domain of MICA.
  • Binding of anti-MICA agent to cells transfected with MICA mutants can be measured and compared to the ability of anti-MICA agent to bind wild-type MICA polypeptide (e.g., any one or more of any of SEQ ID NOS: 22-26).
  • a reduction in binding between an anti-MICA agent and a mutant MICA polypeptide means that there is a reduction in binding affinity (e.g., as measured by known methods such FACS testing of cells expressing a particular mutant, or by Biacore testing of binding to mutant polypeptides) and/or a reduction in the total binding capacity of the anti-MICA agent (e.g., as evidenced by a decrease in Bmax in a plot of anti- MICA agent concentration versus polypeptide concentration).
  • a significant reduction in binding indicates that the mutated residue is directly involved in binding to the anti-MICA agent or is in close proximity to the binding protein when the anti-MICA agent is bound to MICA.
  • a significant reduction in binding means that the binding affinity and/or capacity between an anti-MICA antibody and a mutant MICA polypeptide is reduced by greater than 40 %, greater than 50 %, greater than 55 %, greater than 60 %, greater than 65 %, greater than 70 %, greater than 75 %, greater than 80 %, greater than 85 %, greater than 90% or greater than 95% relative to binding between the antibody and a wild type MICA polypeptide. In certain embodiments, binding is reduced below detectable limits.
  • a significant reduction in binding is evidenced when binding of an anti-MICA antibody to a mutant MICA polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%) of the binding observed between the anti-MICA antibody and a wild-type MICA polypeptide.
  • anti-MICA antibodies exhibit significantly lower binding for a mutant MICA polypeptide in which a residue in a segment corresponding to residues 1-88 (optionally 1-85), residues 89-181 (optionally 86-181 ), or residues 182-274 (or a subsequence thereof) in a wild-type MICA polypeptide (e.g., comprising a sequence of any of SEQ ID NOS: 22-26) is substituted with a different amino acid.
  • a mutant MICA polypeptide in which a residue in a segment corresponding to residues 1-88 (optionally 1-85), residues 89-181 (optionally 86-181 ), or residues 182-274 (or a subsequence thereof) in a wild-type MICA polypeptide (e.g., comprising a sequence of any of SEQ ID NOS: 22-26) is substituted with a different amino acid.
  • anti-MICA antibodies exhibit significantly lower binding for a mutant MICA polypeptide in which a residue in a segment corresponding to residues 1-88 (optionally 1-85), residues 89-181 (optionally 86-181 ), or residues 182-274 (or a subsequence thereof) in a wild-type MICA polypeptide (e.g., comprising a sequence of any of SEQ ID NOS: 22-26) is substituted with a different amino acid.
  • a mutant MICA polypeptide in which a residue in a segment corresponding to residues 1-88 (optionally 1-85), residues 89-181 (optionally 86-181 ), or residues 182-274 (or a subsequence thereof) in a wild-type MICA polypeptide (e.g., comprising a sequence of any of SEQ ID NOS: 22-26) is substituted with a different amino acid.
  • 6E4 binds to the a1 domain at the lateral side of MICA away from the NKG2D binding surface, consistent with the finding that 6E4 does not block MICA-NKG2D interactions.
  • Antibodies 9C10 and 12A10 display loss of binding to MICA mutants 12 and 13 having substitutions N56A, K57S, T58A (mutant 12); and R61A and R64A (mutant 13), respectively (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 9C10 and 12A10 therefore includes one or more of residues N56 and K57, and/or one or more of residues T58, R61 , and R64. These residues are within the a1 domain of MICA (the epitope may further include residues within the a2 or a3 domains).
  • 9C10 and 12A10 bind to the a1 domain at the lateral side of MICA near the NKG2D binding surface with possible partial overlap, consistent with the finding that 9C10 and 12A10 block MICA- NKG2D interactions.
  • Antibody 20C6 displays loss of binding to each of MICA mutants 16, 17, 21 , 60, 27 and 28 having substitutions: K81A and D82A (mutant 16); Q83A and K84A (mutant 17); H109A Y11 1A and L1 16A (mutant 21 ); D1 13A (mutant 60); S133A R134S and T137A (mutant 27); and MHOS N141A R143S N144A (mutant 28), respectively (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 20C6 therefore includes one or more of residues K81 and D82, one or more of residues Q83 and K84, one or more of residues H109, Y11 1 and L1 16, residue D113, one or more of residues S133, R134 and T137, and/or one or more of residues M140, N 141 , R143 and N144. These residues are within the a1 and a2 domains of MICA (the epitope may further include residues within the a3 domains).
  • Antibody 10A7 has partial overlap of epitope with 20C6. 10A7 displays loss of binding to each of MICA mutants 16, 17, 21 , 60, 26 and 28 having substitutions: K81A and D82A (mutant 16); Q83A and K84A (mutant 17); H109A, Y11 1A and L1 16A (mutant 21 ); D1 13A (mutant 60); Q131A, S132A and Q136S (mutant 26); and MHOS, N141A, R143S and N144A (mutant 28), respectively (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 10A7 therefore includes one or more of residues K81 and D82, one or more of residues Q83 and K84, one or more of residues H109, Y11 1 abd L1 16, residue D113, one or more of residues Q131 , S132 and Q136, and/or one or more of residues M140, N 141 , R143 and N144. These residues are within the a1 and a2 domains of MICA (the epitope may further include residues within the a3 domains).
  • 20C6 and 10A7 bind to the a1 and a2 domains at the lateral side of MICA near the NKG2D binding surface with possible partial overlap. This is consistent with the finding that 20C6 blocks MICA NKG2D interactions.
  • Antibodies 19E9, 18E8 and 10F3 display loss of binding to MICA mutants 19, 20, 23 and 24 having substitutions: E100A, D101S, N102A (mutant 19); S103A, T104S, R105A (mutant 20); N121A, E123S (mutant 23); T124A and E126A (mutant 24), respectively (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 19E9, 18E8 and 10F3 therefore includes one or more of residues E100, D101 and N102, one or more of residues S103, T104, and R105, one or more of residues N 121 , and E123, and/or one or more of residues T124 and E126.
  • residues are within the a2 domain of MICA (the epitope may further include residues within the a1 or a3 domains).
  • 19E9, 18E8 and 10F3 bind to the a2 domain at the lateral side of MICA near the NKG2D binding surface, consistent with the finding that 19E9, 18E8 and 10F3 block MICA-NKG2D interactions.
  • Antibody 15F9 displays loss of binding to MICA mutants 1 , 18, 19, 20, 61 and 36, having the substitutions: R6A and N8A (mutant 1 ); E97A and H99A (mutant 18); E100A, D101 S and N102A (mutant 19); S103A, T104S and R105A (mutant 20); E1 15A (mutant 61 ); L178A, R179S and R180A (mutant 36); respectively (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 15F9 therefore includes one or more of residues R6 and N8, one or more of residues E97 and H99, one or more of residues E100, D101 and N102, one or more of residues S103, T104 and R105, residue E1 15, and/or one or more of residues L178, R179 and R180. These residues are within the a2 domain of MICA (the epitope may further include residues within the a1 or a3 domains).
