Anti-NKG2A Antibodies and Compositions

Abstract

The present invention relates to anti-NKG2A antibodies and methods of using them to enhance immunity and treat cancer in patients in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 195,470, filed June 1, 2021. The disclosure of that priority application is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been filed electronically in ASCII format, and is incorporated herein by reference in its entirety. The electronic copy of the Sequence Listing, created on May 26, 2021, is named 022675_P1065_SL.txt and is 35,631 bytes in size.

[0003] 2. Background of the Invention The NKG2 protein is a C-type lectin receptor that dimerizes with CD94 on the cell surface. NKG2A is an inhibitory family member expressed on natural killer (NK) cells and a subset of CD8+ T cells. Binding of the NKG2A / CD94 heterodimer to the nonclassical MHC class I molecule HLA-E triggers phosphorylation of immunoreceptor tyrosine kinase-based inhibitory motifs (ITIMs) in the cytoplasmic tail of NKG2A, resulting in the transmission of inhibitory signals and the consequent inhibition of NK and T cell activation.

[0004] HLA-E is expressed on the cells of many solid tumors and hematological cancers. By overexpressing HLA-E, cancer cells can enhance inhibitory signaling by the NKG2A / CD94 / HLA-E complex, thus providing protection from the cytotoxic activity of NK and T cells, resulting in poorer patient outcomes.

[0005] Given the role of NKG2A in cancer, new and improved anti-cancer therapies that target NKG2A are needed.

[0006] Summary of the Invention The present disclosure provides anti-NKG2A antibodies that can enhance the activation of NK cells and T cells. The antibodies can be used to stimulate immune responses in patients in need thereof, such as patients with cancer or immunodeficiency. Also provided are pharmaceutical compositions comprising one or more of these antibodies, and the use of the antibodies and pharmaceutical compositions for the treatment of cancer. The antibodies and compositions described herein can be used in a method of treating cancer in a patient; can be used for the manufacture of a medicament for the treatment of cancer in a patient; or can be for use in the treatment of cancer in a patient. Compared to currently available treatments for such cancers, including antibody treatments, it is believed that the antibodies and compositions described herein can provide superior clinical responses, either alone or in combination with another cancer therapeutic agent.

[0007] In some embodiments, the disclosure provides anti-NKG2A antibodies, or antigen-binding portions thereof, that compete or cross-compete for binding to, or bind to, the same epitope on human NKG2A as antibodies 24208, 23765, 23686, 23566, 23925, or 24135. In certain embodiments, the anti-NKG2A antibodies or antigen-binding portions are defined by the six CDRs, the heavy and light chain variable domains, or the heavy and light chain amino acid sequences of the antibody.

[0008] In some embodiments, the disclosure provides an anti-NKG2A antibody, or antigen-binding portion thereof, comprising: a) the heavy chain of the antibody i) heavy chain complementarity determining regions (H-CDRs)-1-3, each comprising the amino acid sequences of SEQ ID NOs: 5-7; ii) a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3; iii) a VH comprising the amino acid sequence of SEQ ID NO:3; or iv) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 61; and b) the light chain of said antibody is i) light chain complementarity determining regions (L-CDRs)-1-3, each comprising the amino acid sequences of SEQ ID NOs: 8 to 10; ii) a light chain variable domain (VL) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4; iii) a VL comprising the amino acid sequence of SEQ ID NO: 4; or iv) A light chain (LC) comprising the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 62.

[0009] In some embodiments, the disclosure provides an anti-NKG2A antibody, or antigen-binding portion thereof, comprising: a) the heavy chain of the antibody i) H-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 15 to 17; ii) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; iii) a VH comprising the amino acid sequence of SEQ ID NO: 13; or iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 61; and b) the light chain of the antibody i) L-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 18 to 20; ii) a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14; iii) a VL comprising the amino acid sequence of SEQ ID NO: 14; or iv) A light chain comprising the amino acid sequences of SEQ ID NOs: 14 and 62.

[0010] In some embodiments, the disclosure provides an anti-NKG2A antibody, or antigen-binding portion thereof, comprising: a) the heavy chain of the antibody i) H-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 25 to 27; ii) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23; iii) a VH comprising the amino acid sequence of SEQ ID NO: 23; or iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and SEQ ID NO: 61; and b) the light chain of the antibody i) L-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 28 to 30; ii) a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 24; iii) a VL comprising the amino acid sequence of SEQ ID NO: 24; or iv) A light chain comprising the amino acid sequences of SEQ ID NOs: 24 and 62.

[0011] In some embodiments, the disclosure provides an anti-NKG2A antibody, or antigen-binding portion thereof, comprising: a) the heavy chain of the antibody i) H-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 35 to 37; ii) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33; iii) a VH comprising the amino acid sequence of SEQ ID NO: 33; or iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and SEQ ID NO: 61; and b) the light chain of the antibody i) L-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 38 to 40; ii) a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 34; iii) a VL comprising the amino acid sequence of SEQ ID NO: 34; or iv) A light chain comprising the amino acid sequences of SEQ ID NOs: 34 and 62.

[0012] In some embodiments, the disclosure provides an anti-NKG2A antibody, or antigen-binding portion thereof, comprising: a) the heavy chain of the antibody i) H-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 45 to 47; ii) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 43; iii) a VH comprising the amino acid sequence of SEQ ID NO: 43; or iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and SEQ ID NO: 61; and b) the light chain of the antibody i) L-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 48 to 50; ii) a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44; iii) a VL comprising the amino acid sequence of SEQ ID NO: 44; or iv) A light chain comprising the amino acid sequences of SEQ ID NOs: 44 and 62.

[0013] In some embodiments, the disclosure provides an anti-NKG2A antibody, or antigen-binding portion thereof, comprising: a) the heavy chain of the antibody i) H-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 55 to 57; ii) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53; iii) a VH comprising the amino acid sequence of SEQ ID NO: 53; or iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and SEQ ID NO: 61; and b) the light chain of the antibody i) L-CDR-1-3, each containing the amino acid sequences of SEQ ID NOs: 58 to 60; ii) a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 54; iii) a VL comprising the amino acid sequence of SEQ ID NO: 54; or iv) A light chain comprising the amino acid sequence of SEQ ID NO: 54 and SEQ ID NO: 62.

[0014] The present disclosure also provides isolated nucleic acid molecules, vectors, and host cells comprising nucleotide sequences encoding the heavy chain or antigen-binding portion thereof, the light chain or antigen-binding portion thereof, or both, of the anti-NKG2A antibodies or antigen-binding portions thereof described herein. Additionally, the present disclosure provides methods for producing the anti-NKG2A antibodies or antigen-binding portions thereof described herein by culturing the host cells, as well as methods for producing antibody compositions by mixing the antibodies or antigen-binding portions thereof described herein.

[0015] Other features, objects and advantages of the present invention will become apparent in the following detailed description. However, it should be understood that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0016] [Figure 1-1] Figure 1 is a set of graphs showing the binding profiles of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to human and cynomolgus NKG2A and NKG2C expressed on transiently transfected CHO-C cells. Antibody binding was measured by flow cytometry (MFI: mean fluorescence intensity). [Figure 1-2] Figure 1 is a set of graphs showing the binding profiles of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to human and cynomolgus NKG2A and NKG2C expressed on transiently transfected CHO-C cells. Antibody binding was measured by flow cytometry (MFI: mean fluorescence intensity). [Figure 1-3]Figure 1 is a set of graphs showing the binding profiles of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to human and cynomolgus NKG2A and NKG2C expressed on transiently transfected CHO-C cells. Antibody binding was measured by flow cytometry (MFI: mean fluorescence intensity). [Figure 1-4] Figure 1 is a set of graphs showing the binding profiles of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to human and cynomolgus NKG2A and NKG2C expressed on transiently transfected CHO-C cells. Antibody binding was measured by flow cytometry (MFI: mean fluorescence intensity). [Figure 2-1] FIG. 2 is a set of graphs showing the ability of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to block binding of HLA-E to CHO-S cells transiently transfected with human NKG2A (panel A), cynomolgus NKG2A (panel B), human NKG2C (panel C), or cynomolgus NKG2C (panel D). [Figure 2-2] FIG. 2 is a set of graphs showing the ability of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to block binding of HLA-E to CHO-S cells transiently transfected with human NKG2A (panel A), cynomolgus NKG2A (panel B), human NKG2C (panel C), or cynomolgus NKG2C (panel D). [Diagram 3] Figure 3 shows the crystal structure of human NKG2A / CD94 in complex with HLA-E / β2m (Protein Structure Database: 3CDG). HLA-E / β2m and CD94 are shown as ribbons. NKG2A is shown as a surface representation. Key binding epitope residues for specific anti-human NKG2A antibodies (as discussed in Example 6) are highlighted in black as sticks. [Figure 4-1]4 is a set of graphs showing binding of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to the extracellular domain (ECD) of human NKG2A with one or three amino acid substitutions to residues at the corresponding position(s) in human NKG2C. MFI: mean fluorescence intensity; Mock: mock-transfected cells. [Figure 4-2] 4 is a set of graphs showing binding of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to the extracellular domain (ECD) of human NKG2A with one or three amino acid substitutions to residues at the corresponding position(s) in human NKG2C. MFI: mean fluorescence intensity; Mock: mock-transfected cells. [Figure 4-3] 4 is a set of graphs showing binding of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to the extracellular domain (ECD) of human NKG2A with one or three amino acid substitutions to residues at the corresponding position(s) in human NKG2C. MFI: mean fluorescence intensity; Mock: mock-transfected cells. [Figure 4-4] 4 is a set of graphs showing binding of six anti-human NKG2A antibodies, a reference antibody, and a control antibody to the extracellular domain (ECD) of human NKG2A with one or three amino acid substitutions to residues at the corresponding position(s) in human NKG2C. MFI: mean fluorescence intensity; Mock: mock-transfected cells. [Figure 5-1] Figure 5 is a set of graphs showing lysis of K562-HLA-E cells mediated by either NK92 cells (panel A) or primary NK cells (panel B) after incubation with a series of anti-human NKG2A antibodies. Data are presented as mean ± SEM, n=3. Correlation of the data presented in panels A and B is shown in panel C. The dotted line indicates the level of lysis in samples not treated with antibody ("untreated") or treated with a monalizumab analog. [Figure 5-2]Figure 5 is a set of graphs showing lysis of K562-HLA-E cells mediated by either NK92 cells (panel A) or primary NK cells (panel B) after incubation with a series of anti-human NKG2A antibodies. Data are presented as mean ± SEM, n=3. Correlation of the data presented in panels A and B is shown in panel C. The dotted line indicates the level of lysis in samples not treated with antibody ("untreated") or treated with a monalizumab analog. [Figure 6] 6 is a pair of graphs showing lysis of K562-HLA-E cells mediated by NK-2 cells after incubation with the indicated anti-NKG2A monoclonal antibodies, a reference antibody, an IgG4 control ("control") antibody, and an IgG1-LALA control antibody. Data are normalized to the untreated control in panel A. [Figure 7] 7 is a pair of graphs showing lysis of K562-HLA-E cells mediated by primary NK cells after incubation with the indicated anti-NKG2A monoclonal antibodies, a reference antibody, an IgG4 control ("control") antibody, and an IgG1-LALA control antibody. Data are normalized to untreated controls. [Figure 8] Figure 8 is a graph showing lysis of K562-HLA-E cells mediated by γδ T cells after incubation with the indicated anti-NKG2A antibodies, a reference antibody, and a control antibody ("Control"). Data are normalized to untreated controls. [Figure 9] Figure 9 is a pair of graphs showing the EC50 (50% effective concentration) (top) and efficacy (bottom) of primary human NK cell-mediated killing of K562-HLA-E cancer cells after incubation with the indicated anti-NKG2A antibodies. EC50 values ​​as well as efficacy data were extracted from the NK cell killing assays and summarized for comparison. [Figure 10] FIG. 10 is a pair of graphs showing the expression of endogenous HLA-E on the surface of six different tumor cell lines (Panel A) and the effect of antibody 24208 on NK-mediated killing of these six tumor cell lines (Panel B). [Figure 11] FIG. 11 is a pair of graphs showing the concentration of MIP (macrophage inflammatory protein)-1β upon co-culture of primary human NK cells treated with antibody 24208 or IgG1LALA with A549HLAE trimer cells (top) or JIMT-1HLAE trimer cells (bottom).

