Antibodies against hla-e and use thereof

Abstract

Antibodies, or antigen-binding portions thereof, bispecific antibodies, single chain variable regions and chimeric antigen receptors (CARs) against a complex comprising HLA-E and a peptide comprising the amino acid sequence VMAPRTLFL (SEQ ID NO: 10), as well as pharmaceutical compositions comprising same and methods of producing same are provided. Also provided are methods of treating cancer comprising administering the antibodies or compositions of the invention. Methods of treating cancer, combination treatments, and patient selection are also provided.

Description

ANTIBODIES AGAINST HLA-E AND USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 446,966, filed February 20, 2023, the contents of which are all incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (NIBN-P-042-PCT.xml; Size: 37,371 bytes; and Date of Creation: February 08, 2024) is herein incorporated by reference in its entirety.FIELD OF INVENTION

[0003] The present invention is in the field of monoclonal antibodies and modulating the immune response to cancer.BACKGROUND OF THE INVENTION

[0004] Immunotherapy of cancer is an emerging field of research potentially leading to novel treatment approaches of cancer patients. Besides T cells, there are great hopes on exploiting the antitumor activity of natural killer (NK) cells. NK cells are innate immune cells that efficiently recognize and eliminate tumor cells. Their activation is determined by a delicate balance of activating receptors that are counteracted by signals delivered by inhibitory receptors.

[0005] HLA-E is a non-classical HLA class I molecule that is monomorphic in the human population. HLA-E is known to bind peptides derived from the leader sequence of HLA-class I molecules (HLA-A, -B, -C, and -G) and to present them to NK cells through the interaction with the inhibitory receptor CD94 / NKG2A, thus inhibiting NK- directed cytotoxicity against cells expressing normal levels of HLA-class 1 molecules. Interestingly, many tumors overexpress HLA- E, resulting in HLA-E- mediated immune suppression in cancer.

[0006] The expression of the HLA-class I molecule, HLA-G, in healthy tissues is mainly restricted to the placenta and rarely found in non-transformed adult tissues. Yet, HLA-G is frequently expressed in various human malignancies in an oncofetal antigen-like fashion, which makes it analmost tumor-specific target. Interestingly, HLA-E molecules loaded with the HLA-G derived leader peptide VMAPRTLFL (SEQ ID NO: 10) (HLA-E[pHLA-G]) are the strongest natural ligands for the inhibitory NKG2A / CD94 receptor. However, (HLA-E[pHLA-G]) is also known to interact with the activating receptor NKG2C / CD94, complicating the use of the HLA-E[pHLA-G]- NKG2A axis as an inhibitory checkpoint at the tumor site. There is an urgent need for new antibody-based therapies that target this immune checkpoint in cancer.SUMMARY OF THE INVENTION

[0007] The present invention provides monoclonal antibodies, or antigen -binding portions thereof, bispecific antibodies, single chain variable regions (scFvs) and chimeric antigen receptors against a complex comprising HLA-E and a peptide comprising the amino acid sequence VMAPRTLFL (SEQ ID NO: 10), as well as pharmaceutical compositions comprising same and methods of producing same. Also provided are methods of treating cancer comprising administering the antibodies or compositions of the invention. Methods of treating cancer, combination treatments, and patient selection are also provided.

[0008] According to a first aspect, there is provided a monoclonal antibody or antigen binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:CDR-H 1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (NYYVH), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 (WIYSGNFFTKFNENFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 (YGNYYFDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 (KASQDVSTAVA), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 (WASTRHT), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 (QQHYSTPWT).

[0009] According to another aspect, there is provided a monoclonal antibody, or antigen binding fragment, that binds an epitope comprising a fragment of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is I or F.

[0010] According to another aspect, there is provided a monoclonal antibody, or antigen binding fragment, that blocks interaction of Killer cell lectin-like receptor subfamily C, member 1 (NKG2A) with HLA-E.[Oi l] According to some embodiments, the antibody or antigen binding fragment comprises a heavy chain comprising the amino acid sequence: MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTNYYVHWVKQRP GQGLEWIGWIYSGNFFTKFNENFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCASYGN YYFDYWGQGTTLTVSS (SEQ ID NO: 7).