  • 15F9 binds to the a2 domain at the lateral side of MICA below the NKG2D binding surface. 15F9 blocks sMICA- NKG2D interactions.
  • Antibody 16A8 has loss of binding to MICA mutants 45, 46, and 47, having substitutions: S224A, H225S and D226A (mutant 45); T227A, Q228S and Q229A (mutant 46); W230A and D232A (mutant 47), respectively (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 16A8 therefore includes one or more of residues W230 and/or D232, one or more of residues T227, Q228 and Q229, one or more of residues S224, H225 and D226.
  • the epitope of 16A8 is primarily within the a3 domain of MICA. 16A8 binds to the a3 domain away from the NKG2D binding surface.
  • Antibody 14B4 displays loss of binding to MICA mutant 46 having the substitutions: T227A, Q228S and Q229A substitutions (amino acid positions are with reference to SEQ ID NO: 22).
  • the principal epitope of 14B4 therefore includes one or more of residues T227, Q228 and Q229, and had a partial overlap with antibody 16A8.
  • the epitope of 14B4 is primarily within the a3 domain of MICA.
  • 14B4 binds to the a3 domain away from the NKG2D binding surface (however 14B4 is a functionally blocking antibody). Accordingly, an anti-MICA antibody can be characterized by loss of binding to one or more particular mutant MICA polypeptides having one or more (e.g. 2, 3, or 4) amino acid substitution(s) (compared to a wild-type MICA polypeptide of SEQ ID NO : 22) at:
  • (L) a residue selected from the group consisting of H109, Y11 1 , L1 16;
  • a R6, N8, Q48, W49, E51 , D52, V53, L54, N56, K57, T58, R61 , R64, K81 , D82, Q83, K84, E97, H99, E100, D101 , N102, S103, T104, R105, H109, Y11 1 , D1 13, E 115, L1 16, N121 , E123, T124, E126, Q131 , S132, S133, R134, Q136, T137, M140, N141 , R143, N144, L178, R179, R180, S224, H225, D226, T227, Q228, Q229, W230 or D232 substitution may be specified as being a R6A, N8A, W14A, Q48A, W49S, E51S, D52A, V53S, L54A, N56A, K57S, T58A, R61A, R64A, K81A, D82A, Q83
  • an anti-MICA antibody can be characterized by loss of binding to a mutant MICA polypeptide having the amino acid substitutions Q48A and W49S (mutant 10); and to a mutant MICA polypeptide having the amino acid substitutions E51 S, D52A, V53S and L54A (mutant 1 1 ).
  • an anti-MICA antibody can be characterized by loss of binding to a mutant MICA polypeptide having the amino acid substitutions N56A, K57S, T58A (mutant 12); and to a mutant MICA polypeptide having the amino acid substitutions R61A and R64A (mutant 13).
  • an anti-MICA antibody can be characterized by loss of binding to: a mutant MICA polypeptide having the amino acid substitutions K81A and D82A (mutant
  • mutant MICA polypeptide having the amino acid substitutions H109A Y1 11A and L116A mutant 21
  • a mutant MICA polypeptide having the amino acid substitutions D1 13A mutant 60
  • a mutant MICA polypeptide having the amino acid substitutions S133A R134S and T137A mutant 27
  • a mutant MICA polypeptide having the amino acid substitutions M140S, N141A, R143S, N144A mutant 28.
  • an anti-MICA antibody can be characterized by loss of binding to: a mutant MICA polypeptide having the amino acid substitutions K81A and D82A (mutant
  • mutant MICA polypeptide having the amino acid substitutions H109A Y1 11A and L116A mutant MICA polypeptide having the amino acid substitutions D1 13A (mutant 60); a mutant MICA polypeptide having the amino acid substitutions Q131A, S132A and Q136S (mutant 26); and a mutant MICA polypeptide having the amino acid substitutions M140S, N141A, R143S and N144A (mutant 28).
  • an anti-MICA antibody can be characterized by loss of binding to: a mutant MICA polypeptide having the amino acid substitutions E100A, D101S, N102A (mutant 19); a mutant MICA polypeptide having the amino acid substitutions S103A, T104S, R105A (mutant 20); a mutant MICA polypeptide having the amino acid substitutions N121A, E123S (mutant 23); and a mutant MICA polypeptide having the amino acid substitutions T124A and E126A (mutant 24).
  • an anti-MICA antibody can be characterized by loss of binding to: a mutant MICA polypeptide having the amino acid substitutions R6A and N8A, a mutant MICA polypeptide having the amino acid substitutions E97A and H99A, a mutant MICA polypeptide having the amino acid substitutions E100A, D101 S and N102A, a mutant MICA polypeptide having the amino acid substitutions S103A, T104S and R105A, a mutant MICA polypeptide having the amino acid substitution E1 15A, and a mutant MICA polypeptide having the amino acid substitutions L178A, R179S and R180A.
  • an anti-MICA antibody can be characterized by loss of binding to: a mutant MICA polypeptide having the amino acid substitutions S224A, H225S and D226A, a mutant MICA polypeptide having the amino acid substitutions T227A, Q228S and Q229A, and a mutant MICA polypeptide having the amino acid substitutions W230A and D232A.
  • an anti-MICA antibody can be characterized by loss of binding to a mutant MICA polypeptide having the amino acid substitutions T227A, Q228S and Q229A.
  • amino acid sequence of the heavy and light chain variable region of exemplary antibodies 6E4, 20C6, 16A8, 9C10, 19E9 (and its humanized variants 19E9-1 , 19E9-2 and 19E9-3), 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 and 6E1 are shown in Table 1.
  • the antibody binds essentially the same epitope or determinant as any of the monoclonal antibodies 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F11 or 6E1 ; optionally the antibody comprises an antigen binding region (e.g. hypervariable region) of antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1.
  • an antigen binding region e.g. hypervariable region
  • antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1 can be characterized by the amino acid sequences thereof (e.g. CDR sequences) and/or nucleic acid sequence encoding it.
  • the monoclonal antibody comprises the Fab or F(ab') 2 portion.
  • an antibody comprises the three CDRs of the heavy chain variable region of 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1 , and the three of the CDRs of the light chain variable region of the respective 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F11 or 6E1 antibody.
  • any one or more of said light or heavy chain CDRs may contain one, two, three, four or five or more amino acid modifications (e.g. substitutions, insertions or deletions).
  • CDRs may for example be specified to be according to Kabat.