[0017] Detailed Description of the Invention The present disclosure provides novel antagonistic anti-human NKG2A antibodies that can be used to inhibit NKG2A activity in patients, such as cancer patients. Unless otherwise specified, "NKG2A" as used herein refers to human NKG2A. The human NKG2A polypeptide sequence is available at UnipProt Accession No. P26715 (NKG2A_HUMAN) (SEQ ID NO: 63), as shown below: [ka]

[0018] As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer containing two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FRs). Each VH and VL is composed of three CDRs (H-CDR herein refers to the CDR of the heavy chain, and L-CDR herein refers to the CDR of the light chain) and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers for FR and CDR regions within a heavy or light chain may be according to the IMGT® definition (Eu numbering; Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)), or according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); or Honegger and Pluckthun, J. Mol. Biol. 309(3):657-70 (2001).

[0019] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line that contains a nucleotide sequence(s) encoding an antibody, where the nucleotide sequence(s) is not naturally associated within the cell.

[0020] The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, by reason of its origin or source of derivation, (1) is not associated with naturally associated components with which it is naturally associated, (2) is free from other proteins from the same species, (3) is expressed by cells from a different species, and / or (4) is not naturally occurring. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell of its natural origin will be "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques that are well known in the art.

[0021] The term "affinity" refers to a measure of the attraction between an antigen and an antibody. The intrinsic attraction of an antibody for an antigen is typically measured by the binding affinity equilibrium constant (K D ) The antibody has a binding K D is said to specifically bind to an antigen when its K is 1 μM or less, e.g., 100 nM or less, or 10 nM or less. D Binding affinity constants can be measured by surface plasmon resonance (Biacore™) using, for example, an IBIS MX96 SPR system from IBIS Technologies or a Carterra LSA SPR platform, or by biolayer interferometry using, for example, an Octet™ system from ForteBio.

[0022] The term "epitope" as used herein refers to a portion (determinant) of an antigen that specifically binds to an antibody or related molecule, such as a bispecific binding molecule. Epitope determinants generally consist of chemically active surface molecular groups, such as amino acids or sugar or sugar side chains, and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be "linear" or "conformational". In linear epitopes, all of the interaction points between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur in a line along the primary amino acid sequence of the protein. In conformational epitopes, the interaction points occur between amino acid residues on the protein that are separated from each other within the primary amino acid sequence. Once a desired epitope on an antigen has been determined, it is possible to generate antibodies against that epitope using techniques well known in the art. For example, antibodies against linear epitopes can be generated, for example, by immunizing animals with peptides that have amino acid residues of the linear epitope. Antibodies to a conformational epitope can be generated, for example, by immunizing an animal with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies to a particular epitope can also be generated, for example, by immunizing an animal with a target molecule of interest (e.g., NKG2A) or a relevant portion thereof, followed by screening for binding to the epitope.

[0023] It can be determined whether an antibody binds to the same epitope as an anti-NKG2A antibody of the present disclosure or competes for binding with it by using methods known in the art, including but not limited to competitive assays, epitope binning, and alanine scanning. In some embodiments, the anti-NKG2A antibody of the present disclosure is allowed to bind to NKG2A under saturating conditions, and then the ability of the test antibody to bind to NKG2A is measured. If the test antibody can bind to NKG2A simultaneously with the reference anti-NKG2A antibody, the test antibody binds to a different epitope than the reference anti-NKG2A antibody. However, if the test antibody cannot bind to NKG2A simultaneously, the test antibody binds to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope that binds to the anti-NKG2A antibody of the present disclosure. This experiment can be performed, for example, using ELISA, radioimmunoassay, Biacore™, surface plasmon resonance, Biolayer interferometry, or flow cytometry. To test whether an anti-NKG2A antibody cross-competes with another anti-NKG2A antibody, the above competition method can be used in two ways, i.e., to determine whether a known antibody blocks a test antibody and vice versa. Such cross-competition experiments can be performed, for example, using an IBIS MX96 or Carterra LSA surface plasmon resonance instrument or an Octet™ system.

[0024] The term "human antibody" refers to an antibody in which the sequences of the variable domain and constant region are derived from human sequences. The term encompasses antibodies having sequences derived from human genes, but which have been modified, for example, to reduce immunogenicity, increase affinity, and / or increase stability. In addition, the term encompasses antibodies recombinantly produced in non-human cells, which may confer glycosylation that is not typical of human cells. The term also encompasses antibodies produced in transgenic non-human organisms (e.g., Omni Rat® rats) that have human antibody genes.

[0025] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human NKG2A or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" include: (i) Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment: a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of the VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be recombinantly linked by a synthetic linker that allows them to be made as a single protein chain, where the VL and VH domains pair to form a monovalent molecule (also known as single chain Fv (scFv)). Within the present disclosure are also antigen-binding molecules that include VH and / or VL. In the case of VH, the molecule may also include one or more of the CH1, hinge, CH2, or CH3 regions. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies are also encompassed, such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, creating two antigen-binding sites.

[0026] Antibody portions, such as Fab fragments and F(ab')2 fragments, can be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies. Moreover, antibodies, antibody portions, and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, such as those described herein.

[0027] The class (isotype) and subclass of an anti-NKG2A antibody can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA or Western blot, as well as other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant regions of the heavy and / or light chains of the antibody and comparing their amino acid sequences to known amino acid sequences of various classes and subclasses of immunoglobulins to determine the class and subclass of the antibody.

[0028] Unless otherwise specified, numbering of amino acid residues of all antibodies referred to in this disclosure is under the IMGT® numbering scheme (EU numbering).

[0029] Anti-NKG2A antibody The present disclosure provides antibodies and antigen-binding portions thereof directed against NKG2A. In certain aspects, the antibodies disclosed herein are human antibodies generated from transgenic animals (e.g., rats) capable of producing antibodies encoded by rearranged human antibody genes. In certain embodiments, the human antibodies may contain specific mutations, e.g., reverting primer-derived mutations to germline sequences (see, e.g., "simplex-corrected" mutant sequences in Table 1).

[0030] In some embodiments, the anti-NKG2A antibodies of the present disclosure have "LALA" mutations (L234A / L235A) in the Fc region. These mutations prevent the antibody from binding to human FcγR (Fc gamma receptor). Such antibodies are advantageous because they have low levels of secondary effector function and therefore do not deplete or target effector T cells to other non-malignant tumors.

[0031] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has the following binding domains for binding to human NKG2A: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 61, and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 62; b) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 13 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 14 and 62; c) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 23 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 24 and 62; d) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 33 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 34 and 62; e) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 43 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 44 and 62; or f) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 53 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 54 and 62 or binds to the same epitope of human NKG2A as an antibody comprising said antibody.

[0032] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a heavy chain CDR3 (H-CDR3) amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, or 57.

[0033] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a heavy chain CDR1-3 (H-CDR1-3) comprising the amino acid sequence of SEQ ID NO:5-7, 15-17, 25-27, 35-37, 45-47, or 55-57, respectively.

[0034] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a heavy chain variable domain (VH) amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, or 53.

[0035] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a VH comprising the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, or 53.

[0036] In some embodiments, the anti-NKG2A antibody has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3, 13, 23, 33, 43, or 53, and a heavy chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:61.

[0037] In some embodiments, the anti-NKG2A antibody comprises a VH amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, or 53, and a heavy chain constant region amino acid sequence of the amino acid sequence of SEQ ID NO:61.

[0038] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a light chain CDR3 (L-CDR3) amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, or 60.

[0039] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a light chain CDR1-3 (L-CDR1-3) comprising the amino acid sequence of SEQ ID NO: 8-10, 18-20, 28-30, 38-40, 48-50, or 58-60, respectively.

[0040] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a light chain variable domain (VL) amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, or 54.

[0041] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a VL comprising the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, or 54.

[0042] In some embodiments, the anti-NKG2A antibody has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4, 14, 24, 34, 44, or 54; and a light chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:62.

[0043] In some embodiments, the anti-NKG2A antibody comprises a VL amino acid sequence of SEQ ID NO:4, 14, 24, 34, 44, or 54, and a light chain constant region amino acid sequence of SEQ ID NO:62.

[0044] In certain embodiments, the anti-NKG2A antibody comprises any one of the heavy chains described above and any one of the light chains described above.

[0045] In some embodiments, the anti-NKG2A antibody or antigen-binding portion of the disclosure comprises: a) SEQ ID NOs: 5 to 10, respectively; b) SEQ ID NOs: 15 to 20, respectively; c) SEQ ID NOs: 25 to 30, respectively; d) SEQ ID NOs: 35 to 40, respectively; e) SEQ ID NOs: 45 to 50, respectively; or f) SEQ ID NOs: 55 to 60, respectively It includes the amino acid sequences of H-CDR1-3 and L-CDR1-3.

[0046] In some embodiments, the anti-NKG2A antibody or antigen-binding portion of the disclosure comprises: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; or f) SEQ ID NOs: 53 and 54, respectively In some embodiments, the VH and VL are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., 90% identical) to the amino acid sequence of

[0047] In some embodiments, the anti-NKG2A antibody or antigen-binding portion of the disclosure comprises: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; or f) SEQ ID NOs: 53 and 54, respectively The VH and VL comprise the amino acid sequence of

[0048] In some embodiments, the anti-NKG2A antibodies of the disclosure: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 61, and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 62; b) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 13 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 14 and 62; c) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 23 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 24 and 62; d) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 33 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 34 and 62; e) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 43 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 44 and 62; or f) a heavy chain comprising the amino acid sequences of SEQ ID NOs: 53 and 61 and a light chain comprising the amino acid sequences of SEQ ID NOs: 54 and 62 Includes.

[0049] The disclosure also provides anti-NKG2A antibodies, or antigen-binding portions thereof, that compete or cross-compete for binding with, or bind to the same epitope as, antibodies 24208, 23765, 23686, 23566, 23925, or 24135.

[0050] In some embodiments, an anti-NKG2A antibody or antigen-binding portion of the disclosure comprises the amino acid sequence of H-CDR1-3 and L-CDR1-3 of antibody 24208, 23765, 23686, 23566, 23925, or 24135.

[0051] In some embodiments, an anti-NKG2A antibody or antigen-binding portion of the disclosure comprises a VH and VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the VH and VL of antibodies 24208, 23765, 23686, 23566, 23925, or 24135, respectively.

[0052] In some embodiments, an anti-NKG2A antibody or antigen-binding portion of the disclosure comprises a VH and a VL that are the VH and VL of antibody 24208, 23765, 23686, 23566, 23925, or 24135, respectively.

[0053] In some embodiments, the anti-NKG2A antibody of the disclosure is antibody 24208, 23765, 23686, 23566, 23925, or 24135, or an antibody having the same amino acid sequence as said antibody.

[0054] The class of the anti-NKG2A antibody obtained by the methods described herein may be changed or switched to another class or subclass. In some embodiments of the present disclosure, the nucleic acid molecule encoding the VL or VH is isolated using methods well known in the art, and thus does not contain a nucleic acid sequence encoding a CL or CH, respectively. The nucleic acid molecule encoding the VL or VH is then operably linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class of immunoglobulin molecule. This may be achieved using a vector or nucleic acid molecule that includes a CL or CH sequence as described above. For example, an anti-NKG2A antibody that was originally IgM may be class switched to an IgG. Furthermore, class switching may be used to convert one IgG subclass to another subclass, for example from IgG1 to IgG2. The κ light chain constant region may be changed to, for example, a γ light chain constant region, or vice versa. An exemplary method for producing an antibody of the present disclosure having a desired Ig isotype includes isolating a nucleic acid molecule encoding a heavy chain of an anti-NKG2A antibody and a nucleic acid molecule encoding a light chain of an anti-NKG2A antibody, obtaining the variable domain of the heavy chain, ligating the coding sequence of the variable domain of the heavy chain to a coding sequence of a constant region of a heavy chain of the desired isotype, expressing the light and heavy chains encoded by the ligated sequences in cells, and harvesting the anti-NKG2A antibody having the desired isotype.