[0012] According to some embodiments, the antibody or antigen binding fragment comprises a light chain comprising an amino acid sequence:MESQIQAFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQK PGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFG GGTKLEIK (SEQ ID NO: 8).

[0013] According to some embodiments, the antibody comprises an IgGl constant region.

[0014] According to some embodiments, the antibody or antigen binding fragment is humanized.

[0015] According to some embodiments, the antibody or antigen binding fragment thereof binds to a fragment of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is I or F; blocks interaction between NKG2A and HLA-E or both.

[0016] According to some embodiments, the peptide and the HLA-E are complexed together.

[0017] According to some embodiments, the SEQ ID NO: 9 is VMAPRTLFL (SEQ ID NO: 10).

[0018] According to some embodiments, the antibody or antigen binding fragment thereof does not block interaction of NKG2C with HLA-E.

[0019] According to another aspect, there is provided a bispecific antibody comprising: (i) the antigen binding fragment of the invention; and (ii) an antigen binding fragment specific to a receptor expressed by an immune cell.

[0020] According to another aspect, there is provided a single chain variable fragment (scFv) comprising a variable region from a heavy chain and a variable region from a light chain separated by a peptide linker, wherein the variable region from a heavy chain comprises CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1 (NYYVH), CDR-H2 comprising the amino acid sequence as set forth in SEQ ID NO: 2 (WIYSGNFFTKFNENFKG), and CDR-H3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 (YGNYYFDY), and wherein the variable region from a light chain comprises CDR-L1 comprising the amino acid sequence as set forth in SEQ ID NO: 4 (KASQDVSTAVA), CDR-L2 comprising the amino acid sequence as set forth in SEQ IDNO: 5 (WASTRHT), and CDR-L3 comprising the amino acid sequence as set forth in SEQ ID NO: 6 (QQHYSTPWT).

[0021] According to some embodiments, the linker is a GGGGS (SEQ ID NO: 32) linker.

[0022] According to some embodiments, the variable region from a heavy chain is N-terminal to the variable region from a light chain.

[0023] According to some embodiments, the scFv further comprises a signal peptide of an IgG heavy chain, optionally wherein the signal peptide of an IgG heavy chain comprises the amino acid sequence MGWSRIFLFLLSIIAGVHC (SEQ ID NO: 25).

[0024] According to some embodiments, the variable region from a light chain is N-terminal to the variable region from a heavy chain.

[0025] According to some embodiments, the scFv further comprises a signal peptide of an IgG light chain, optionally wherein the signal peptide of an IgG light chain comprises the amino acid sequence MESQIQAFVFVFLWLSGVDG (SEQ ID NO: 26).

[0026] According to some embodiments, the scFv further comprises a signal peptide comprising the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 34).

[0027] According to another aspect, there is provided a chimeric antigen receptor (CAR) comprising an antigen recognition domain comprising the antigen binding fragment of an antibody of the invention or an scFv of the invention.

[0028] According to some embodiments, the CAR further comprises a transmembrane domain and an intracellular cell activation domain.

[0029] According to some embodiments, the intracellular activation domain comprises CD3-zeta chain.

[0030] According to some embodiments, the transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 29 and the CD3-zeta chain comprises the amino acid sequence as set forth in SEQ ID NO: 30.

[0031] According to another aspect, there is provided a cell comprising any one of: (i) a monoclonal antibody or antigen binding fragment of the invention, (ii) the bispecific antibody of the invention, (iii) a scFv of the invention, (iv) a CAR of the invention, and (v) any combination thereof.

[0032] According to some embodiments, any one of: the monoclonal antibody or antigen binding fragment, the bispecific antibody, and the CAR, are anchored to the plasma membrane of the cell.

[0033] According to some embodiments, the cell further comprises a monoclonal antibody or antigen binding fragment specific to a receptor expressed by an immune cell.

[0034] According to some embodiments, the cell is a cytotoxic immune cell selected from an NK cell and a T cell.