  • an antibody that binds human MICA comprises:
  • an antibody that binds human MICA comprises: the heavy chain CDRs 1 , 2 and 3 (H-CDR1 , H-CDR2, H-CDR3) amino acid sequences as shown in Table 2, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; and the respective light chain CDRs 1 , 2 and 3 (L-CDR1 , L-CDR2, L-CDR3) amino acid sequences as shown in Table 2, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid.
  • an antibody that binds human MICA comprises: a heavy chain variable region comprising the heavy chain CDRs 1 , 2 and 3 (H-CDR1 , H-CDR2, H- CDR3) amino acid sequences as shown in Table 2, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; and a light chain variable region comprising the respective light chain CDRs 1 , 2 and 3 (L-CDR1 , L-CDR2, L- CDR3) amino acid sequences as shown in Table 2, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid.
  • an antibody that binds human MICA comprises: a heavy chain variable region which is at least 60%, 70%, 80%, 85%, 90% or 95% identical to the heavy chain variable region having an amino acid sequence of Table 1 ; and a light chain variable region which is at least 60%, 70%, 80%, 85%, 90% or 95% identical to the respective light chain variable region having an amino acid sequence of Table 1.
  • any of the CDRs 1 , 2 and 3 of the heavy and light chains may be characterized by a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, and/or as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR or set of CDRs of the VH/VL of Table 1 or as set out in Table 2.
  • an antibody competes for binding to a MICA polypeptide with any one or any combination of monoclonal antibodies 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 and 6E1 (e.g. an antibody having the VH and the VL amino acid sequence listed in the Table 1 below).
  • an antibody that binds human MICA comprises: the heavy chain CDRs 1 , 2 and 3 (H-CDR1 , H-CDR2, H-CDR3) amino acid sequences of the 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F11 or 6E1 VH domain as shown in Table 1 , optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; and the light chain CDRs 1 , 2 and 3 (L- CDR1 , L-CDR2, L-CDR3) amino acid sequences of the respective 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F11 or 6E1 VL domain shown in Table 1 , optionally wherein one, two, three or more amino acids in
  • an antibody that binds human MICA comprises: the heavy chain CDRs 1 , 2 and 3 (H-CDR1 , H-CDR2, H-CDR3) amino acid sequences of the anti-a3 domain antibody commercialized as reference BAM03 (Novus Biologicals) or its derivative referred to as PDI (PDI VH domain of SEQ ID NO: 158 as shown in Table 1 , CDRs underlined), optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; and the light chain CDRs 1 , 2 and 3 (L- CDR1 , L-CDR2, L-CDR3) amino acid sequences of the respective BAM03 or PDI (PDI VL domain of SEQ ID NO: 159 as shown in Table 1 , CDRs underlined), optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid.
  • the agent is an antibody that is a function-conservative variant of 19E9 or of an antibody having a heavy chain variable region of SEQ ID NO: 43 and a light chain variable region of SEQ ID NO: 44, optionally further wherein the antibody is a human lgG1 isotype.
  • the agent is an antibody that is a function-conservative variant of 14B4 or 16A8.
  • the agent is an antibody that is a function-conservative vari- ant of BAM03 or PD1.
  • the sequences of the CDRs can be determined according to any desired numbering scheme, for example AbM (Oxford Molecular's AbM antibody modelling software definition), IMGT, Kabat or Chothia definitions systems. Table 1
  • an antibody that binds human MICA comprises: the Kabat heavy chain CDRs 1 , 2 and 3 (H-CDR1 , H-CDR2, H-CDR3) amino acid sequences of antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9,14B4, 1 D5, 13A9, 15F1 1 or 6E1 as shown in Table 2, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; and the Kabat light chain CDRs 1 , 2 and 3 (L-CDR1 , L-CDR2, L-CDR3) amino acid sequences of the respective 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9, 14B4, 1 D5, 13A9, 15F1 1 or 6E1 as shown in Table 2, optionally wherein one, two, three or more amino acids in a C
  • the H-CDR2 of antibody 1 D5 may comprise the amino acid sequence YIEPYNVVPXYNPKFKG SEQ ID NO : 157, wherein X is any amino acid, optionally wherein X is methionine (M), alanine (A), leucine (L) or valine (V).
  • all of the antibodies 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9,14B4, 1 D5, 13A9, 15F1 1 and 6E1 are capable of mediating ADCC toward MICA-expressing cells and thus directing the lysis of such cells.
  • some of the antibodies block the interaction of MICA with NKG2D while others do not, and some block MICA shedding from the cell surface (e.g., tumor cell surface) while others do not.
  • the 20C6 and 10A7 mAbs block the interaction of recombinant non-glycosylated MICA (e.g., MICA * 001-BirA tagged protein) with bivalent NKG2D-Fc recombinant protein but they do not block the interaction of cell surface MICA with cell surface NKG2D (they do not block the NKG2D-mediated killing of Raji-MICA * 08 by NK92).
  • MICA non-glycosylated MICA
  • Antibody 19E9 is an example of a blocking anti-MICA antibody in both assays, and with an epitope not directly on the NKG2D binding site.
  • Antibodies 14B4 and 16A8 bind to the a3 domain.
  • 16A8 is an example of an antibody that does not block the NKG2D-MICA interaction, while 14B4 is capable of blocking the NKG2D-MICA interaction despite its epitope being on the a3 domain suggesting that MICA conformation is altered upon 14B4 binding.
  • Antibodies 1 D5, 13A9, 15F1 1 and 6E1 also have their epitope on the a3 domain at the same or proximal site as 14B4 and 16A8 and have been reported to decrease MICA shedding from the tumor cell surface (thereby reducing MICA-mediated down-modulation of NKG2D) without blocking the NKG2D-MICA interaction.
  • the epitope for antibody 20C6 is near the NKG2D binding site but not overlapping and while this antibody blocks the interaction of soluble MICA * 001-BirA protein with NKG2A-Fc, it does not block cell surface MICA interaction with cell surface NKG2D).
  • NK92/Raji- MICA * 08 lysis is a functional assay used to assess whether an antibody may block cell surface MICA interaction with cell surface NKG2D, block ability of cell surface MICA to induce NKG2D signalling and/or block ability of cell surface MICA to induce NKG2D downmodulation.
  • An antibody that is blocking in NK92/Raji-MICA * 08 lysis (cytotoxicity) functional assay may be designated as blocking cell surface MICA interaction with cell surface NKG2D, blocking ability of cell surface MICA to induce NKG2D signalling and/or blocking ability of cell surface MICA to induce NKG2D downmodulation.
  • Table 3 Summary of NKG2D-MICA blocking capacity of anti-MICA antibodies assessed by surface plasmon resonance or by a functional cytotoxicity assay
  • Fragments and derivatives of antibodies can be produced by techniques that are known in the art. “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region.