[0055] The anti-NKG2A antibodies of the disclosure can be IgG, IgM, IgE, IgA, or IgD molecules, but are typically of the IgG subclasses, e.g., IgG1, IgG 2a , or IgG 2bIn some embodiments, the antibody is of the isotype subclass IgG1, IgG2, or IgG4.

[0056] In some embodiments, the anti-NKG2A antibody may comprise at least one mutation in the Fc region. Many different Fc mutations are known, and these mutations alter the effector function of the antibody. For example, in some embodiments, the anti-NKG2A antibody comprises at least one mutation in the Fc region that reduces effector function, such as a mutation at one or more of positions 228, 233, 234, and 235, where the amino acid positions are numbered according to the IMGT® numbering system.

[0057] In some embodiments, for example, when the antibody is of the IgG1 subclass, one or both of the amino acid residues at positions 234 and 235 may be mutated, for example from leucine to alanine (L234A / L235A). These mutations reduce the effector function of the Fc region of IgG1 antibodies. The amino acid positions are numbered according to the IMGT® numbering system.

[0058] In some embodiments, for example, if the antibody is of the IgG4 subclass, it may contain the mutation S228P, where the amino acid positions are numbered according to the IMGT® numbering system, which is known to reduce undesired Fab arm exchange.

[0059] In some embodiments, the anti-NKG2A antibodies or antigen-binding portions of the disclosure are antagonistic.

[0060] In some embodiments, the anti-NKG2A antibody or antigen-binding portion has a K of 15, 10, 7, 5, 4, 3, 2, or 1 nM or less as measured by surface plasmon resonance. D In some embodiments, the anti-NKG2A antibody or antigen-binding portion binds to human NKG2C at a KD K at least 40, 30, 20, 10, or 5 times lower than D and binds to human NGK2A.

[0061] In some embodiments, the anti-NKG2A antibody or antigen-binding portion does not specifically bind or binds very weakly to human NKG2C, cynomolgus NKG2A, cynomolgus NKG2C, or any combination thereof. In some embodiments, the anti-NKG2A antibody or antigen-binding portion specifically binds to human NKG2C, cynomolgus NKG2A, cynomolgus NKG2C, or any combination thereof.

[0062] In some embodiments, the anti-NKG2A antibody or antigen-binding portion binds to human NKG2A expressed on CHO-S cells at a concentration of, e.g., 30 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, or 0.5 ng / mL or less.

[0063] In some embodiments, the anti-NKG2A antibody or antigen-binding portion blocks binding of HLA-E to human NKG2A / CD94 heterodimers expressed on CHO-S cells, e.g., at concentrations of 10 μg / mL, 3 μg / mL, 1 μg / mL, 0.4 μg / mL, 0.1 μg / mL, 40 ng / mL, 14 ng / mL or less.

[0064] In some embodiments, the anti-NKG2A antibody or antigen binding portion enhances NK-92 cell-mediated killing of K562 cells transfected with HLA-E at concentrations of, for example, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.1 μg / mL, 1.7 μg / mL, 0.85 μg / mL, or 0.41 μg / mL or less. In certain embodiments, for example, at one of the concentrations, killing is enhanced by 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500% or more compared to untreated cells. In certain embodiments, the anti-NKG2A antibody or antigen binding portion enhances NK-92 cell-mediated killing of K562 cells transfected with HLA-E more efficiently than monalizumab.

[0065] In some embodiments, the anti-NKG2A antibody or antigen-binding portion enhances primary NK cell-mediated killing of K562 cells transfected with HLA-E at concentrations of, e.g., 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.1 μg / mL, 1.7 μg / mL, 0.85 μg / mL, or 0.41 μg / mL or less. In certain embodiments, e.g., at one of the concentrations, killing is enhanced by 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500% or more compared to untreated cells. In certain embodiments, the anti-NKG2A antibody or antigen-binding portion enhances primary NK cell-mediated killing of K562 cells transfected with HLA-E more efficiently than monalizumab, BMS-NKG2A.9 and / or BMS-NKG2A.11.

[0066] In some embodiments, the anti-NKG2A antibody or antigen-binding portion enhances γδ T cell-mediated killing of K562 cells transfected with HLA-E at concentrations of, e.g., 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.1 μg / mL, 1.7 μg / mL, 0.85 μg / mL, or 0.41 μg / mL or less. In certain embodiments, for example, at one of the concentrations, killing is enhanced by 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500% or more compared to untreated cells. In certain embodiments, the anti-NKG2A antibody or antigen-binding portion enhances γδ T cell-mediated killing of K562 cells transfected with HLA-E more efficiently than monalizumab.

[0067] In some embodiments, the binding of the anti-NKG2A antibody or antigen-binding portion to human NKG2A depends on residue S170 (optionally in combination with S167 and / or I168) or residue E197 of human NKG2A.

[0068] In some embodiments, the anti-NKG2A antibody or antigen-binding portion binds to an epitope on human NKG2A that includes amino acid residue S170. In certain embodiments, the epitope further includes amino acid residues S167 and 1168. In some embodiments, the epitope (which may include amino acid residues S167, 1168, and / or S170) does not include amino acid residues E179 and / or M189.

[0069] The present disclosure also contemplates anti-NKG2A antibodies or antigen-binding portions having any combination of the above properties.

[0070] In some embodiments, the anti-NKG2A antibodies or antigen-binding portions described herein have at least one (e.g., one, two, three, four, five, six, seven, eight, or all nine) of the following properties: a) a K of 15 nM or less as measured by surface plasmon resonance D and binds to human NKG2A; b) binds to human NKG2A expressed on CHO-S cells; c) blocking the binding of HLA-E to human NKG2A / CD94 heterodimers expressed on CHO-S cells; d) enhances NK-92 cell-mediated killing of K562 cells transfected with HLA-E; e) enhancing primary NK cell-mediated killing of K562 cells transfected with HLA-E; f) enhancing γδ T cell-mediated killing of K562 cells transfected with HLA-E; g) binds to a different epitope on human NKG2A than monalizumab; h) enhances primary NK cell-mediated killing of HT-29, CCRF-CEM, A253, Detroit562, CAL-120 and / or FaDu cells; and i) induce secretion of MIP-1β. For example, in certain embodiments, the anti-NKG2A antibodies or antigen-binding portions described herein may have properties a)-f), a)-e), a)-g), a)-i), or a)-e) and g).

[0071] In some embodiments, the anti-NKG2A antibodies or antigen-binding portions described herein may enhance the activity of NK cells and / or T cells in a patient.

[0072] In some embodiments, the anti-NKG2A antibodies or antigen-binding portions described herein may inhibit tumor growth and / or induce regression of tumor growth in vivo. In some embodiments, the anti-NKG2A antibodies or antigen-binding portions described herein may slow or reverse metastasis in cancer patients. In some embodiments, the anti-NKG2A antibodies or antigen-binding portions described herein may prolong survival of cancer patients. Combinations of any of the above properties are also contemplated.

[0073] In certain embodiments, an antibody or antigen-binding portion thereof of the present disclosure may be part of a larger immunoadhesin molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesin molecules include the use of a streptavidin core region to generate tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)) and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidine tags to generate bivalent, biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include the incorporation, either covalently or non-covalently, of one or more CDRs from an antibody into an immunoadhesin molecule that specifically binds an antigen of interest. In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated non-covalently.

[0074] In another aspect, fusion antibodies or immunoadhesins can be made that include all or a portion of the anti-NKG2A antibody of the present disclosure linked to another polypeptide. In certain embodiments, only the variable domain of the anti-NKG2A antibody is linked to the polypeptide. In certain embodiments, the VH domain of the anti-NKG2A antibody is linked to a first polypeptide, while the VL domain of the anti-NKG2A antibody is linked to a second polypeptide that is associated with the first polypeptide such that the VH and VL domains can interact with each other to form an antigen-binding site. In some embodiments, the VH domain is separated from the VL domain by a linker (e.g., a single chain antibody) such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to a polypeptide of interest. Additionally, fusion antibodies can be made in which two (or more) single chain antibodies are linked to each other. This is useful if one wishes to create a bivalent or multivalent antibody on a single polypeptide chain, or if one wishes to create a bispecific antibody.

[0075] To create a single chain antibody (scFv), the DNA fragment encoding the VH and VL is operably linked to another fragment encoding a flexible linker, e.g., the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:64), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains connected by the flexible linker. See, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). Single chain antibodies can be monovalent, where only one VH and VL are used; bivalent, where two VH and VL are used; or multivalent, where more than two VH and VL are used. Bispecific or multivalent antibodies can be made, for example, that specifically bind to human NKG2A and to another molecule.

[0076] In other embodiments, other modified antibodies can be prepared using nucleic acid molecules encoding anti-NKG2A antibodies. For example, "kappabodies" (Ill et al., Protein Eng. 10:949-57 (1997)), "minibodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) can be prepared using standard molecular biology techniques in accordance with the teachings of the specification.

[0077] The anti-NKG2A antibodies or antigen-binding portions of the present disclosure may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or portion thereof is derivatized such that NKG2A binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody portions of the present disclosure are intended to include the human anti-NKG2A antibodies described herein, both in intact or modified form. For example, the antibodies or antibody portions of the present disclosure may be functionally linked (by chemical bond, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0078] A type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those that are homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available, for example, from Pierce Chemical Co., Rockford, Ill.

[0079] The anti-NKG2A antibodies or antigen-binding portions thereof may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups, which may be useful to improve the biological characteristics of the antibody, e.g., to increase serum half-life.

[0080] The antibody or antigen-binding portion according to the present disclosure may also be labeled. As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into the antibody. In some embodiments, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide that can be detected by a marked avidin (e.g., streptavidin containing a fluorescent marker, or an enzymatic activity that can be detected by optical or colorimetric methods). In some embodiments, the label or marker is a therapeutic agent, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanides, phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents, e.g., gadolinium chelators, toxins, e.g., pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0081] In some embodiments, the antibodies or antigen-binding portions described herein may be conjugated to a cytotoxic agent to form an immunoconjugate. In some embodiments, the antibodies or antigen-binding portions described herein may be conjugated to a radioisotope.

[0082] In certain embodiments, the antibodies of the present disclosure may exist in neutral form (including zwitterionic forms) or as positively or negatively charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharma- ceutically acceptable salt.

[0083] Anti-NKG2A antibody composition The present disclosure also provides combination therapies (e.g., compositions) comprising one, two, three, four, or more of the anti-NKG2A antibodies or antigen-binding portions thereof described herein. In certain embodiments, the combination therapy (e.g., composition) comprises two anti-NKG2A antibodies or antigen-binding portions. The combination therapy may take the form of, for example, a method of treatment using the antibodies or antigen-binding portions, or a pharmaceutical composition comprising the antibodies or antigen-binding portions.

[0084] In some embodiments, the disclosure provides a composition comprising a first anti-NKG2A antibody, or antigen-binding portion thereof, and a second anti-NKG2A antibody, or antigen-binding portion thereof, wherein the first and second antibodies are: - antibodies 23765 and 23686, respectively; - antibodies 23765 and 24208, respectively; - antibodies 23765 and 23566, respectively; - antibodies 23765 and 23925, respectively; - antibodies 23765 and 24135, respectively; - antibodies 23686 and 24208, respectively; - antibodies 24686 and 23566, respectively; - antibodies 23686 and 23925, respectively; - antibodies 23686 and 24135, respectively; - antibodies 24208 and 23566, respectively; - antibodies 24208 and 23925, respectively; - antibodies 24208 and 24135, respectively; - antibodies 23566 and 23925, respectively; - antibodies 23566 and 24135, respectively; or - antibodies 23925 and 24135, respectively.

[0085] In some embodiments, the composition comprises an antibody or antigen-binding portion thereof that binds to the same epitope as the first and second antibodies or competes for binding with the first and second antibodies.

[0086] In some embodiments, the composition comprises an antibody or antigen-binding portion thereof comprising the H-CDR1-3 amino acid sequence and the L-CDR1-3 amino acid sequence of the first antibody, and an antibody or antigen-binding portion thereof comprising the H-CDR1-3 amino acid sequence and the L-CDR1-3 amino acid sequence of the second antibody.