[0035] According to another aspect, there is provided a pharmaceutical composition comprising any one of: (i) an antibody or antigen binding fragment of the invention, (ii) the bispecific antibody of the invention, (iii) a scFv of the invention, (iv) a CAR of the invention, (v) a cell of the invention, and (vi) any combination thereof, and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0036] According to another aspect, there is provided a method of blocking binding of Killer cell lectin-like receptor subfamily C, member 1 (NKG2A) to a complex comprising HLA-E and a peptide comprising SEQ ID NO: 9, the method comprising contacting the complex with any one of: (i) an antibody or antigen binding fragment of the invention, (ii) the bispecific antibody of the invention, (iii) a scFv of the invention, (iv) a CAR of the invention, (v) a cell of the invention, (vi) the pharmaceutical composition of the invention, and (vii) any combination thereof, thereby blocking binding.

[0037] According to some embodiments, the blocking comprises increasing activation of an NK cell expressing the NKG2A.

[0038] According to some embodiments, the antibody or antigen binding fragment thereof does not inhibit activation of NKG2C in an NK cell expressing the NKG2A.

[0039] According to another aspect, there is provided a method of treating an HLA-E expressing disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby treating an HLA-E expressing cancer.

[0040] According to some embodiments, the HLA-E expressing disease comprises surface expression of HLA-E above a predetermined threshold or comprises circulating soluble HLA-E above a predetermined threshold.

[0041] According to some embodiments, the HLA-E expressing disease is HLA-E expressing cancer.

[0042] According to some embodiments, the cancer is selected from: hematologic cancer, gynecologic cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, skin cancer, prostate cancer, head and neck, stomach cancer, rectal cancer, and colorectal cancer.

[0043] According to some embodiments, the cancer is hematologic cancer or head and neck cancer.

[0044] According to some embodiments, the hematologic cancer is selected from acute myeloid leukemia (AML), and multiple myeloma.

[0045] According to some embodiments, the treating comprises increasing activation of an NK cell expressing NKG2A.

[0046] According to some embodiments, the treating does not inhibit activation of NKG2C in an NK cell expressing NKG2C.

[0047] According to another aspect, there is provided a nucleic acid molecule encoding an antibody or antigen binding fragment of the invention, the bispecific antibody of the invention, a scFv of the invention or a CAR of the invention.

[0048] According to some embodiments, the nucleic acid molecule is an expression vector.

[0049] According to some embodiments, the nucleic acid molecule comprises nucleotide sequences of three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:CDR-H1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 16 (AACTACTATGTACAC), CDR-H2 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 17(GGATTTATTCTGGAAATTTTTTTACTAAGTTCAATGAGAACTTCAAGGGC), CDR-H3 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 18 (TATGGCAACTACTACTTTGACTAC), CDR-L 1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 19(AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC), CDR-L2 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 20 (TGGGCATCCACCCGGCACACT), and CDR-L3 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 21(CAGCAACATTATAGCACTCCGTGGACG).

[0050] According to some embodiments, the nucleic acid molecule comprises SEQ ID NO: 23 and SEQ ID NO: 24.

[0051] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0052] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figures 1A-1F: Recognition of recombinant HLAE-peptide complexes by mAb 4D7. (1A) Bar graph demonstrating the recognition of recombinant HLAE-peptide complexes by the 4D7 mAb. ELISA wells were coated with 2 ug / ml of recombinant HLAE-peptide complex; following blocking, 4D7 or murine IgGl isotype control (2 ug / ml) were added, followed by HRP-goat-anti- mouse light chain and TMB. Absorption was measured at ODesonm. (IB) The binding of 4D7 Ab to recombinant HLA-E[pHLA G]protein was measured using a ProteOn XPR36 instrument (Bio-Rad Laboratories, CA, USA). (1C-1D) Modeling the binding mode comparison of mAb 4D7 to NKG2A in (1C) the presence and (ID) the absence of VMAPRTLFL (SEQ ID NO: 10) peptide. (IE) Table of center weighted scores for binding free energy of 4D7 mAb to HLAE complexed with peptides as well as HLAE without complexed peptide. (IF) Histomgrams showing staining of human tumor cell lines with mAb 4D7. Cells were incubated with mAb 4D7 or matched isotype control (murine IgGl; 10 pg / ml); after washing, cells were incubated with 2ndAb (APC-conjugated F(ab)2-Goat- anti-mouse IgG, 2pg / ml). Samples were assessed by the CytoFLEX flow-cytometer. Top line in each histogram box shows the isotype control and the bottom line shows 4D7.