  • antibody fragments include Fab, Fab', Fab'-SH, F (ab') 2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or“single chain polypeptide”), including without limitation (1 ) single-chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Included, inter alia, are a nanobody, domain antibody, single domain antibody or a“dAb”.
  • Anti-MICA antibodies that mediate ADCC can optionally comprise one or more amino acid modifications (e.g. amino acid substitutions) in their Fc domain which increase binding to human CD16.
  • the modifications will not substantially decrease or abolish the ability of the an anti-MICA antibody to bind to neonatal Fc receptor (FcRn), e.g. human FcRn.
  • Typical modifications include modified human lgG1-derived constant regions comprising at least one amino acid modification (e.g. substitution, deletions, insertions), and/or altered types of glycosylation, e.g., hypofucosylation.
  • Such modifications can affect interaction with Fc receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16).
  • FcyRI CD64
  • FcyRIIA CD32A
  • FcyRIII CD 16
  • a modification may, for example, increase binding of the Fc domain to FcyRI I la on effector (e.g. NK) cells and/or decrease binding to FcyRIIB.
  • an anti-MICA antibody comprises an Fc domain comprising at least one amino acid modification (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the mol- ecule has enhanced binding affinity for human CD16 relative to a molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 239, 298, 330, 332, 333 and/or 334 (e.g.
  • compositions comprising a NKG2A neutralizing agent such as an anti-NKG2A antibody and/or an antibody that binds MICA.
  • a pharmaceutical composition containing a NKG2A neutralizing agent such as an anti-NKG2A antibody and an antibody that binds MICA, and optionally further a pharmaceutically acceptable carrier.
  • a NKG2A neutralizing agent and/or an anti-MICA antibody can be incorporated in a pharmaceutical formulation in a concentration from 1 mg/ml to 500 mg/ml, wherein said formulation has a pH from 2.0 to 10.0.
  • the NKG2A neutralizing agent and the anti-MICA agent can be comprised in the same or separate pharmaceutical formulations.
  • the formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants.
  • the pharmaceutical formulation is an aqueous formulation, i.e., formulation comprising water. Such formulation is typically a solution or a suspension.
  • the pharmaceutical formulation is an aqueous solution.
  • aqueous formulation is defined as a formulation comprising at least 50 %w/w water.
  • the term“aqueous solution” is defined as a solution comprising at least 50 %w/w water
  • the term“aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.
  • the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.
  • the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.
  • the pharmaceutical formulation comprises an aqueous solution of such an antibody, and a buffer, wherein the antibody is present in a concentration from 1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.
  • the pH of the formulation is in the range selected from the list consisting of from about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.
  • the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.
  • Each one of these specific buffers constitutes an alternative embodiment of the invention.
  • the formulation further comprises a pharmaceutically acceptable preservative.
  • the formulation further comprises an isotonic agent.
  • the formulation also comprises a chelating agent.
  • the formulation further comprises a stabilizer.
  • the formulation further comprises a surfactant.
  • Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine).
  • additional ingredients should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.
  • compositions according to the invention may be through any appropriate route of administration, for example, intravenous. Suitable antibody formulations can also be determined by examining experiences with other already developed therapeutic monoclonal antibodies.
  • kits for example kits which include:
  • a pharmaceutical composition containing an anti-MICA antibody and instructions to administer said anti-MICA antibody with a NKG2A neutralizing agent such as an anti-NKG2A antibody.
  • kits optionally also can include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient having cancer.
  • a kit optionally can include instructions to administer said NKG2A neutralizing agent simultaneously, separately, or sequentially with said anti-MICA antibody.
  • a kit optionally can include instructions for use in the treatment of a MICA-expressing cancer.
  • a kit optionally can include instructions for use in the treatment of a colorectal cancer, for example.
  • the kit also can include a syringe.
  • kits include multiple packages of the single-dose pharmaceutical compositions each containing an effective amount of the NKG2A neutralizing agent, and/or the anti-MICA antibody, for a single administration in accordance with the methods provided above.
  • Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits.
  • a kit may provide one or more pre-filled syringes containing an amount of the anti-NKG2A or an anti-MICA antibody.
  • the present invention provides a kit for treating a cancer or a tumor in a human patient, optionally wherein said cancer or tumor is a MICA-positive tumor or cancer (and optionally further an HLA-E-positive tumor or cancer), the kit comprising: (a) a dose of an anti-NKG2A antibody comprising the H-CDR1 , H-CDR2 and H- CDR3 domains of a heavy chain variable region having the sequence set forth in any of SEQ ID NOS: 2-6, and the L-CDR1 , L-CDR2 and L-CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO: 7; and/or
  • an anti-MICA antibody optionally wherein the anti-MICA antibody is capable of inhibiting the MICA-mediated down-modulation of NKG2D on NK and/or T cells, optionally wherein the anti-MICA antibody is capable of directing ADCC toward a MICA- expressing tumor cell, and/or optionally wherein the anti-MICA antibody is capable of inducing or increasing immune cell (e.g. T cell) infiltration into a tumor (e.g. a MICA- expressing tumor); and
  • the present invention provides a kit for treating a cancer or a tumor in a human patient, optionally wherein said cancer or tumor is characterized by presence of MICA polypeptide and/or MICA-expressing cells, the kit comprising:
  • a dose of an anti-NKG2A antibody comprising the heavy chain H-CDR1 , H-CDR2 and H-CDR3 domains having the sequences of SEQ ID NOS: 14-16, and the light chain L- CDR1 , L-CDR2 and L-CDR3 domains having the sequences of SEQ ID NOS: 17-19, respectively; and/or
  • an anti-MICA antibody optionally wherein the anti-MICA antibody is capable of inhibiting the MICA-mediated down-modulation of NKG2D on NK and/or T cells, optionally wherein the anti-MICA antibody is capable of directing ADCC toward a MICA- expressing tumor cell, and/or optionally wherein the anti-MICA antibody is capable of inducing or increasing immune cell (e.g. T cell) infiltration into a tumor (e.g. a MICA- expressing tumor); and
  • Described are methods useful in the diagnosis, prognosis, monitoring and treatment of a cancer particularly colorectal cancer, renal cell carcinoma, lung cancer (e.g. non-small cell lung carcinoma), melanoma, ovarian cancer, endometrial cancer, pancreatic cancer or a head and neck cancer.
  • a cancer particularly colorectal cancer, renal cell carcinoma, lung cancer (e.g. non-small cell lung carcinoma), melanoma, ovarian cancer, endometrial cancer, pancreatic cancer or a head and neck cancer.
  • MICA-expressing cells e.g. MICA-expressing tumor cells and/or MICA-expressing MDSC.
  • Colorectal cancer (CRC) as used herein refers to colon cancer, rectal cancer, and cancer of both the colon and rectal areas.
  • a CRC is an adenocarcinoma.