[0087] In some embodiments, the composition comprises an antibody, or antigen-binding portion thereof, comprising a VH and a VL having amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH amino acid sequence and the VL amino acid sequence, respectively, of the first antibody, and an antibody, or antigen-binding portion thereof, comprising a VH and a VL having amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH amino acid sequence and the VL amino acid sequence, respectively, of the second antibody.

[0088] In some embodiments, the composition comprises an antibody, or antigen-binding portion thereof, that comprises the VH amino acid sequence and the VL amino acid sequence of the first antibody, and an antibody, or antigen-binding portion thereof, that comprises the VH amino acid sequence and the VL amino acid sequence of the second antibody.

[0089] In some embodiments, the composition comprises an antibody, or antigen-binding portion thereof, that comprises the HC amino acid sequence and the LC amino acid sequence of the first antibody, and an antibody, or antigen-binding portion thereof, that comprises the HC amino acid sequence and the LC amino acid sequence of the second antibody.

[0090] In certain embodiments, the composition comprises: a) an antibody comprising H-CDR1-3, each of which comprises the amino acid sequence of SEQ ID NO: 5 to 7, 15 to 17, 25 to 27, 35 to 37, 45 to 47, or 55 to 57; b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, or 53; c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, or 53; d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 3 and 61, 13 and 61, 23 and 61, 33 and 61, 43 and 61, or 53 and 61; e) an antibody comprising L-CDR1-3, each of which comprises an amino acid sequence of SEQ ID NO: 8 to 10, 18 to 20, 28 to 30, 38 to 40, 48 to 50, or 58 to 60; f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, or 54; g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, or 54; h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 4 and 62, 14 and 62, 24 and 62, 34 and 62, 44 and 62, or 54 and 62; i) an antibody in which the H-CDR1-3 and the L-CDR1-3 comprise the amino acid sequences of SEQ ID NOs: 5 to 10, 15 to 20, 25 to 30, 35 to 40, 45 to 50, or 55 to 60, respectively; j) an antibody comprising a VH and a VL that comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of amino acid sequences 3 and 4, 13 and 14, 23 and 24, 33 and 34, 43 and 44, or 53 and 54, respectively; k) an antibody comprising a VH and a VL comprising the amino acid sequences 3 and 4, 13 and 14, 23 and 24, 33 and 34, 43 and 44, or 53 and 54, respectively; and l) an antibody comprising an HC and an LC comprising the amino acid sequences of 3 and 61, and 4 and 62; 13 and 61, and 14 and 62; 23 and 61, and 24 and 62; 33 and 61, and 34 and 62; 43 and 61, and 44 and 62; or 53 and 61, and 54 and 62. The antibody may comprise one, two or more antibodies or antigen-binding portions thereof selected from the group consisting of:

[0091] In some embodiments, the anti-NKG2A antibody compositions described herein may inhibit tumor growth and / or induce tumor regression in vivo. In some embodiments, the anti-NKG2A antibody compositions described herein may slow or reverse metastasis in cancer patients. In some embodiments, the anti-NKG2A antibody compositions described herein may extend survival of cancer patients.

[0092] The present disclosure also provides a method for producing an anti-NKG2A antibody composition described herein, comprising the steps of providing a first anti-NKG2A antibody or antigen-binding portion and a second anti-NKG2A antibody or antigen-binding portion, and mixing the two antibodies or portions.

[0093] bispecific binding molecules The present disclosure also provides bispecific binding molecules having the binding specificity of an anti-NKG2A antibody described herein (e.g., comprising the six CDRs of an antigen-binding portion, e.g., VH and VL). In some embodiments, the bispecific binding molecule further has binding specificity for another distinct anti-NKG2A antibody (e.g., another anti-NKG2A antibody described herein), or an antibody whose activity targets a different protein, such as a cancer antigen or another cell surface molecule, whose activity mediates a disease condition, such as cancer. Such bispecific binding molecules are known in the art, and examples of various types of bispecific binding molecules are provided elsewhere herein.

[0094] Nucleic Acid Molecules and Vectors The present disclosure also provides nucleic acid molecules and sequences encoding the anti-NKG2A antibodies or antigen-binding portions thereof described herein. In some embodiments, different nucleic acid molecules encode the amino acid sequences of the heavy and light chains of the anti-NKG2A antibodies or antigen-binding portions. In other embodiments, the same nucleic acid molecule encodes the amino acid sequences of the heavy and light chains of the anti-NKG2A antibodies or antigen-binding portions.

[0095] Reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand with its complementary sequence. The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or any type of nucleotide in modified form. The term includes single-stranded or double-stranded forms.

[0096] In some embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof, or a nucleotide sequence encoding a light chain or an antigen-binding portion thereof, or both, of an anti-NKG2A antibody, or antigen-binding portion thereof, described herein.

[0097] The present disclosure also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to one or more of the nucleotide sequences listed herein, for example, to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 51, and 52, or to a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 13, 14, 23, 24, 33, 34, 43, 44, 53, and 54. The term "sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison can be over a span of at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. Many different algorithms are known in the art that can be used to measure percent identity of nucleotide sequences. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs in the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, including, for example, the programs FASTA2 and FASTA3, provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences (see, e.g., Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); and Pearson, J. Mol. Biol. 276:71-84 (1998); incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used.For example, percent sequence identity between nucleic acid sequences can be determined using FASTA and its default parameters (word size 6 and NOPAM counting for the scoring matrix), or using Gap and its default parameters as provided in GCG version 6.1, incorporated herein by reference.

[0098] In some embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 51 and 52. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NOs: 1 and 2, 11 and 12, 21 and 22, 31 and 32, 41 and 42, or 51 and 52.

[0099] In any of the above embodiments, the nucleic acid molecule may be isolated. A nucleic acid molecule referred to herein as "isolated" or "purified" is a nucleic acid that is (1) separated from the genomic DNA or cellular RNA nucleic acid from which it is obtained; and / or (2) is not naturally occurring.

[0100] In a further aspect, the disclosure provides vectors suitable for expressing one or both chains of an antibody or antigen-binding portion thereof as described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, a vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0101] The present disclosure provides vectors comprising a nucleic acid molecule encoding the heavy chain, the light chain, or both the heavy and light chains of an anti-NKG2A antibody or an antigen-binding portion thereof as described herein. In certain embodiments, the vectors of the present disclosure comprise a nucleic acid molecule as described herein. The present disclosure further provides vectors comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and a probe thereof. The vector may further comprise an expression control sequence.

[0102] The term "expression control sequence" as used herein refers to polynucleotide sequences required to cause expression and processing of ligated coding sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize mRNA in the cytoplasm; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; in eukaryotes, such control sequences generally include promoter sequences and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential for expression and processing; and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0103] In some embodiments, a nucleic acid molecule as described herein comprises a nucleotide sequence encoding a VH domain derived from an anti-NKG2A antibody or antigen-binding portion as described herein joined in-frame to a nucleotide sequence encoding a heavy chain constant region derived from any source. Similarly, a nucleic acid molecule as described herein can comprise a nucleotide sequence encoding a VL domain derived from an anti-NKG2A antibody or antigen-binding portion as described herein joined in-frame to a nucleotide sequence encoding a light chain constant region derived from any source.

[0104] In further aspects of the disclosure, nucleic acid molecules encoding VH and / or VL can be "converted" into full-length antibody genes. In some embodiments, nucleic acid molecules encoding VH or VL domains are converted into full-length antibody genes by insertion into an expression vector already encoding a heavy chain constant (CH) region or a light chain constant (CL) region, respectively, such that the VH segment is operably linked to the CH segment(s) in the vector and / or the VL segment is operably linked to the CL segment in the vector. In another aspect, nucleic acid molecules encoding VH and / or VL domains are converted into full-length antibody genes using standard molecular biology techniques, e.g., by linking the nucleic acid molecules encoding the VH and / or VL domains to nucleic acid molecules encoding the CH and / or CL regions. The nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed from a cell into which they have been introduced and anti-NKG2A antibodies can be isolated.

[0105] In some embodiments, the framework region(s) are mutated such that the resulting framework region(s) have the amino acid sequence of the corresponding germline gene. Mutations can be made in the framework region or constant region, for example, to extend the half-life of the anti-NKG2A antibody. See, for example, PCT Publication No. WO00 / 09560. Mutations in the framework region or constant region can be made to alter the immunogenicity of the antibody and / or to provide a site for covalent or non-covalent binding to another molecule. According to the present disclosure, the antibody can have mutations in one or more CDR regions or framework regions of any of the variable domains, or in the constant region.

[0106] Host Cells and Methods for Producing Antibodies and Antibody Compositions The present disclosure also provides antibody compositions and methods for producing antibodies and antigen-binding portions thereof described herein. In some embodiments, the present disclosure relates to a method for producing an anti-NKG2A antibody or antigen-binding portion thereof as described herein, comprising the steps of: providing a host cell (e.g., a recombinant host cell) that contains a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof, and a nucleotide sequence encoding a light chain or antigen-binding portion thereof, of an anti-NKG2A antibody or antigen-binding portion described herein; culturing the host cell under conditions suitable for expression of the antibody or antigen-binding portion; and isolating the resulting antibody or antigen-binding portion. The antibody or antigen-binding portion produced by such expression in such a recombinant host cell is referred to herein as a "recombinant" antibody or antigen-binding portion. The present disclosure also provides progeny cells of such host cells, as well as the antibodies or antigen-binding portions produced thereby.

[0107] The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. By definition, a recombinant host cell does not exist in nature. The present disclosure provides host cells, which may comprise a vector, e.g., as described herein. The present disclosure also provides host cells, which comprise, e.g., a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof, a nucleotide sequence encoding a light chain or antigen-binding portion thereof, or both, of an anti-NKG2A antibody or antigen-binding portion thereof described herein. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still within the scope of the term "host cell" as used herein.

[0108] Nucleic acid molecules encoding anti-NKG2A antibodies and antigen-binding portions thereof, as well as vectors containing these nucleic acid molecules, can be used for transfection of suitable mammalian, plant, bacterial or yeast host cells. Transformation can be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) into liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

[0109] Antibodies expressed by different cell lines or in transgenic animals will likely have different glycosylation patterns from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present disclosure, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modification(s).

[0110] Pharmaceutical Compositions Another aspect of the present disclosure is a pharmaceutical composition comprising as an active ingredient (or as the only active ingredient) an anti-NKG2A antibody or antigen-binding portion thereof, an antibody composition, or a bispecific binding molecule of the present disclosure. The pharmaceutical composition may further comprise a pharma-ceutical acceptable excipient. In some embodiments, the pharmaceutical composition is intended for the amelioration, prevention, and / or treatment of cancer, such as, for example, the cancers described herein. In certain embodiments, the cancer is in tissues such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas.

[0111] A pharmaceutical composition of the present disclosure will comprise one or more anti-NKG2A antibodies, antigen-binding portions, antibody compositions, or bispecific binding molecules of the present disclosure, such as one or two anti-NKG2A antibodies, antigen-binding portions, or bispecific binding molecules. In some embodiments, the composition comprises a single anti-NKG2A antibody or antigen-binding portion thereof of the present disclosure. In another aspect, the composition comprises two separate anti-NKG2A antibodies or antigen-binding portions thereof of the present disclosure.

[0112] In some embodiments, a pharmaceutical composition may comprise at least one anti-NKG2A antibody or antigen-binding portion thereof of the present disclosure, e.g., an anti-NKG2A antibody or portion, as well as one or more additional antibodies that target one or more associated cell surface receptors, e.g., one or more cancer-associated receptors.

[0113] In some embodiments, a pharmaceutical composition may comprise at least one anti-NKG2A antibody or antigen-binding portion thereof of the present disclosure, e.g., an anti-NKG2A antibody or portion, and one or more additional agents selected from, e.g., an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, and a tyrosine kinase inhibitor.

[0114] Generally, the antibodies, antigen-binding portions, and bispecific binding molecules of the disclosure are suitable for administration as a formulation in association with one or more pharma- ceutically acceptable excipient(s), e.g., as described below.

[0115] The term "excipient" is used herein to describe any ingredient other than the compound(s) of the present disclosure. The choice of excipient(s) will depend largely on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and similar substances that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include an isotonicity agent, such as a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffers that enhance the shelf life or effectiveness of the antibody.