[0054] Figure 2: Bar graph depicting the effect of single amino acid mutation on 4D7 mAb binding. The cell line 721.221 transfected with the HLA-G gene (721.221 HLA-G) was briefly treated with an acidic buffer to strip endogenous peptides bound to membrane-associated HLA-E molecules. Then, 45 exogenous peptides, with single AA mutation of the VMAPRTLFL peptide (SEQ ID NO: 10), were loaded, as well as the VMAPRTLFL (SEQ ID NO: 10) peptide itself. Cells were then stained with commercial Ab to HLA-E or with mAb 4D7. To exemplify the difference between the various tested peptides, the staining results were processed as follows. First, the Geo MFI staining results with 4D7 of cells without an added peptide (“No Pep.”, unloaded cells) were divided by theirstaining results with the commercial antibody directed to HLA-E. Then the staining ratios (4D7 / commercial anti-HLAE) of the cells incubated with the various peptides were normalized to the ratio of the unloaded cells, in which the staining fold ratio was 1. Thus, fold change from the ratio 1 of the normalized 4D7 staining is presented, enabling the comparison between the effect of loading HLA-E with the various one-AA mutations of SEQ ID NO: 10 on the staining with 4D7. The X axis shows the single AA mutations (e.g., R5E means amino acid substitution from R to E, at position 5 resulting in the peptide VMAPETLFL, SEQ ID NO: 22). Loading with the original SEQ ID NO: 10 was tested twice (WT-1, WT-2). “No pep” = acid-washed cells without added exogenous peptides; staining results divided by staining with commercial anti-HLAE Ab were normalized to 1.

[0055] Figures 3A-3C: Effect of mAb 4D7 on primary human NK activity summarized for four to five healthy donors (CD107a degranulation assay, done in triplicate for each donor). Bar graphs showing the average effect of 4D7 mAb on primary human NK activity from five NKG2C-negative donors or four NKG2C-positive donors in a CD107a degranulation assay. Primary NK (effector cells) were incubated with various target cell lines. (3A) NKG2A- positive / NKG2C-negative, (3B) NKG2A-negative / NKG2C-negative, or (3C) NKG2A- negative / NKG2C-positive NK cells were incubated with target cells (Effector:Target=l:3) in the presence of either isotype control or 4D7 mAb. Four hours later cells were stained for CD 107a and NK markers, analyzed by flow cytometer, and the percentage of CD107a-positive NK cells was calculated. The percentage of CD 107a positive NK cells was calculated and normalized (for each donor and each cell type, average degranulation background assessed with isotype control was normalized to 1, and results with 4D7 were normalized accordingly). Bars, ±SD. Two-way ANOVA test showed p *** < 0.001 and p **** < 0.0001. Statistical analysis and design were performed using Prism GraphPad v8.

[0056] Figures 4A-4C: Bar graphs demonstrating the effect of 4D7 mAb on primary human NK activity from NKG2c-positive donor in CD 107a degranulation assay. Primary NK cells were incubated with various target cell lines. (4A) NKG2A-positive and NKG2C-negative, (4B) NKG2A-negative and NKG2C-negative and (4C) NKG2A-negative and NKG2C-positive NK cells were incubated with target cells (E:T=1:3) in the presence of either isotype control or 4D7 mAb. Four hours later cells were stained for CD107a and NK markers, analyzed by flow cytometer and the percentage of CD107a-positive NK cells was calculated.

[0057] Figures 5A-5C: Effect of mAb 4D7 on primary human NK activity summarized for 6 MM patients (CD107a degranulation assay, done in triplicate for each donor). Primary NKcells derived from BM of MM patients were incubated with autologous BM containing MM blasts, allogeneic BM containing MM blasts and various target cell lines. Degranulation results are shown for (5A) NKG2A+NKG2C-, (5B) NKG2A-NKG2C+ and (5C) NKG2A-NKG2C- NK subsets. NK cells were incubated with target cells ( E:T-I :3) in the presence of either isotype control (mlgGl) or mAb 4D7. Four hours later incubation, cells were stained for cell-membrane CD 107a and for the NK receptors CD16, CD56, NKG2A, and NKG2C (mAb concentration: 2ug / ml); analyzed in a flow cytometer, and the percentage of CD 107a positive NK cells was calculated and normalized (for each donor and each cell type, average degranulation background assessed with isotype control was normalized to 1, and results with 4D7 were normalized accordingly). Bars, ±SD. Two-way ANOVA test showed p** <0.01, and p **** < 0.0001. Statistical analysis and design were performed using Prism GraphPad v8.