  • kidney tumors, hepatocellular carcinomas, thyroid tumors, and adrenal tumors also express MICA and accordingly can also advantageous be treated in accordance with the methods and compositions of the disclosure.
  • HLA-E polypeptides were expressed in MICA-positive tumors treated with anti-MICA antibody. Accordingly, in one embodiment, provided is use of an agent that neutralizes the inhibitory activity of human NKG2A, for the treatment of a cancer in an individual having a MICA-positive cancer. In one embodiment, an individual has received prior treatment with an antibody that binds MICA.
  • an individual treated according to the disclosure has a solid tumor characterized by MICA and/or MICB expressing cells, e.g. malignant cells or immunosuppressive cells such as MDSC.
  • MICA and/or MICB expressing cells are present in tumor tissue and/or tumor-adjacent tissue.
  • a NKG2A-neutralizing agent e.g., an anti- NKG2A antibody; an anti-NKG2A antibody in combination with an anti-MICA antibody
  • a method for treating or preventing a cancer or tumors in an individual having a MICA-positive tumor or cancer comprising administering to the individual an agent that binds and neutralizes NKG2A.
  • the disclosure provides a method for the treatment or prevention of a MICA-positive cancer in an individual, the method comprising: administering to the individual a NKG2A neutralizing agent.
  • the disclosure provides a method for the treatment or prevention of a MICA- positive cancer in an individual, the method comprising: administering to the individual a NKG2A neutralizing agent and an antibody that binds MICA. In one embodiment, the disclosure provides a method for the treatment or prevention of a HLA-E-positive cancer in an individual, the method comprising: administering to the individual a NKG2A neutralizing agent and an antibody that binds MICA.
  • a method of treating a tumor in an individual comprising administering to the individual an effective amount of a NKG2A- neutralizing agent and an antibody or antibody fragment that binds MICA.
  • a method of treating a tumor in an individual comprising (i) identifying an individual who has a MICA-positive tumor, and (ii) administering to the individual an effective amount of a NKG2A-neutralizing agent and an antibody or antibody fragment that binds MICA.
  • a method for assessing whether an individual is suitable for treatment with an agent that inhibits NKG2A comprising assessing tumor expression of MICA in a biological sample from an individual.
  • a determination that the individual has a tumor that expresses MICA polypeptide (or comprises MICA-expressing cells) indicates that the patient has a cancer that can be treated with an agent that inhibits NKG2A (e.g., in combination with an anti-MICA antibody).
  • a MICA-positive tumor or cancer is a tumor or cancer known to be generally characterized by presence of MICA-expressing cells in the tumor or tumor environment. Accordingly, an individual having a cancer can be treated with the NKG2A neutralizing agent with or without a prior detection step to assess expression of MICA on cells in the tumor microenvironment (e.g. on tumor cells, on MDSC).
  • the treatment methods can comprise a step of detecting a MICA nucleic acid or polypeptide in a biological sample of a tumor from an individual (e.g., in cancer tissue, tissue proximal to or at the periphery of a cancer, cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue).
  • the method comprises determining the level of expression of a MICA nucleic acid or polypeptide in a biological sample and comparing the level to a reference level corresponding to a healthy individual.
  • a determination that a biological sample comprises cells expressing MICA nucleic acid or polypeptide at a level that is elevated compared to a reference level indicates that the patient has a cancer that could be treated with a NKG2A neutralizing agent.
  • detecting a MICA polypeptide in a biological sample comprises detecting MICA polypeptide expressed on the surface of a malignant cell.
  • a determination that a biological sample comprises cells (e.g. malignant cells) that express MICA nucleic acid or polypeptide indicates that the patient has a cancer that can be treated with a NKG2A neutralizing agent.
  • a MICA polypeptide can be specified as being expressed in a substantial number of cells taken from a given individual, for example MICA polypeptide can be present on at least 5%, 10%, 20% 30%, 40%, 50°%, 60%, 70%, 80%, or more of the tumor cells or the cells in tumor tissue or tumor-adjacent tissue sample (e.g. biopsy) taken from the individual.
  • MICA polypeptide can be present on at least 5%, 10%, 20% 30%, 40%, 50°%, 60%, 70%, 80%, or more of the tumor cells or the cells in tumor tissue or tumor-adjacent tissue sample (e.g. biopsy) taken from the individual.
  • Determining whether an individual has a cancer characterized by cells that express a MICA polypeptide can for example comprise obtaining a biological sample (e.g. by performing a biopsy) from the individual that comprises cells from the cancer environment (e.g. tumor or tumor adjacent tissue), bringing said cells into contact with an antibody that binds an MICA polypeptide, and detecting whether the cells express MICA on their surface.
  • determining whether an individual has cells that express MICA comprises conducting an immunohistochemistry assay.
  • the disclosure provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising:
  • MICA polypeptide e.g. MICA-expressing cells
  • detecting MICA polypeptide or MICA-expressing cells within the tumor environment comprises obtaining from the individual a biological sample that comprises cancer tissue and/or tissue proximal to or at the periphery of a cancer (e.g., cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue), and detecting levels of MICA polypeptide or MICA-expressing cells.
  • a biological sample that comprises cancer tissue and/or tissue proximal to or at the periphery of a cancer (e.g., cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue), and detecting levels of MICA polypeptide or MICA-expressing cells.
  • adjunctive or combined administration includes simultaneous administration of the compounds in the same or different dosage form, or separate administration of the compounds (e.g., sequential administration).
  • a NKG2A- neutralizing agent can be used in combination with an antibody that binds MICA.
  • an anti-NKG2A antibody and an anti-MICA antibody can be simultaneously administered in a single formulation.
  • the NKG2A-neutralizing agent and anti- MICA antibody can be formulated for separate administration and are administered concurrently or sequentially.
  • the disclosure provides a method for the treatment or prevention of a cancer (e.g. CRC) in an individual having a MICA-positive tumor, the method comprising:
  • identifying an individual who has a MICA-positive tumor optionally obtaining a biological sample comprising tumor cells from the individual and determining whether the tumor is MICA-positive,
  • any of the treatment regimens and methods described herein may be used with or without a prior step of detecting the expression of HLA-E on cells in a biological sample obtained from an individual (e.g. a biological sample comprising cancer cells, cancer tissue or cancer-adjacent tissue).
  • the cancer treated with the methods disclosed herein is a cancer characterized by HLA-E, optionally high levels of HLA-E.
  • a patient having a cancer can be treated with the NKG2A neutralizing agent with or without a prior detection step to assess expression of HLA-E on the surface of tumor cells.
  • the treatment methods can comprises a step of detecting a HLA-E nucleic acid or polypeptide in a biological sample of a tumor (e.g. on a tumor cell) from an individual. A determination that a biological sample expresses HLA-E (e.g.