[0116] The pharmaceutical compositions of the present disclosure and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably manufactured under GMP (Good Manufacturing Practice) conditions.

[0117] The pharmaceutical composition of the present disclosure may be prepared, packaged, or sold in bulk as a single unit dose or as a number of single unit doses.As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dose of active ingredient that would be administered to a subject, or a convenient fraction of such a dose, such as a half or a third of such a dose.

[0118] A formulation of a pharmaceutical composition suitable for parenteral administration typically comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffering agents (preferably pH 3-9), although in some applications they may be more suitably formulated as sterile nonaqueous solutions or as dry forms for use in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. Other parenterally administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form or in a liposomal preparation.

[0119] Therapeutic Uses of Antibodies and Compositions of the Disclosure In some embodiments, the anti-NKG2A antibodies and antigen-binding portions thereof, anti-NKG2A antibody compositions, and bispecific binding molecules of the present disclosure are used to enhance or activate the immune system in a patient (e.g., a mammal, such as a human) in need thereof, for example, by enhancing the activity of NKG2A-positive NK cells or T cells. In certain embodiments, the patient is immunosuppressed. In certain embodiments, a physician can boost the anti-cancer activity of the patient's own immune system by administering an anti-NKG2A antibody or antigen-binding portion thereof, composition, or bispecific binding molecule as described herein. For example, a physician can boost the anti-tumor activity in a patient by administering an anti-NKG2A antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure, alone or in combination (sequentially or simultaneously) with other therapeutic agents.

[0120] In certain embodiments, the antibodies or antigen-binding portions thereof, compositions, or bispecific binding molecules of the present disclosure are for use in the treatment of cancer, which may be present in one or more tissues, such as the skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas.

[0121] In some embodiments, cancers that may be treated by the anti-NKG2A antibodies, antigen-binding portions, compositions, and bispecific binding molecules of the disclosure include, for example, melanoma (e.g., advanced or metastatic melanoma), basal cell skin cancer, glioblastoma, glioma, gliosarcoma, astrocytoma, meningioma, neuroblastoma, adrenocortical carcinoma, squamous cell carcinoma of the head and neck, oral cancer, salivary gland cancer, nasopharyngeal cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer, and squamous cell lung cancer), esophageal cancer, esophagogastric junction cancer, gastric cancer, gastrointestinal cancer, primary peritoneal cancer, liver cancer, hepatocellular carcinoma, biliary tract cancer, colon cancer, rectal cancer, colorectal cancer, ovarian cancer, fallopian tube cancer, bladder cancer, upper urinary tract cancer, urothelial cancer, These may include renal cell carcinoma, kidney cancer, genitourinary tract cancer, cervical cancer, prostate cancer, fibrosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, histiocytoma, pancreatic cancer, uterine cancer, appendix cancer, advanced Merkel cell carcinoma, multiple myeloma, sarcoma, choriocarcinoma, erythroleukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, mast cell leukemia, small lymphocytic lymphoma, Burkitt lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, monocytic lymphoma, HTLV-associated T-cell leukemia / lymphoma, mesothelioma, and solid tumors. The cancer may be, for example, early stage, intermediate stage, late stage, locally advanced, or metastatic, and may be recurrent or refractory to other therapies (e.g., other anti-NKG2A therapeutics, or checkpoint inhibitors), or for which no standard treatment is available.

[0122] In some embodiments, conditions treated by the anti-NKG2A antibodies, antigen-binding portions, compositions, and bispecific binding molecules of the disclosure may include, for example, head and neck cancer, breast cancer (e.g., HER2-positive breast cancer), colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndromes, multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, myeloproliferative neoplasms, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

[0123] In some embodiments, the antibodies or antigen-binding portions, compositions, or bispecific binding molecules of the disclosure are for use in treating an immune disorder.

[0124] In some embodiments, the antibodies or antigen-binding portions, compositions, or bispecific binding molecules may be used to treat patients who are or are at risk of being immunocompromised (e.g., due to chemotherapy or radiation therapy). In some embodiments, the antibodies or antigen-binding portions, compositions, or bispecific binding molecules may be used to expand stem cells following stem cell transplantation in a patient.

[0125] In some embodiments, the antibody or antigen-binding portion, composition, or bispecific binding molecule is for use in treating viral and / or parasitic infections (e.g., where a pathogen suppresses the host immune response). The pathogen can be, for example, an immunodeficiency virus, hepatitis (types A, B, or C), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-cell lymphotropic virus (HTLV), human cytomegalovirus (HCMV), dengue virus, molluscum contagiosum virus, poliovirus, rabies virus, John Cunningham (JC) virus, arboviral encephalitis virus, simian immunodeficiency virus (SIV), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, or Pseudomonas aeruginosa.

[0126] "Treat," "treating," and "treatment" refer to a method of alleviating or eliminating a biological disorder and / or one of its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include references to curative treatment, symptomatic treatment, and prophylactic treatment.

[0127] "Therapeutically effective amount" refers to that amount of a therapeutic agent administered that will relieve to some extent one or more of the symptoms of the disorder being treated. A therapeutically effective amount of an anti-cancer therapeutic agent may result, for example, in delayed tumor growth, tumor shrinkage, extended survival, elimination of cancer cells, slowed or reduced disease progression, reversal of metastasis, or other clinical endpoint desired by a health care professional.

[0128] The anti-NKG2A antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules described herein can be administered alone or in combination with one or more other drugs or antibodies (or in any combination thereof). Thus, the pharmaceutical compositions, methods and uses described herein also encompass embodiments in combination (co-administration) with other active agents, as detailed below.

[0129] As used herein, with reference to the anti-NKG2A antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the present disclosure together with one or more other therapeutic agents, the terms "co-administered," "co-administered," and "in combination with" are intended to mean, refer to, and include the following: a) simultaneous administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the disclosure and a therapeutic agent(s) to a patient in need of treatment, where such components, when formulated together into a single dosage form, release said components to said patient substantially simultaneously; b) substantially simultaneous administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the disclosure and a therapeutic agent(s) to a patient in need of treatment, where such components are formulated apart from one another into separate dosage forms which are taken by said patient at substantially the same time, whereupon said components are released to said patient at substantially the same time; c) sequential administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent(s) to a patient in need of treatment, where such components are formulated separately from one another into separate dosage forms which are taken sequentially by the patient with a significant time between administrations, whereby the components are released to the patient at substantially different times; and d) Sequential administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent(s) to a patient in need of treatment, where such components are formulated together into a single dosage form that releases the components in a controlled manner whereby they are released to the patient at the same and / or different times, simultaneously, sequentially, and / or overlappingly, where each portion may be administered either by the same route or by different routes.

[0130] The anti-NKG2A antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be administered without additional therapeutic treatment, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with the anti-NKG2A antibodies or antigen-binding portions thereof, antibody compositions, or bispecific molecules of the present disclosure may include at least one additional therapeutic treatment (combination therapy), such as another immunostimulant, anti-cancer agent (e.g., a chemotherapeutic agent, antineoplastic agent, an anti-angiogenic agent, or a tyrosine kinase inhibitor), or a vaccine (e.g., a tumor vaccine).

[0131] In some embodiments, the antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule may be co-administered or formulated with another medicament / drug for the treatment of cancer. The additional therapeutic treatment may include, for example, an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, and / or radiation therapy. In some embodiments, the additional therapeutic treatment may include a different anti-cancer antibody.

[0132] Pharmaceutical preparations comprising an anti-NKG2A antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule described herein and at least one other agent (e.g., a chemotherapeutic agent, an anti-neoplastic agent, or an anti-angiogenic agent) may be used as a combination treatment for simultaneous, separate, or sequential administration in the treatment of cancer. The other agent may be any agent suitable for treating the particular cancer in question, for example an agent selected from the group consisting of alkylating agents, such as platinum derivatives, for example cisplatin, carboplatin, and / or oxaliplatin; plant alkaloids, for example paclitaxel, docetaxel, and / or irinotecan; antitumor antibiotics, for example doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; topoisomerase inhibitors, for example topotecan; antimetabolites, for example fluorouracil and / or other fluorinated pyrimidines; FOLFOX; osimertinib; cyclophosphamide; anthracyclines; dacarbazine; gemcitabine; or any combination thereof. In some embodiments, the anti-NKG2A antibodies, or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules described herein re-establish responsiveness to other agents.

[0133] The anti-NKG2A antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be used in combination with other anti-cancer therapies, such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T-cell therapy. In the case of a vaccine, it may be, for example, a protein, peptide, or DNA vaccine containing one or more antigens relevant to the cancer being treated, or a vaccine containing dendritic cells together with the antigen. Suitable cytokines include, for example, IL-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor. One example of a class of enzyme inhibitors with anti-cancer activity is an indoleamine-2,3-dioxygenase (IDO) inhibitor, such as 1-methyl-D-tryptophan (1-D-MT). Also contemplated is adoptive T-cell therapy, which refers to a variety of immunotherapy techniques that involve expanding or engineering a patient's own T cells to recognize and attack the patient's tumor.

[0134] It is also contemplated that the anti-NKG2A antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be used in adjunctive therapy with tyrosine kinase inhibitors, which are synthetic, primarily quinazoline-derived, low molecular weight molecules that interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation, e.g., by competing with the intracellular Mg-ATP binding site.

[0135] In some embodiments, the antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule is selected from the group consisting of A2AR, A1AR, A2BR, A3AR, ADA, ALP, AXL, BTLA, B7-H3, B7-H4, CTLA-4, CD116, CD123, CD27, CD28, CD39, CD40, CD47, CD55, CD73, CD122, CD137, CD160, CGEN-15049, CHK1, CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and / or CEACAM-5), EGFR, FLT3, NKG2AL, GAL9, GITR, HVEM, LA The present invention may be used in combination with medicines / drugs that mediate immune system activation, including, but not limited to, agents that modulate the expression or activity of G-3, LILRB1, LY108, LAIR1, MET, ICOS, IDO, IL2R, IL4R, KIR, LAIR1, PAP, PD-1 / PD-L1 / PD-L2, OX40, STING, TIGIT, TIM-3, TGFR-β, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10, TNFR2, VEGFR, VEGF, VISTA, LILRB2, CMTM6 and / or 2B4. In certain embodiments, the agent is a small molecule inhibitor. In certain embodiments, the agent is an antibody or an antigen-binding fragment thereof that binds to one of the above molecules. The anti-NKG2A antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the disclosure may also be used in combination with cytokines (e.g., IL-1, IL-2, IL-12, IL-15, or IL-21), epidermal growth factor receptor inhibitors, vascular endothelial growth factor inhibitors, and the like.

[0136] In some embodiments, the antibody or antigen binding molecule thereof, antibody composition, or bispecific binding molecule may be used in combination with trastuzumab, pembrolizumab, nivolumab, durvalumab, ibrutinib, cetuximab, avelumab, rituximab, margetuximab, tafasitamab, bevacizumab, trastuzumab, deruxtecan, trifluridine, tipiracil, trifluridine / tipiracil, irinotecan, gemcitabine, oxaliplatin, bendamustine, FOLFOX (mFOLFOX6), or any combination thereof.

[0137] In certain embodiments, the antibodies or antigen binding molecules thereof, antibody compositions, or bispecific binding molecules may be used in combination with an agent that modulates CD94 expression or activity, an agent that modulates HLA-E expression or activity, or both.

[0138] The present disclosure also contemplates the use of the sequences of the anti-NKG2A antibodies or antigen-binding portions described herein (e.g., the six CDR sequences or the VH and VL sequences) in the preparation of chimeric antigen receptors, which may be for use in CAR-T technology.

[0139] It is understood that the antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the present disclosure can be used in methods of treatment as described herein, can be for use in treatments as described herein, and / or can be for use in the manufacture of a medicament for treatments as described herein.

[0140] Dose and Route of Administration The antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be administered in an amount effective for treating the condition in question, i.e., at the dosage and for the period of time needed to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the specific condition being treated, the age, sex, and weight of the patient, and whether the antibody is administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments.

[0141] The dosage regimen may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit forms. As used herein, dosage unit forms refer to physically discrete units suitable as unitary doses for the patient / subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The details of dosage unit forms of the present disclosure are generally dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of hypersensitivity in individuals.