[0058] Figure 6: Micrograph images showing staining of 5pm cuts from formalin-fixed paraffin- embedded (FFPE) blocks of two patient-derived xenografts (head and neck cancers). Following antigen retrieval, cuts were stained with either murine IgGl subtype control or with 4D7 mAb. Staining with haematoxylin was performed to better define tumoral and non-tumoral tissue. PDX1 refers to a patient with an HLAE-positive tumor and PDX-2 refers to a patient with an HLAE- negative tumor.

[0059] Figures 7A-7D: (7A) Schematic representation of four CAR constructs of the invention. (7B-7C) Dot plots of APC surface fluorescence from BW cells transduced with the four CAR constructs and imaged with (7B) anti-Myc antibody or (7C) recombinant fusion protein containing HLA-E / peptide complex. Non-transduced cells are used as a negative control. (7D) Bar graph of IL-2 secretion from BW cells after culture in PBS or wells coated with a recombinant fusion protein complex of HLA-E and target peptide SEQ ID NO: 10 or HLA-E and control peptide SEQ ID NO: 15.DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention is directed to an antibody or antigen binding fragment and a pharmaceutical composition that binds a complex comprising HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLFL (SEQ ID NO: 10). Methods of blocking binding of the complex comprising HLA-E and the peptide comprising SEQ ID NO: 10 with Killer cell lectin-like receptor subfamily C, member 1 (NKG2A), and methods for treating cancer are also provided.

[0061] The invention is at least partially based on the finding that a monoclonal antibody (mAb) directed against a complex comprising HLA-E and the leader peptide of HLA-G (SEQ ID NO: 10), herein termed as HLA-E[pHLA-G], effectively and specifically binds HLA-E[pHLA-G], compared to its binding to a complex comprising HLA-E and a leader peptide of heat shock protein 60 (HSP60) or HLA-E with no peptide (Fig. 1A-1E). The inventors further characterized the epitope within the complex, to which the mAb binds, and demonstrated that the epitope comprises residues 1, 4 and 5, as set forth in SEQ ID NO: 10, demonstrating the selectivity of the mAb to the peptide of SEQ ID NO: 10 (Fig. 2). Even though HLA-E[pHLA’G]is known to bind the inhibitory receptor killer cell lectin-like receptor subfamily C, member 1 (NKG2A), as well as the activating receptor NKG2C, both of which are present on NK cells, it was surprisingly found that the developed mAb was able to block the inhibitory activity of NK cells, mediated by NKG2A, without reducing the activation of NK cells mediated by the NKG2C receptor (Fig. 3-5). Finally, it was demonstrated that the mAb was able to bind HLA-E positive xenograft (Figure 6), highlighting its therapeutic potential as a checkpoint inhibitor in HLA-E positive cancer.Antibodies

[0062] By a first aspect there is provided an antibody or antigen binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (NYYVH), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 (WIYSGNFFTKFNENFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 (YGNYYFDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 (KASQDVSTAVA), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 (WASTRHT), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 (QQHYSTPWT).

[0063] By another aspect, there is provided an antibody or antigen binding fragment that binds an epitope comprising a fragment of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is I or F.

[0064] By another aspect, there is provided an antibody or antigen binding fragment that blocks interaction of NKG2A with HLA-E and does not block interaction of NKG2C with HLA-E.

[0065] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that include at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically hasa tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form an antibody binding site. An antibody may be oligoclonal, polyclonal, monoclonal, chimeric, camelised, CDR-grafted, multi- specific, bi-specific, catalytic, humanized, fully human, anti- idiotypic and antibodies that can be labeled in soluble or bound form as well as fragments, including epitope-binding fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences. An antibody may be from any species. The term antibody also includes binding fragments, including, but not limited to Fv, Fab, Fab', F(ab')2 single stranded antibody (svFC), dimeric variable region (Diabody) and disulphide -linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Antibody fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof. The skilled artisan will further appreciate that other fusion products may be generated including but not limited to, scFv- Fc fusions, variable region (e.g., VL and VH)~ Fc fusions and scFv-scFv-Fc fusions.