  • the method comprises determining the level of expression of a HLA-E nucleic acid or polypeptide in a biological sample and comparing the level to a reference level (e.g. a value, weak cell surface staining, etc.). A determination that a biological sample expresses an HLA-E nucleic acid or polypeptide at a level that is increased compared to the reference level may indicate that the individual has a cancer that can be treated with an agent that inhibits NKG2A.
  • a reference level e.g. a value, weak cell surface staining, etc.
  • Determining whether an individual has cancer cells that express an HLA-E polypeptide can for example comprise obtaining a biological sample (e.g. by performing a biopsy) from the individual that comprises cancer cells, bringing said cells into contact with an antibody that binds an HLA-E polypeptide, and detecting whether the cells express HLA-E on their surface.
  • determining whether an individual has cancer cells that express HLA-E comprises conducting an immunohistochemistry assay.
  • determining whether an individual has cancer cells that express HLA-E comprises conducting a flow cytometry assay.
  • the disclosure provides a method for the treatment or prevention of a cancer (e.g. CRC) in an individual having a MICA-positive tumor, the method comprising: a) detecting HLA-E polypeptide (e.g. HLA-E-expressing tumor cells) in the tumor environment, optionally within the tumor and/or within adjacent tissue, and b) upon a determination that the tumor environment comprises HLA-E polypeptides (e.g. HLA- E-expressing tumor cells), optionally at a level that is elevated compared to a reference level, administering to the individual a NKG2A neutralizing agent.
  • a cancer e.g. CRC
  • HLA-E polypeptide e.g. HLA-E-expressing tumor cells
  • HLA-E polypeptides e.g. HLA- E-expressing tumor cells
  • the disclosure provides a method for the treatment or prevention of a cancer (e.g. CRC) in an individual having a MICA-positive tumor, the method comprising:
  • HLA-E polypeptide e.g. HLA-E-expressing tumor cells
  • HLA-E polypeptides e.g. HLA-E-expressing tumor cells
  • administering to the individual an agent that neutralizes the inhibitory activity of NKG2A in combination with an antibody that binds MICA.
  • HLA-E polypeptides were expressed in tumors treated with an- ti-MICA antibody. Accordingly, in one embodiment, provided is use of an agent that neutral- izes the inhibitory activity of human NKG2A, for the treatment of a cancer in an individual having a MICA-positive cancer and/or having received prior treatment with an antibody that binds MICA.
  • the cancer is a MICA-positive cancer.
  • the individual has a cancer that has progressed despite prior treatment with the antibody that binds MICA.
  • said treatment comprises: a) determining whether HLA-E polypeptide is expressed by malignant cells from the individual, and b) upon the determina- tion that malignant cells express HLA-E polypeptide, administering to the individual an agent, optionally an antibody, that neutralizes the inhibitory activity of human NKG2A (and optionally further an antibody that binds MICA).
  • said treatment comprises adminis- tering to the individual an effective amount of each of: (a) an agent, optionally an antibody, that neutralizes the inhibitory activity of human NKG2A, and (b) an antibody that binds MICA.
  • the disclosure provides a method for the treatment or prevention of a cancer (e.g. CRC) in an individual having a MICA-positive tumor, the method comprising:
  • step (b) obtaining, from the individual treated in step (a), a biological sample that comprises tumor cells, and detecting HLA-E polypeptide (e.g. HLA-E-expressing tumor cells), optionally detecting HLA-E polypeptides (e.g. HLA-E-expressing tumor cells) within the tumor and/or within adjacent tissue in a sample from the individual, and
  • HLA-E polypeptide e.g. HLA-E-expressing tumor cells
  • HLA-E polypeptides e.g. HLA-E-expressing tumor cells
  • the tumor comprises HLA-E polypeptides (e.g. HLA-E- expressing tumor cells), administering to the individual an agent that neutralizes the inhibitory activity of NKG2A.
  • the agent that neutralizes the inhibitory activity of NKG2A is administered in combination with an antibody that binds MICA.
  • Determining whether HLA-E polypeptide is expressed by cells may comprise obtaining from the individual a biological sample that comprises tumor cells, bringing said cells into contact in vitro with an antibody that binds a HLA-E polypeptide, and detecting cells that express HLA-E.
  • the present disclosure also provides the use of a NKG2A neutralizing agent and an antibody that binds MICA for the manufacture of a pharmaceutical composition for the treatment of cancer, in particular a MICA-positive cancer or tumor, in accordance with the methods disclosed herein.
  • the present disclosure also provides a NKG2A neutralizing agent, for use in treating a human individual who has a cancer, wherein the individual has a tumor that is MICA- positive, wherein said NKG2A neutralizing agent is administered in combination with an antibody that binds MICA, in accordance with the methods disclosed herein.
  • the present disclosure also provides a NKG2A neutralizing agent, for use in treating a human individual who has a cancer, wherein the individual has a tumor that is MICA-positive, wherein said NKG2A neutralizing agent is administered following administration of or a course of therapy with an antibody that binds MICA, in accordance with the methods disclosed herein.
  • the present disclosure also provides an agent that is an antibody that binds MICA, for use in treating a human individual who has a MICA-positive cancer, wherein said antibody that binds MICA is administered in combination with a NKG2A neutralizing agent.
  • NKG2A neutralizing agent is an antibody that binds a human NKG2A protein, optionally a human or humanized anti-NKG2A antibody;
  • NKG2A neutralizing agent is an antibody that inhibits binding of NKG2A to HLA-E;
  • NKG2A neutralizing agent comprises the heavy chain H-CDR1 , H-CDR2 and H-CDR3 domains having the sequences of SEQ ID NOS: 14-16, and the light chain L-CDR1 , L-CDR2 and L-CDR3 domains having the sequences of SEQ ID NOS: 17-19, respectively; the agent for use as described above, wherein said NKG2A neutralizing agent is monalizumab;
  • said antibody that binds MICA is an antibody that binds to MICA expressed at the surface of a cell;
  • said antibody that binds MICA is an antibody that binds to the a1 a2 platform domain of MICA or to the a3 domain of MICA;
  • the agent for use as described above wherein said NKG2A neutralizing agent and said antibody that binds MICA are administered simultaneously, separately, or sequentially; the agent for use as described above, wherein said NKG2A neutralizing agent and said antibody that binds MICA are formulated for separate administration and are administered concurrently or sequentially; and/or
  • NKG2A neutralizing agent is administered at a dose ranging from 0.1 to 10 mg/kg and said antibody that binds MICA is administered at a dose ranging from 1 to 20 mg/kg.
  • the NKG2A-neutralizing agent and anti-MICA antibody can be administered separately, together or sequentially, or in a cocktail.
  • the NKG2A-neutralizing agent is administered prior to the administration of the anti-MICA antibodies.
  • the NKG2A-neutralizing agent can be administered approximately 0 to 30 days prior to the administration of the anti-MICA antibodies.
  • antibody NKG2A-neutralizing agent is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days prior to the administration of the anti-MICA antibodies.