[0142] Thus, one skilled in the art will understand, based on the disclosure provided herein, that the dose and dosage regimen will be adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, and the effective amount that provides a detectable therapeutic benefit to the patient can be determined, as can the time requirements for administering each agent that provides a detectable therapeutic benefit to the patient. Thus, although specific doses and dosage regimens are exemplified herein, these examples do not in any way limit the dose and dosage regimen that may be provided to a patient in practicing the present disclosure.

[0143] It is noted that the dosage values ​​may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that the specific dosage regimen for any particular subject should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the embodied compositions. Furthermore, dosage regimens including compositions of the present disclosure may be based on a wide variety of factors, including the type of disease, the patient's age, weight, sex, health status, severity of the condition, route of administration, and the specific antibody used. Thus, dosage regimens may vary widely, but can be routinely determined using standard methods. For example, the dose may be adjusted based on pharmacodynamic or pharmacokinetic parameters, which may include clinical effects such as toxic effects and / or clinical laboratory values. Thus, the present disclosure encompasses intrapatient dose escalation as determined by those skilled in the art. It will be appreciated that the determination of appropriate dosages and regimens is well known in the relevant art and is within the scope of those of ordinary skill in the art once armed with the teachings disclosed herein.

[0144] An effective amount of tumor therapy may be measured by its ability to stabilize disease progression and / or ameliorate symptoms in a patient, preferably reversing disease progression, for example by reducing tumor size. The ability of an antibody, antigen-binding portion, antibody composition, or bispecific binding molecule of the present disclosure to inhibit cancer may be evaluated by in vitro assays, for example as described in the Examples, as well as in appropriate animal models predicting efficacy in human tumors. In each particular situation, for example administered as a single bolus or administered as a continuous infusion, an appropriate dosage regimen will be selected to provide the optimal therapeutic response, with possible dosage adjustments as dictated by the exigencies of each case.

[0145] The antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure may be administered by any method accepted in the art for administration of peptides, proteins, or antibodies, which are typically suitable for parenteral administration. As used herein, "parenteral administration" includes any route of administration characterized by administration through physical breaks in the subject's tissue and breaks in the tissue, generally resulting in administration into the bloodstream, intramuscularly, or directly into an internal organ. Thus, parenteral administration includes, but is not limited to, administration by injection, application through a surgical incision, application through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is considered to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intracapsular, intravenous, intraarterial, intrathecal, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion. Particular embodiments include intravenous and subcutaneous routes.

[0146] Diagnostic Uses and Compositions The antibodies and antigen-binding moieties of the present disclosure are also useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies and antigen-binding moieties can be used to detect and / or measure NKG2A levels in a sample from a patient (e.g., a tissue sample, or a bodily fluid sample, such as inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistochemistry. The present disclosure further encompasses kits (e.g., diagnostic kits) that include the antibodies and antigen-binding moieties described herein.

[0147] Products and Kits The present disclosure also provides articles of manufacture, e.g., kits, including one or more containers (e.g., single-use or multi-use containers) containing a pharmaceutical composition of an anti-NKG2A antibody or antigen-binding portion thereof, composition, or bispecific binding molecule described herein, optionally an additional biologically active molecule (e.g., another therapeutic agent), and instructions for use. The antibody or antigen-binding portion, composition, or bispecific binding molecule, and optionally an additional biologically active molecule, may be packaged separately in suitable packaging, such as vials or ampoules made from non-reactive glass or plastic. In certain embodiments, the vials or ampoules hold concentrated stocks (e.g., 2x, 5x, 10x, or more) of the antibody or antigen-binding portion, composition, or bispecific binding molecule, and optionally the biologically active molecule. In certain embodiments, articles of manufacture, such as kits, include medical devices (e.g., syringes and needles) for administration of the antibody or antigen-binding portion, composition, or bispecific binding molecule, and / or the biologically active molecule; and / or suitable diluents (e.g., sterile water and normal saline). The present disclosure also includes methods for producing the above-mentioned articles.

[0148] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure will have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In case of conflict, the present specification (including definitions) will control.

[0149] Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein.

[0150] Further, unless otherwise required by context, singular terms include plural terms and plural terms include the singular terms. Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0151] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0152] In order that this disclosure may be better understood, the following examples are set forth, which are for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0153] Working Example Example 1. Cloning of anti-NKG2A antibodies from rat B cells Materials and Methods Antibodies against human NKG2A were isolated from the antibody repertoire derived from OmniRat® rats, a transgenic rat line from Ligand Pharmaceuticals that produces antibodies with fully human idiotypes (Osborn et al., J Immunol. 190(4):1481-90 (2013)). Cloning of rat-derived antibody genes from single-cell sorted antibody-secreting B cells (ASC) was performed by Simplex™ antibody discovery technology (Meijer et al., J Mol Biol 358(3):764-72 (2006)).

[0154] Antibody repertoire constructs encoding fully human immunoglobulins in the IgG1-LALA format (see below) were transfected into HEK293 cells. Cell supernatants were screened for binding to NKG2A expressed on the surface of CHO cells using flow cytometry in a high-throughput format. NKG2A-reactive clones were analyzed by DNA sequencing and the DNA sequences encoding the antibodies were extracted. Selected antibody clones were expressed and functionally tested as described below.

[0155] Missense mutations in the amino termini of the heavy and light chains introduced by the use of degenerate primers during Simplex™ cloning of the antibody-encoding cDNA fragments were corrected back to germline sequences. Table 1 shows the nucleotide sequences of the heavy and light chain variable domains of the germline antibodies designated 23765, 23686, 24208, 23566, 23925 and 24135. The correction process included correction of the amino terminal sequences to germline as well as optimization of codon usage. Targets for matching to human germline sequences were identified by blast homology searches of the heavy and light chain variable regions.

[0156] The protein sequences of the variable domains, constant regions and complementarity determining regions (CDRs) of antibodies 24208, 23765, 23686, 23566, 23925 and 24135 are shown in Tables 2, 3 and 4, respectively.

[0157] result Table 1 shows the nucleotide sequences encoding the variable domains of antibodies 24208, 23765, 23686, 23566, 23925, and 24135.

[0158] [Table 1] TIFF2024520577000003.tif231165

[0159] Table 2 shows the deduced amino acid sequences of antibodies 24208, 23765, 23686, 23566, 23925 and 24135. The CDRs are in bold / underlined.

[0160] [Table 2]

[0161] Table 3 shows the amino acid sequences of the heavy and light chain constant regions (CH and CL, respectively). "IgG1LALA" refers to the presence of "LALA" mutations (L234A / L235A, numbered according to the IMGT® numbering system) in the heavy chain, which are known to reduce the effector function of the Fc region of IgG1 antibodies (Hezareh et al., J Virol. 75(24):12161-68 (2001); Hessell et al., Nature 449(7158):101-04 (2007)).

[0162] [Table 3]

[0163] Table 4 shows the heavy and light chain CDR amino acid sequences of antibodies 24208, 23765, 23686, 23566, 23925 and 24135, where the CDRs are defined according to the IMGT® system.

[0164] [Table 4]

[0165] Table 5 shows sequence information for antibodies 24208, 23765, 23686, 23566, 23925 and 24135. Unless otherwise stated, sequences are amino acid sequences.

[0166] [Table 5]

[0167] Example 2. Cloning of anti-NKG2A reference antibody analogs This example lists the sources of amino acid sequences and final antibody formats used for the generation of anti-NKG2A reference antibody analogs.

[0168] Materials and Methods The amino acid sequences of the heavy and light chain variable domains of the antibody analogs in Table 6 were obtained from the listed patent publications. The protein sequences were back translated into DNA sequences using human codon usage. The corresponding DNA sequences were gene synthesized and cloned into expression vectors containing human heavy or light chain constant domains, resulting in the expression of full-length antibodies. The human antibody isotypes selected for expression are listed in the antibody format column, along with additional mutations introduced within the Fc region. CHO cells were transfected with the resulting expression plasmids using a standard protein expression system. Antibody supernatants were purified using standard Protein A purification column chromatography.

[0169] [Table 6]

[0170] Example 3. Measurement of antibody binding kinetics to human NKG2A / CD94 and NKG2C / CD94 heterodimers This example measures the binding of anti-NKG2A antibodies to recombinant human NKG2A / CD94 and NKG2C / CD94 heterodimers (extracellular domains) as measured by surface plasmon resonance (SPR).

[0171] Materials and Methods Kinetic binding analysis of anti-NKG2A monoclonal antibodies (mAbs) was performed by surface plasmon resonance (SPR) using a continuous flow microspotter (CFM, Wasatch Microfluidics, Salt Lake City, USA) in combination with an IBIS MX96SPR instrument (IBIS Technologies, The Netherlands). Human histidine-tagged NKG2A, NKG2C and FLAG-tagged extracellular domains (ECDs) of CD94 were co-expressed using the Expi293™ expression system and purified by standard Ni-NTA chromatography followed by anti-FlagM2 affinity gel (A2220, Sigma). Binding kinetics were measured by capturing monoclonal antibodies on a Ga-hu-IgG Fc SensEye® for 15 min using a CFM. After spotting, the SensEye® was placed on an IBIS MX96 biosensor and the immobilized monoclonal antibody was fixed by SensEye Fixlt kit. Kinetic analysis was performed by applying a series of kinetic titrations in which increasing concentrations of antigen from 1.6 nM to 400 nM were injected in two-fold dilutions. After each cycle of antigen injection, the surface was regenerated with 100 mM H3PO4 (pH 3). Antigen binding and dissociation were measured for 15 min. The recorded binding response was expressed as the binding rate constant (k on or k a ), dissociation rate constant (k off or k d ) and affinity (K D ) The data were fitted to a simple 1:1 binding model using Scrubber 2.0 software for the calculation of constants. Binding kinetic parameters were determined as the average of three independent measurement points.

[0172] result The binding affinity and kinetic parameters of anti-NKG2A monoclonal antibodies binding to NKG2A / CD94 and NKG2C / CD94 heterodimers are shown in Table 7. Binding affinity (K D) were 30-50 times stronger than their binding affinity to NKG2C / CD94. Monoclonal antibody 23686 had similar binding affinity to NKG2A / CD94 and NKG2C / CD94. None of the monoclonal antibodies bound to CD94 homodimers (data not shown).

[0173] In summary, anti-NKG2A monoclonal antibodies 24208, 23925, 23566 and 24135 have high binding specificity for NKG2A compared to NKG2C. Monoclonal antibody 23686 binds to NKG2A and NKG2C with similar affinity.

[0174] [Table 7]

[0175] Example 4. In vitro binding of anti-human NKG2A antibodies to CHO-S cells transiently transfected with human or cynomolgus NKG2A and NKG2C This example describes the in vitro binding of six anti-human NKG2A monoclonal antibodies to human and cynomolgus monkey NKG2A and NKG2C receptors expressed on transiently transfected CHO-S cells. Reference antibody analogs and isotype controls were included for comparison.

[0176] Materials and Methods CHO-S cells were transiently transfected with constructs for human NKG2A and human CD94, human NKG2C and human CD94, cynomolgus NKG2A and cynomolgus CD94, or cynomolgus NKG2C and cynomolgus CD94. Mock-transfected CHO-S cells were included in the assay as negative controls. Antibodies were tested for binding to the transfected cells at 3-fold dilutions from 30 μg / mL to 0.5 ng / mL antibody and at 0 μg / mL antibody and incubated for 30 min at 4° C. After a washing step, cells were incubated with fluorescent goat anti-human IgG (heavy and light chain) secondary antibody (A-21445, Invitrogen) for 30 min at 4° C. in the dark. After a second wash, cells were examined by high-throughput flow cytometry (IQue screener, Sartorius). Data was transferred to Excel and graphs were generated using Graph Pad Prism® software.

[0177] result FIG. 1 shows dose-response curves of antibodies binding to human or cynomolgus NKG2A and NKG2C expressed on transiently transfected CHO-S cells. All antibodies bound strongly and dose-dependently to human NKG2A. Antibody 23686 bound to all four different constructs. Four antibodies, namely 23566, 23925, 24135 and 24208, showed very weak binding to human NKG2C. Antibody 23765 bound to cynomolgus NKG2A but only weakly to human and cynomolgus NKG2C. Monalizumab analogs bound to both cynomolgus NKG2A and cynomolgus NKG2C.