[0066] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.

[0067] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody comprises a CDR-H1 of SEQ ID NO: 1. In some embodiments, the antibody comprises a CDR-H2 of SEQ ID NO: 2. In some embodiments, the antibody comprises a CDR-H3 of SEQ ID NO: 3. In some embodiments, the antibody comprises a CDR-L1 of SEQ ID NO: 4. In some embodiments, the antibody comprises a CDR-L2 of SEQ ID NO: 5. In some embodiments, the antibody comprises a CDR-L3 of SEQ ID NO: 6.

[0068] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain comprising an amino acid sequence:MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTNYYVHWVKQRP GQGLEWIGWIYSGNFFTKFNENFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCASYGN YYFDYWGQGTTLTVSS (SEQ ID NO: 7). In some embodiments, the antibody or antigen binding fragment comprises a light chain comprising an amino acid sequence: MESQIQAFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQK PGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFG GGTKLEIK (SEQ ID NO: 8). In some embodiments, the antibody or antigen binding fragmentcomprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 7, and a light chain comprises an amino acid sequence as set forth in SEQ ID NO: 8.

[0069] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a multi- specific antibody. In some embodiments, the multi- specific antibody is a bispecific antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody is a humanized antibody. As used herein, a “humanized” antibody refers to an antibody with a human backbone, but which has CDRs that are derived or taken from a nonhuman antibody. In some embodiments, during humanization the CDRs may become altered but are generally still derived from the CDRs of the non-human antibody. In some embodiments, the antigen binding fragment is a single chain antibody. In some embodiments, antigen binding fragment is a single domain antibody. In some embodiments, the antibody is a therapeutic antibody.

[0070] In some embodiments, the antibody or antigen binding fragment comprises IgGl heavy chain. In some embodiments, the antibody or antigen binding fragment comprises any one of an IgGl, IgG2, IgG3 or IgG4 heavy chain. In some embodiments, the antibody or antigen binding fragment comprises a kappa light chain. In some embodiments, the antibody or antigen binding fragment comprises a lambda light chain. In some embodiments, the antibody or antigen binding fragment comprises IgGl kappa.

[0071] In some embodiments, the antibody or antigen binding fragment that binds an epitope comprising a fragment of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is I or F. In some embodiments, the antibody or antigen binding fragment has an increased binding efficacy to a complex comprising HLA-E and a peptide comprising SEQ ID NO: 9, wherein X is I or F. In some embodiments, the antibody or antibody fragment binds SEQ ID NO: 9, wherein X is I or F. In some embodiments, the antibody or antibody fragment binds HLA-E. In some embodiments, the antibody or antibody fragment binds SEQ ID NO: 9. In some embodiments, HLA-E is human HLA-E. In some embodiments, HLA-E is mammalian HLA-E. In some embodiments, HLA-E is primate HLA- E. In some embodiments, HLA-E is murine HLA-E. As used herein, HLA-E, also known as MHC class I antigen E, is a protein that in humans is encoded by the HLA-E gene. In some embodiments, HLA-E is a non-classical MHC class I molecule. In some embodiments, the functional homolog of HLA-E in mice is named Qa-lb, or H2-T23. In some embodiments, HLA-E is a heterodimer comprising a heavy chain and a light chain. In some embodiments, the light chain of HLA-Ecomprises P-2 microglobulin. In some embodiments, the heavy chain of HLA-E is anchored to the cell membrane.

[0072] In some embodiments, HLA-E comprises a human HLA-E. In some embodiments, HLA-E comprises HLA-E*01:01 allele. In some embodiments, HLA-E comprises HLA-E*01:03 allele. In some embodiments, HLA-E comprises HLA-E*01:01 allele, HLA-E*01:03 allele, or any combination thereof. In some embodiments, HLA-E A) 1 :03 comprises UniProt accession number: P13747. In some embodiments, HLA-E comprises UniProt accession number: P13747. In some embodiments, HLA-E comprises the amino acid sequence: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDND AASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQW MHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEH QRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLR WKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSE SHSL (SEQ ID NO: 12). In some embodiments, HLA-E*01:03 comprises SEQ ID NO: 12. In some embodiments, HLA-E*01:01 comprises substitution of the amino acid glycine (G) at position 128, as set forth in SEQ ID NO: 12, to arginine (R). In some embodiments, HLA-E comprises the amino acid sequence:MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDND AASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQW MHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEH QRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITL TWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLR WKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSE SHSL (SEQ ID NO: 13). In some embodiments, HLA-E*01:01 comprises SEQ ID NO: 13.