  • a NKG2A-neutralizing agent is administered concurrently with the administration of the anti- MICA antibodies.
  • a NKG2A-neutralizing agent is administered after the administration of the anti-MICA antibodies.
  • a NKG2A-neutralizing agent can be administered approximately 0 to 30 days after the administration of the anti-MICA antibodies.
  • a NKG2A-neutralizing agent is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days after the administration of the anti-MICA antibodies.
  • Exemplary treatment protocols for treating a human with an anti-NKG2A antibody include, for example, administering to the patient an effective amount of an antibody that inhibits NKG2A, wherein the method comprises at least one administration cycle in which at least one dose of the anti-NKG2A antibody is administered at a dose of 1-10 mg/kg body weight.
  • the administration cycle is between 2 weeks and 8 weeks.
  • Exemplary treatment protocols for treating a human with an anti-NKG2A antibody include, for example, administering to the patient an effective amount of each of an antibody that inhibits NKG2A and an anti-MICA antibody, wherein the method comprises at least one administration cycle in which at least one dose of the anti-NKG2A antibody is administered at a dose of 0.1-10 mg/kg body weight or 1-10 mg/kg body weight and at least one dose of the anti-MICA antibody is administered at a dose of 1-20 mg/kg body weight.
  • the administration cycle is between 2 weeks and 8 weeks.
  • the method comprises at least one administration cycle, wherein the cycle is a period of eight weeks or less, wherein for each of the at least one cycles, two, three or four doses of the anti-NKG2A antibody are administered at a dose of 1-10 mg/kg body weight.
  • each cycle further comprises the administration of two, three or four doses of the anti-MICA antibody at a dose of 1-20 mg/kg body weight.
  • Exemplary treatment protocols for an anti-NKG2A antibody such as a humZ270 comprise at least one administration cycle in which at least one dose of the anti-NKG2A antibody is administered at a dose of about 10 mg/kg, optionally 2-10 mg/kg, optionally 4-10 mg/kg, optionally 6-10 mg/kg, optionally 2-6 mg/kg, optionally 2-8 mg/kg, or optionally 2-4 mg/kg body weight.
  • at least 2, 3, 4, 5, 6, 7 or 8 doses of the anti- NKG2A antibody are administered.
  • the administration cycle is between 2 weeks and 8 weeks.
  • a dose (e.g. each dose) of the anti-NKG2A antibody e.g.
  • monalizumab is administered at a fixed dose of 7.5 mg, 22.5 mg, 75 mg, 225 mg or 750 mg, optionally administered every two or four weeks.
  • the administration cycle is 8 weeks.
  • the administration cycle is 8 weeks and comprises administering one dose of the anti-NKG2A antibody every two weeks (i.e. a total of four doses).
  • an anti-MICA antibody can be administered in an amount that induces or increases immune cell (e.g. CD8 T cell, NK cell) infiltration into a tumor (e.g. a MICA-expressing tumor).
  • immune cell e.g. CD8 T cell, NK cell
  • a tumor e.g. a MICA-expressing tumor
  • an anti-MICA antibody can be administered in an amount that mediates ADCC towards MICA-expressing cells (e.g. tumor cells), for example the amount provides (e.g. achieves upon administration and/or maintains until the subsequent administration) a concentration in circulation (and/or in a tumor tissue) that is at least the EC 50 , the EC 70 or the EC-ioo for ADCC, e.g. as assessed in an in vitro ADCC assay, or for example an amount that corresponds to a concentration that results in detectable ADCC as assessed in an in vitro cell cytotoxicity assay.
  • MICA-expressing cells e.g. tumor cells
  • the amount provides (e.g. achieves upon administration and/or maintains until the subsequent administration) a concentration in circulation (and/or in a tumor tissue) that is at least the EC 50 , the EC 70 or the EC-ioo for ADCC, e.g. as assessed in an in vitro ADCC assay, or for example an amount that corresponds to a concentration
  • the amount is an amount that modulates the interaction between a MICA polypeptide (e.g. a MICA polypeptide expressed at the surface of a tumor cell and NKG2D polypeptides expressed at the surface of an immune cell (e.g. a T cell, an NK cell).
  • a MICA polypeptide e.g. a MICA polypeptide expressed at the surface of a tumor cell and NKG2D polypeptides expressed at the surface of an immune cell (e.g. a T cell, an NK cell).
  • the amount is an amount that prevents MICA-mediated down-modulation of NKG2D at the surface of immune cells (e.g. CD8 T cells, NK cells).
  • the amount is an amount that increases NKG2D expression at the surface of immune cells.
  • the amount is an amount that prevents shedding of MICA from the surface of tumor cells.
  • An amount having a specified biological effect can be specified to be an amount that provides a concentration in circulation (and/or in a tumor tissue) that corresponds to at least the EC 50 (or for example the EC 70 or EC100) as determined in an in vitro assay for the particular biological effect.
  • the EC 50 e.g. for binding, mediating ADCC, depleting cells, for example
  • agent e.g. anti-MICA antibody
  • EC 70 with respect to a biological activity, refers to the efficient concentration of an agent which produces 70% of its maximum response or effect with respect to such biological activity.
  • EC100 with respect to the biological activity, refers to the efficient concentration of an agent which produces its substantially maximum response or effect with respect to such biological activity.
  • Determining the at least the EC 50 , the EC 70 or the EC 10 o for ADCC can for example be carried out using a as shown for example using a classical 4 hour 51 Cr-release assay. Briefly, the cytolytic activity of human NK cell line KHYG-1 (DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, product ref. ACC 725) transfected with human CD16 is assessed in a classical 4-h 51 Cr-release assay in 96 well plates.
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, product ref. ACC 725
  • C1 R-MICA cells are labelled with 51 Cr, then mixed with KHYG-transfected with hCD16 at an effector/target ratio equal to 20, in the presence of antibody at indicated concentrations and of 10 pg/ml F(ab’) 2 ON72 antibody (Beckman Coulter Inc.) to block any NKG2D- mediated cytotoxicity). After brief centrifugation and 4 hours of incubation at 37°C, 50mI_ supernatant are removed, and the 51 Cr release is measured with a TopCount NXT beta detector (PerkinElmer Life Sciences, Boston, MA).
  • a treatment according to the disclosure results in a decrease of soluble (e.g., shed) MICA in circulation in an individual.
  • the treatment results in an increase in NKG2D receptor expression on the surface of immune effector cells (e.g. T cells, NK cells).
  • a treatment according to the disclosure results in an increase in biological activity of effector cells that express NKG2D (e.g. NK and/or T cells), for example as assessed by increase proliferation of cells, pro-inflammatory cytokine production and/or markers of cytotoxicity, etc.