[0178] Example 5. In vitro blocking of HLA-E binding to NKG2A and NKG2C by anti-human NKG2A antibodies This example describes in vitro blocking of HLA-E binding to human and cynomolgus monkey NKG2A and NKG2C transiently expressed on CHO-S cells by six anti-human NKG2A monoclonal antibodies. Reference antibody analogs and isotype controls were included for comparison.

[0179] Materials and Methods CHO-S cells were transiently transfected with constructs for the expression of human NKG2A and human CD94, human NKG2C and human CD94, cynomolgus NKG2A and cynomolgus CD94, or cynomolgus NKG2C and cynomolgus CD94. Mock-transfected CHO-S cells were included in the assay as negative controls. PE (phycoerythrin)-conjugated HLA-E pentamers were tested for binding to the heterodimers in the presence of anti-human NKG2A antibodies at 3-fold serial dilutions from 10 μg / mL to 14 ng / mL and 0 μg / mL of antibody. Antibodies were allowed to bind for 30 min at 4° C., after which HLA-E pentamers were added at a dilution of 1:50. Fluorescence intensity of HLA-E binding to transfected cells was measured by high-throughput flow cytometry (IQue screener, Sartorius). Data was transferred to Excel and graphs were generated using Graph Pad Prism® software.

[0180] result As shown in Figure 2, all antibodies dose-dependently inhibited HLA-E binding to human NKG2A (panel A). Only 23686 and the monalizumab analog (IgG4) completely blocked HLA-E binding to cynomolgus monkey NKG2A (panel B). HLA-E binding to human NKG2C was blocked by 23686 and less potently by 23566 (panel C). Only the monalizumab analog (IgG4) and 23686 completely blocked HLA-E binding to cynomolgus monkey NKG2C (panel D).

[0181] Example 6. Binding of anti-human NKG2A antibodies to NKG2A epitopes This example illustrates the in vitro binding of anti-human NKG2A monoclonal antibodies to different NKG2A / CD94 heterodimers in which one residue from the human NKG2A extracellular domain is replaced with a residue from human NKG2C at the corresponding position. Six different constructs encoding heterodimers with one amino acid substitution and one construct encoding a heterodimer with three amino acid substitutions were transiently transfected into CHO-S cells, and mock-transfected CHO-S cells served as negative controls. Reference antibody analogs and isotype control antibodies were included for comparison.

[0182] Materials and Methods CHO-S cells were transiently transfected with nucleic acid constructs encoding human NKG2A and human CD94 extracellular domains with one of the following NKG2A amino acid substitutions: S167A, I168S, S170L, M189I, E197K, I225M, or the triple substitutions S167A+I168S+S170L (corresponding to residues on the receptor that are part of the binding site for HLA-E) (Kaiser et al., Proc Natl Acad Sci USA (2008) 105(18):6696-701). Figure 3 shows the four amino acid substitutions highlighted on the crystal structure of the human NKG2A / CD94 heterodimer in complex with HLA-E (Protein Structure Database: 3CDG).

[0183] Six anti-human NKG2A antibodies were tested for binding to the various expressed heterodimers. Antibodies were tested for binding to transfected cells at 3-fold serial dilutions from 1 μg / mL antibody and 0 μg / mL antibody and incubated for 30 min at 4° C. After a washing step, cells were incubated with fluorescent goat anti-human IgG (heavy + light chain) secondary antibody (A-21445, Invitrogen) for 30 min at 4° C. in the dark. After a second wash, cells were examined by high-throughput flow cytometry (IQue Screener, Sartorius). Data were transferred to Excel and graphs were generated using Graph Pad Prism® software. Reference antibody analogs and isotype controls were included for comparison.

[0184] result As shown in Figure 4, four antibodies (23566, 23925, 24135, and 24208) showed reduced binding to heterodimers with NKG2A S170L substitutions as well as to heterodimers with NKG2A triple substitutions (S167A, I168S, S170L), indicating that for these four antibodies, binding to human NKG2A is dependent on at least the S170 residue. The reduction in binding to 23925 and 24208 was greater for the triple substitution than for the single S170L substitution, indicating an additive effect of the simultaneous mutations S167A and I168S. Antibody 23765 and the monalizumab analog showed reduced binding to heterodimers with NKG2A E197K substitutions, demonstrating that glutamic acid at position 197 is required for proper binding of these two antibodies to human NKG2A. Monalizumab also showed reduced binding to the heterodimer with the M189I substitution, indicating some influence of the methionine at position 189.

[0185] Example 7. Screening of anti-NKG2A antibodies in NK cell-mediated killing of K562-HLA-E cells A panel of anti-NKG2A antibodies was evaluated for their ability to enhance killing of HLA-E-transfected K562 chronic myeloid leukemia cells (K562-HLA-E) by the NK cell line NK-92 or by primary NK cells derived from healthy donors.

[0186] Materials and Methods K562-HLAE cells express HLA-B * NK-92 cells were loaded with 0701 peptide and depleted of IL-2 overnight. The next day, NK cells were isolated from healthy donors and incubated with anti-NKG2A antibody at a concentration of 25 μg / mL with NK-92 or primary NK cells, after which calcein-loaded K562-HLA-E target cells were added and incubated for 90 min. The killing capacity of NK-92 or primary NK cells was measured by the release of calcein into the supernatant. Specific lysis was subtracted from spontaneous lysis (calcein-loaded 562-HLA-E cells only) and normalized to maximum lysis (lysis of calcein-loaded K562-HLA-E cells by Triton X-100).

[0187] result Figure 5 shows the specific lysis of K562-HLA-E cells after treatment with a series of anti-NKG2A antibodies. It is clear that the levels of lysis after treatment with the various anti-NKG2A antibodies strongly varied, indicating that some antibodies had no function in this assay, while others strongly enhanced the killing of K562-HLA-E cells to levels higher than the monalizumab analog (IgG4) (Panels A and B). A positive correlation was observed between the levels of killing using NK-92 cells and primary NK cells (Panel C).

[0188] Example 8. Functional activity of anti-human NKG2A antibodies in NK-92 cell-mediated killing of K562-HLA-E cells This example describes the in vitro functional evaluation of several anti-NKG2A antibodies (24208, 23925, 23566, 23765, 23686) with the aim of demonstrating dose-dependent antagonist activity. The antibodies were evaluated for their ability to enhance NK-92 cell-mediated killing of K562 cells transfected with HLA-E (K562-HLA-E). A monalizumab analog (IgG4) was included for comparison.

[0189] Materials and Methods K562-HLA-E cells express HLA-B * NK-92 cells were loaded with 0701 peptide and depleted of IL-2 overnight. The next day, NK-92 cells and anti-NKG2A antibodies were incubated with 2-fold titrations of the indicated antibodies starting at 25 μg / mL, after which calcein-loaded K562-HLA-E target cells were added and incubated for 90 min. The killing capacity of NK-92 cells was measured by the release of calcein into the supernatant. Specific lysis was subtracted from spontaneous lysis (calcein-loaded 562-HLAE cells only) and normalized to maximum lysis (lysis of calcein-loaded 562-HLAE cells by Triton X-100).

[0190] result Killing of K562-HLA-E cells upon treatment with anti-NKG2A antibodies is shown in Figure 6. Several anti-NKG2A antibodies showed superior activity compared to monalizumab analogs.

[0191] Example 9. Functional activity of anti-human NKG2A antibodies in primary NK cell-mediated killing of K562-HLA-E cells This example describes the in vitro functional evaluation of several anti-NKG2A antibodies (24208, 23925, 23566, 23765, 23686) with the aim of demonstrating dose-dependent antagonist activity. The antibodies were evaluated for their ability to enhance primary NK cell-mediated killing of K562 cells transfected with HLA-E (K562-HLA-E). A monalizumab analog (IgG4) was included for comparison.

[0192] Materials and Methods K562-HLA-E cells express HLA-B * Primary NK cells were isolated from fresh peripheral blood mononuclear cells derived from healthy donors, loaded with 0701 peptide. The next day, primary NK cells and anti-NKG2A antibodies were incubated with two-fold titrations of the indicated antibodies starting at 25 μg / mL, after which calcein-loaded K562-HLA-E target cells were added (E(effector):T(target) ratio of 1:10) and incubated for 90 min. The killing capacity of primary NK cells was measured by the release of calcein into the supernatant. Specific lysis was subtracted from spontaneous lysis (calcein-loaded 562-HLA-E cells only) and normalized to maximum lysis (lysis of calcein-loaded 562-HLA-E cells by Triton X-100).

[0193] result Killing of K562-HLA-E cells upon treatment with anti-NKG2A antibodies is shown in Figure 7. Several anti-NKG2A antibodies showed superior activity compared to the monalizumab analog (IgG4).

[0194] Example 10 Functional activity of anti-human NKG2A antibodies in gamma delta T cell-mediated killing of K562-HLA-E cells This example describes the in vitro functional evaluation of selected anti-NKG2A antibodies with the aim of demonstrating dose-dependent antagonist activity. Antibodies were evaluated for their ability to enhance γδ T cell-mediated killing of K562 cells transfected with HLA-E (K562-HLA-E). A monalizumab analog (IgG4) was included for comparison.

[0195] Materials and Methods K562-HLA-E cells express HLA-B * 0701 peptide was loaded and NKG2A-positive γδ T cells were isolated. The next day, NKG2A-positive γδ T cells and anti-NKG2A antibodies were incubated with 2-fold titrations of the indicated antibodies starting at 25 μg / mL, after which calcein-loaded K562-HLA-E target cells were added and incubated for 3 h. The killing capacity of γδ T cells was measured by calcein release into the supernatant. Specific lysis was subtracted from spontaneous lysis (calcein-loaded 562-HLA-E cells only) and normalized to maximum lysis (lysis of calcein-loaded 562-HLAE cells by Triton X-100).

[0196] result Killing of K562-HLA-E cells upon treatment with anti-NKG2A antibodies is shown in Figure 8. Several anti-NKG2A antibodies showed superior activity compared to monalizumab analogs.

[0197] Example 11. EC50 and Efficacy of Cancer Cell Killing This example describes the in vitro functional evaluation of anti-NKG2A antibody 24208 to demonstrate cytotoxic activity. The antibodies were evaluated for their ability to enhance primary human NK cell-mediated killing of K562 cells transfected with HLA-E (K562-HLA-E). A monalizumab analog (IgG4) and two BMS analogs, BMS-NKG2A.9 (heavy chain: SEQ ID NO: 65, light chain: SEQ ID NO: 66) and BMS-NKG2A.11 (heavy chain: SEQ ID NO: 65, light chain: SEQ ID NO: 67), were included for comparison.

[0198] Materials and Methods K562-HLA-E cells express HLA-B * Primary human NK cells were loaded with 0701 peptide and depleted of IL2 overnight. The following day, primary human NK cells and anti-NKG2A antibodies were incubated with two-fold titrations of the indicated antibodies starting at 50 μg / mL, after which calcein-loaded K562-HLA-E target cells were added and incubated for 90 min. The killing capacity of primary human NK cells was measured by the release of calcein into the supernatant. Specific lysis was subtracted from spontaneous lysis (calcein-loaded 562-HLA-E cells only) and normalized to maximum lysis (lysis of calcein-loaded 562-HLAE cells by Triton X-100).

[0199] result Killing of K562-HLA-E cells following treatment with anti-NKG2A antibodies is shown in Table 8 and Figure 9. The 24208 anti-NKG2A antibody showed superior activity compared to the monalizumab analogs and the BMS-NKG2A.9 and BMS-NKG2A.11 analogs.

[0200] [Table 8]

[0201] Example 12. NK cell-mediated killing induced by 24208 in selected cell lines expressing endogenous HLA-E This example describes the expression of endogenous HLA-E on the surface of tumor cell lines (HT-29, CCRF-CEM, A253, Detroit562, CAL-120, FaDu) and the effect of 24208 on NK cell-mediated killing of these tumor cell lines in vitro.