[0073] In some embodiments, the antibody or antigen binding fragment binds a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is any amino acid. In some embodiments, X in SEQ ID NO: 9 is phenylalanine (F / Phe). In some embodiments, the antibody or antigen binding fragment binds an epitope comprising a fragment of HLA-E and a peptide comprising the sequence VMAPRTLFL (SEQ ID NO: 10). In some embodiments, SEQ ID NO: 10 is the leader peptide of HLA-G. In some embodiments, the leader peptide of HLA-G comprises SEQ ID NO: 10. In some embodiments, the antibody or antigen binding fragment has an increased binding efficacy to a complex comprising HLA-E and a peptide comprising SEQ ID NO: 10. In some embodiments, Xin SEQ ID NO: 9 is isoleucine (1 / He). In some embodiments, the antibody or antigen binding fragment binds an epitope comprising a fragment of HLA-E and a peptide comprising the sequence VMAPRTLIL (SEQ ID NO: 11). In some embodiments, the antibody or antigen binding fragment has an increased binding efficacy to a complex comprising HLA-E and a peptide comprising SEQ ID NO: 11.

[0074] In some embodiments, the antibody or antigen binding fragment binds an epitope comprising a fragment of HLA-E and a peptide comprising the sequence VX1X2PRX3X4X5X6, wherein Xi, X2, X3, X4, and Xe are any amino acid, and X5 is I or F. In some embodiments, the peptide is VX1X2PRX3X4IX6 (SEQ ID NO: 14). In some embodiments, the peptide is VX1X2PRX3X4FX6 (SEQ ID NO: 33). In some embodiments, the epitope to which the antibody or antigen binding fragment binds to within the complex of HLA-E and SEQ ID NO: 10, comprises at least one of residues 1, 4 and 5, as set forth in SEQ ID NO: 10. In some embodiments, the epitope to which the antibody or antigen binding fragment binds to within the complex of HLA-E and SEQ ID NO: 10, comprises at least two of residues 1, 4 and 5, as set forth in SEQ ID NO: 10. In some embodiments, the epitope to which the antibody or antigen binding fragment binds to within the complex of HLA- E and SEQ ID NO: 10, comprises residues 1, 4 and 5, as set forth in SEQ ID NO: 10. In some embodiments, the epitope comprises at least one of the amino acids selected from: V, P and R in SEQ ID NO: 10. In some embodiments, the epitope comprises the amino acids V, P, R, and any combination thereof, in SEQ ID NO: 10. In some embodiments, the epitope to which the antibody or antigen binding fragment binds to within the complex of HLA-E and SEQ ID NO: 10, does not comprise residues 2, 3, 6, 7, and 9, as set forth in SEQ ID NO: 10. In some embodiments, the epitope does not comprise the amino acids M, A, T, and L in SEQ ID NO: 10. In some embodiments, the epitope does not comprise at least one of the amino acids selected from: M, A, T, and L, within SEQ ID NO: 10. In some embodiments, the epitope does not comprise at least one of the residues selected from residues: 2, 3, 6, 7, and 9, as set forth in SEQ ID NO: 10.

[0075] As used herein, “increased binding efficacy” refers to a specific binding to a target or antigen that is greater than the binding of an isotype control. In some embodiments, increased binding is an increase of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% of binding efficacy. Each possibility represents a separate embodiment of the invention. In some embodiments, increased binding is the presence of binding as compared to an isotype control that has no binding. Binding of an antibody to a specific domain will be well known to a person of skill in the art. Antibody binding can be assayed in any way known to one skilled in the art, including but not limited to: x-ray crystallography, immunoprecipitation,immunoblotting, competition assays, and kinetic exclusion assays. In some embodiments, increased binding efficacy is specific binding.