  • NKG2D e.g. NK and/or T cells
  • an anti-MICA composition of the disclosure can advantageously be administered in an amount that achieves a concentration in circulation that is at least the concentration that achieves 50%, 70%, 80% or 90% receptor saturation or occupancy (e.g., as assessed by titrating the anti-MICA composition on MICA-expressing cells, as assessed at e.g. 24 hours, 1 week, 2 weeks, 3 weeks or 1 month upon administration).
  • the exemplary MICA binding protein used in the Examples is antibody 19E1.
  • the bi- valent mean K D (M) at pH 7.4 for binding to MICA polypeptide by biotin-conjugated mouse antibody 19E9 (on MICA * 001-His) was 3.2 * 10 13 M, while the monovalent affinity was 7.8 * 10 1 ° M (for detailed conditions, see Example 2 of WO2013/117647, the disclosure of which is incorporated herein by reference).
  • Other antibodies can also be used similarly, for example antibodies characterized by a binding affinity (K D ), optionally wherein binding affinity is mon- ovalent or bivalent, for a human MICA and/or MICB polypeptide (e.g.
  • Antibody 19E9 is characterized by an EC 50 , as determined by flow cytometry, of less than 2 pg/ml, for binding to C1 R cells made to express at their surface a MICA protein (e.g., to each of a MICA * 001 cell, a MICA * 004 cell, a MICA * 007 cell, and a MICA * 008 cell, as further described herein).
  • Other antibodies can also be used similarly, for example antibodies having an EC 50 , as determined by flow cytometry, for binding to cells made to express at their surface a MICA protein (e.g.
  • C1 R cells for example ATCC® CRL-2369TM made to express MICA as shown in WO2013/1 17647), that is less than (better affinity) that of 19E9, or that is within 1 -log of that of antibody 19E9.
  • Mycoplasma-free HT-29 tumor cell line was obtained from ATCC (cat. HTB-38) and cultured in HEPES-containing RPMI 1640 (ThermoFisher) complemented with 10% FCS (Eurobio), 50 U/mL penicillin, 50 pg/mL streptomycin, 2 mM GlutaMAX and 1 mM Sodium Pyruvate (all from ThermoFisher), thereafter named complete RPMI.
  • PBMCs Peripheral blood mononuclear cells
  • T and NK cells were enriched from HD PBMCs by magnetic cell depletion of B cells and monocytes using anti-CD19 and anti-CD14 microbeads with the MACS technology (Miltenyi), according to the manufacturer procedures.
  • HT29 spheroids were generated by seeding 104 HT29 cells per well on Nunclon SpheraTM (ThermoFisher) or CostarTM ultra-low attachment (Corning) round bottom 96 wells plates in complete RPMI. 5 days later, spheroids contained 3. 104 cells and cocultures were started by adding 3.105 total or CD19-CD14- sorted HD PBMCs per well, together with stimulatory or inhibitory molecules. For flow cytometry analyses, 6 wells per condition were seeded. OUT and IN compartments were isolated by first pooling these 6 cocultures wells in eppendorf tubes. Spheroids were gently resuspended and left to sediment to the bottom of the eppendorf.
  • Anti-MICA and anti-NKG2A antibodies Anti-MICA and anti-NKG2A antibodies.
  • Anti-NKG2A (monalizumab, hlgG4) and anti-MICA/B (19E9 VH and VL domains with Fc domain of hlgG1 isotype with amino acid modifications to increase binding of human CD16A), along with corresponding isotype controls.
  • CD19-CD14- sorted HD PBMCs were stained with CFSE (ThermoFisher) according to manufacturer procedure. Infiltrated spheroids were isolated at 24h and embedded in Tissue-TekTM OCT compound (Sakura). Spheroid blocs were then 20 pm sliced using CM1520 cryostat (Leica), the slides were mounted with DAPI- containing Fluoromount-G (Thermofisher) and imaged at a 5x magnification using an epifluorescence microscope (AxioTM Imager 2, Zeiss). Quantification of CFSE+ cells infiltrating the spheroids was done using the H-K means plugin of the Icy software.
  • CFSE ThermoFisher
  • CRC Colorectal cancer patients
  • TILs tumor-infiltrating lymphocytes
  • the formalin-fixed spheroids were first embedded in HistogelTM (Thermo Scientific) and then in paraffin. Blocks were sliced in 5 pm-thick sections and immunostainings performed on a Discovery Ultra automaton (Ventana). After pre-treatment with cell conditioning 1 (Ventana), sections were incubated 1 hour at 37°C with an anti-MICA/B antibody or anti-HLA-E (clone MEM-E/02, Exbio) primary Abs at 2pg/ml_ and 1 pg/mL, respectively.
  • Anti-mouse IgG detection system (discovery OmniMap anti-mouse HRP, Ventana) was used for HLA-E staining and an additional amplification step using tyramide was used for MICA/B staining (Discovery Amp HQTM kit, Ventana). After revelation with 3,3- diaminobenzidine and counterstaining with hematoxylin, sections were washed, dehydrated, cleared and mounted using a coverslipper (ClearVue, Thermo Scientific). Stained sections were finally scanned on a slide scanner (S60 NanozoomerTM, Hamamatsu).
  • Example 1 T cells and NK cells infiltrate colon cancer cell line-derived spheroids
  • Immunohistochemistry (IHC) analyses showed strong expression of NKG2D ligand MICA/B on tumor cells in both control and cocultured spheroids.
  • CD8 T cells upregulated NKG2D inside the spheroids during anti- MICA/B treatment ( Figure 4C), indicating a possible engagement of NKG2D on CD8 T cells as well.
  • Anti-MICA/B induced a strong upregulation of the early activation marker CD137 on NK cells (Figure 11), advocating for their increased stimulation.
  • the Fc receptor CD16 was strongly downregulated at the NK cell surface ( Figure 1 J), exhibiting the engagement of this receptor during ADCC.
  • NKG2D and NKG2A pathways showed interesting interactions in our system, prompting us to test the efficacy of anti-MICA/B and anti-NKG2A combination.
  • Example 5 MICA expression on cancer cells by IHC in FFPE samples
  • Figure 6 shows MICA expression of cancer cells as a proportion score. It can be seen that each of CRC, HNSCC (head and neck squamous cell carcinoma), kidney tumors, HCC (hepatocellular carcinoma), adrenal tumor, melanoma and pancreatic tumors all showed patients with MICA expression, and all these tumor types had patients with a score of 4 indicating a particularly high number of MICA-expressing cells (>75% MICA positive cells).

Abstract

L'invention concerne des méthodes pour le traitement ou la prévention du cancer faisant appel à des composés qui neutralisent la NKG2A humaine en association avec des composés qui se lient à la MICA humaine.
PCT/EP2019/071045 2018-08-14 2019-08-05 Traitement du cancer colorectal par une association d'un anticorps anti-mica et d'un anticorps anti-nkg2a WO2020035345A1 (fr)

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