[0202] Materials and Methods Expression of endogenous HLA-E in six different cell lines (HT-29, CCRF-CEM, A253, Detroit562, CAL-120, and FaDu) was examined by flow cytometry. Human primary NK cells isolated from healthy individuals were transfected with endogenous HLA-E (HLA-B) at a ratio of 10:1. * Cells were co-cultured with six different calcein-labeled target cells expressing 24208 (loaded with 0701 peptide) and treated with a single concentration of 24208 or isotype control (IgG1LALA). Calcein release was measured after 1.5 hours and specific lysis was calculated.

[0203] result All six human tumor cell lines were shown to express endogenous HLA-E on their surface (Figure 10, Panel A). Treatment with 24208 induced NK-mediated killing of these tumor cell lines compared to treatment with IgG1LALA (Figure 10, Panel B).

[0204] Example 13. Release of MIP-1β into NK cancer cell co-culture medium This example describes the in vitro functional evaluation of anti-NKG2A antibody 24208 with the aim of demonstrating cytotoxic activity. The antibodies were evaluated for their ability to induce secretion of the proinflammatory cytokine macrophage inflammatory protein-1β (MIP-1β) upon co-culture of primary human NKG2A-positive NK cells with either of the cancer cell lines A549 and JIMT-1.

[0205] Materials and Methods Primary human NKG2A positive NK cells were incubated with 2-fold titrations of 24208 or IgG1LALA (starting at 50 μg / mL) and cocultured with stable HLA-E expressing cancer cells transduced with HLA-E trimers. After 48 h of coculture, MIP-1β concentrations in the coculture supernatants were measured by ELISA.

[0206] result The concentration of MIP-1β in the supernatant of co-cultures of primary human NK cells treated with 24208 or IgG1LALA with A549HLAE-trimer cells or JIMT-1HLAE-trimer cells is shown in Figure 11. The 24208 anti-NKG2A antibody showed superior induction of MIP-1β compared to the IgG1LALA control antibody.

Claims

1. a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 61, and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 62; b) a HC comprising the amino acid sequences of SEQ ID NOs: 13 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 62; c) a HC comprising the amino acid sequences of SEQ ID NOs: 23 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 24 and 62; d) a HC comprising the amino acid sequences of SEQ ID NOs: 33 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 34 and 62; e) a HC comprising the amino acid sequences of SEQ ID NOs: 43 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 44 and 62; or f) HC comprising the amino acid sequences of SEQ ID NOs: 53 and 61 and LC comprising the amino acid sequences of SEQ ID NOs: 54 and 62. binds to the same epitope of human NKG2A as an antibody comprising An anti-NKG2A antibody or an antigen-binding portion thereof.

2. a) the heavy chain of the anti-NKG2A antibody is i) heavy chain complementarity determining regions (H-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 5 to 7, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:3; iii) a VH comprising the amino acid sequence of SEQ ID NO:3; or iv) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 61; and b) the light chain of the anti-NKG2A antibody is i) light chain complementarity determining regions (L-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 8 to 10, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:4; iii) a VL comprising the amino acid sequence of SEQ ID NO:4; or iv) a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 62 The anti-NKG2A antibody or antigen-binding portion of claim 1 , comprising:

3. a) the heavy chain of the anti-NKG2A antibody is i) heavy chain complementarity determining regions (H-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 15 to 17, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 13; iii) a VH comprising the amino acid sequence of SEQ ID NO: 13; or iv) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 13 and 61; and b) the light chain of the anti-NKG2A antibody is i) light chain complementarity determining regions (L-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 18 to 20, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 14; iii) a VL comprising the amino acid sequence of SEQ ID NO: 14; or iv) a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 14 and 62 The anti-NKG2A antibody or antigen-binding portion of claim 1 , comprising:

4. a) the heavy chain of the anti-NKG2A antibody is i) heavy chain complementarity determining regions (H-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 25 to 27, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 23; iii) a VH comprising the amino acid sequence of SEQ ID NO: 23; or iv) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 23 and 61; and b) the light chain of the anti-NKG2A antibody is i) light chain complementarity determining regions (L-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 28 to 30, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 24; iii) a VL comprising the amino acid sequence of SEQ ID NO: 24; or iv) a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 24 and 62 The anti-NKG2A antibody or antigen-binding portion of claim 1 , comprising:

5. a) the heavy chain of the anti-NKG2A antibody is i) heavy chain complementarity determining regions (H-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 35 to 37, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 33; iii) a VH comprising the amino acid sequence of SEQ ID NO: 33; or iv) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 33 and 61; and b) the light chain of the anti-NKG2A antibody is i) light chain complementarity determining regions (L-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 38 to 40, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 34; iii) a VL comprising the amino acid sequence of SEQ ID NO: 34; or iv) a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 34 and 62 The anti-NKG2A antibody or antigen-binding portion of claim 1 , comprising:

6. a) the heavy chain of the anti-NKG2A antibody is i) heavy chain complementarity determining regions (H-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 45 to 47, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 43; iii) a VH comprising the amino acid sequence of SEQ ID NO: 43; or iv) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 43 and 61; and b) the light chain of the anti-NKG2A antibody is i) light chain complementarity determining regions (L-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 48 to 50, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 44; iii) a VL comprising the amino acid sequence of SEQ ID NO: 44; or iv) a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 44 and 62 The anti-NKG2A antibody or antigen-binding portion of claim 1 , comprising:

7. a) the heavy chain of the anti-NKG2A antibody is i) heavy chain complementarity determining regions (H-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 55 to 47, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 53; iii) a VH comprising the amino acid sequence of SEQ ID NO: 53; or iv) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 53 and 61; and b) the light chain of the anti-NKG2A antibody is i) light chain complementarity determining regions (L-CDRs)-1 to -3 comprising the amino acid sequences of SEQ ID NOs: 58 to 60, respectively; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 54; iii) a VL comprising the amino acid sequence of SEQ ID NO: 54; or iv) a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 54 and 62 The anti-NKG2A antibody or antigen-binding portion thereof of claim 1 .

8. An anti-NKG2A antibody, or an antigen-binding portion thereof, comprising: a) SEQ ID NOs: 5 to 10, respectively; b) SEQ ID NOs: 15-20, respectively; c) SEQ ID NOs: 25-30, respectively; d) SEQ ID NOs: 35-40, respectively; e) SEQ ID NOs: 45-50, respectively; or f) SEQ ID NOs: 55 to 60, respectively. The amino acid sequence of H-CDR1-3 and L-CDR1-3 of An anti-NKG2A antibody or an antigen-binding portion thereof.

9. The antibody, a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; or f) SEQ ID NOs: 53 and 54, respectively a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence that are at least 90% identical to the amino acid sequence of 9. The anti-NKG2A antibody or antigen-binding portion of claim 8.

10. The antibody, a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 13 and 14, respectively; c) SEQ ID NOs: 23 and 24, respectively; d) SEQ ID NOs: 33 and 34, respectively; e) SEQ ID NOs: 43 and 44, respectively; or f) SEQ ID NOs: 53 and 54, respectively a heavy chain variable domain and a light chain variable domain comprising the amino acid sequence 9. The anti-NKG2A antibody or antigen-binding portion of claim 8.

11. The anti-NKG2A antibody of claim 1 , wherein the antibody is an IgG.

12. The antibody is IgG １ The anti-NKG2A antibody of claim 11,

13. The anti-NKG2A antibody of claim 1 , wherein the antibody comprises at least one mutation in the Fc region.

14. The antibody is IgG １ 2. The anti-NKG2A antibody of claim 1, comprising a mutation at one or more of heavy chain amino acid positions 234 and 235, numbered according to the IMGT numbering system.

15. 15. The anti-NKG2A antibody of claim 14, wherein one or both of the amino acid residues at positions 234 and 235 are mutated from leucine to alanine.

16. a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 3 and 61, and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 4 and 62; b) a HC comprising the amino acid sequences of SEQ ID NOs: 13 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 62; c) a HC comprising the amino acid sequences of SEQ ID NOs: 23 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 24 and 62; d) a HC comprising the amino acid sequences of SEQ ID NOs: 33 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 34 and 62; e) a HC comprising the amino acid sequences of SEQ ID NOs: 43 and 61 and a LC comprising the amino acid sequences of SEQ ID NOs: 44 and 62; or f) HC comprising the amino acid sequences of SEQ ID NOs: 53 and 61 and LC comprising the amino acid sequences of SEQ ID NOs: 54 and 62. An anti-NKG2A antibody comprising:

17. An anti-NKG2A antibody, or an antigen-binding portion thereof, that binds to an epitope on human NKG2A that includes amino acid residue S170.

18. 18. The anti-NKG2A antibody or antigen-binding portion of claim 17, wherein the epitope further comprises amino acid residues S167 and I168.

19. The anti-NKG2A antibody of claim 17 , wherein the epitope does not include amino acid residues E197, M189, or both.

20. the antibody or antigen-binding portion being a) a K of 15 nM or less as measured by surface plasmon resonance Ｄ binds to human NGK2A at b) binds to human NGK2A expressed on CHO-S cells; c) blocks the binding of HLA-E to human NKG2A / CD94 heterodimers expressed on CHO-S cells; d) enhances NK-92 cell-mediated killing of HLA-E-transfected K562 cells; e) enhances primary NK cell-mediated killing of HLA-E-transfected K562 cells; f) enhancing γδ T cell-mediated killing of HLA-E transfected K562 cells; and g) binds to a different epitope on human NKG2A than monalizumab; The anti-NKG2A antibody or antigen-binding portion of claim 1, having at least one property selected from the following:

21. 21. The anti-NKG2A antibody or antigen-binding portion of claim 20, wherein the antibody or antigen-binding portion has at least two, three, four, five, six, or all of said properties.

22. The antibody or antigen-binding portion thereof, a) binds to human NGK2A with a K D of 15 nM or less as measured by surface plasmon resonance; b) binds to human NGK2A expressed on CHO-S cells; c) blocks the binding of HLA-E to human NKG2A / CD94 heterodimers expressed on CHO-S cells; d) enhances NK-92 cell-mediated killing of HLA-E-transfected K562 cells; e) enhances primary NK cell-mediated killing of HLA-E-transfected K562 cells; f) enhancing γδ T cell-mediated killing of HLA-E transfected K562 cells; and g) binds to a different epitope on human NKG2A than monalizumab; 20. The anti-NKG2A antibody or antigen-binding portion of claim 17, having at least one property selected from the group consisting of:

23. The anti-NKG2A antibody or antigen-binding portion of claim 22, wherein the antibody or antigen-binding portion has at least two, three, four, five, six, or all of said properties.

24. A pharmaceutical composition comprising the anti-NKG2A antibody or antigen-binding portion of any one of claims 1 to 23.

25. 25. The pharmaceutical composition of claim 24, further comprising an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, or a tyrosine kinase inhibitor.

26. 24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain, or a nucleotide sequence encoding the light chain, or both, of the anti-NKG2A antibody or antigen-binding portion of any one of claims 1 to 23.

27. A vector comprising the isolated nucleic acid molecule of claim 26 and an expression control sequence.

28. 27. The isolated nucleic acid molecule of claim 26, wherein the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 51, and 52.

29. 30. A vector comprising the isolated nucleic acid molecule of claim 28 and an expression control sequence.

30. A host cell comprising a nucleotide sequence encoding the heavy chain and a nucleotide sequence encoding the light chain of the anti-NKG2A antibody or antigen-binding portion of any one of claims 1 to 21.

31. Providing a host cell according to claim 30; culturing said host cell under conditions suitable for expression of said antibody or portion; and Isolating the resulting antibody or portion.

2. A method for producing an anti-NKG2A antibody or an antigen-binding portion thereof, comprising:

32. A bispecific binding molecule comprising the antigen-binding domains of one or two distinct anti-NKG2A antibodies according to any one of claims 1 to 21.

33. A pharmaceutical composition according to claim 24 for diagnostic use.

34. A pharmaceutical composition as described in claim 24 for enhancing immune activity.

35. The pharmaceutical composition of claim 24 for treating cancer.

36. 36. The pharmaceutical composition of claim 35, wherein the cancer is in a tissue selected from the group consisting of skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas.

37. The pharmaceutical composition of claim 24 for treating head and neck cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer, acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, myeloproliferative neoplasms, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

38. 25. The pharmaceutical composition of claim 24 for use in combination with an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, or radiation therapy.

39. The pharmaceutical composition of claim 24 for treating an immune disorder.

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