[0076] In some embodiments, the monoclonal antibody, or antigen binding fragment, comprises increased specificity to a complex comprising HLA-E and SEQ ID NO: 9, wherein X is I or F. In some embodiments, the monoclonal antibody, or antigen binding fragment, comprises increased specificity to a complex comprising HLA-E and SEQ ID NO: 10. In some embodiments, the monoclonal antibody, or antigen binding fragment, comprises increased specificity to a complex comprising HLA-E and SEQ ID NO: 11. As used herein, “increased specificity” refers to increased binding efficacy or affinity, compared to a control antigen. In some embodiments, the control antigen comprises a complex of HLA-E and a peptide comprising the sequence QMRPVSRVL (SEQ ID NO: 15). In some embodiments, heat shock protein 60 (HSP60) comprises SEQ ID NO: 15. In some embodiments, increased specificity is an increase of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% of binding efficacy or affinity compared to a control antigen. Each possibility represents a separate embodiment of the invention.

[0077] In some embodiments, the monoclonal antibody, or antigen binding fragmentblocks interaction of NKG2A to a complex comprising HLA-E. In some embodiments the complex comprising HLA-E is the complex of HLA-E with HLA-G leader peptide (HLA-E[pHLA-G]). In some embodiments, the complex comprising HLA-E is the complex of HLA-E with SEQ ID NO: 9, wherein X is I or F. In some embodiments, the complex comprising HLA-E is the complex of HLA- E with SEQ ID NO: 10.

[0078] NKG2, also known as CD159 (Cluster of Differentiation 159) is a receptor present on the cell surface of natural killer cells (NK cells) and / or cytotoxic CD8 T cells. In some embodiments, NKG2 receptor comprises NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, or any combination thereof. In some embodiments, NKG2A comprises UniProt # P26715. In some embodiments, NKG2A dimerizes with CD94, to generate NKG2A / CD94 dimer. In some embodiments, NKG2A / CD94 dimer is an inhibitory receptor. In some embodiments, ligand binding to NKG2A or NKG2A / CD94 dimer prevents activation of the cell expressing the NKG2A or the NKG2A / CD94 dimer. In some embodiments, the cell expressing the NKG2A or the NKG2A / CD94 dimer comprises NK cell. In some embodiments, the cell expressing the NKG2A / CD94 dimer comprises CD8+T cell. In some embodiments, NKG2A contains immunoreceptor tyrosine-based inhibition motif (ITIM) in the intracellular part of the molecule. In some embodiments, NKG2B comprises a splice variant of NKG2A. In some embodiments, NKG2B contains ITIM. In some embodiments, the antibody or antigen binding fragment prevents the binding of the complexcomprising HLA-E and SEQ ID NO: 10 to an inhibitory NKG2 receptor consisting of an ITIM. In some embodiments, the inhibitory receptor comprises NKG2A, NKG2B or any combination thereof. In some embodiments, the inhibitory receptor comprises NKG2A. In some embodiments, the antibody or antigen binding fragment prevents the recruitment of at least one of: Src homology 2 domain containing phosphatase SHP-1, and SHP-2, by the ITIM within the NKG2 receptor. In some embodiments, the antibody or antigen binding fragment abolishes the inhibition of cytotoxicity, mediated by the ITIM of NKG2A or NKG2A / CD94 dimer.

[0079] In some embodiments, the monoclonal antibody, or antigen binding fragment disclosed herein, does not block interaction of NKG2C to a complex comprising HLA-E. In some embodiments, the monoclonal antibody, or antigen binding fragment, does not prevent activation of a NK cell or CD8+T cell via NKG2C.

[0080] NKG2C is one of the activator receptors of NKG2, comprising NKG2C, NKG2E and its splice variant NKG2H. NKG2-C type II integral membrane protein or NKG2C is a protein that in humans is encoded by the KLRC2 gene. In some embodiments, NKG2C comprises UniProt # P26717. In some embodiments, NKG2C interacts and form a dimer with CD94, generating CD94 / NKG2C dimer. In some embodiments, the CD94 / NKG2C dimer binds to HLA-E. In some embodiments, binding of CD94 / NKG2C to a complex comprising HLA-E leads to NK cell activation. In some embodiments, the activator receptor, comprising NKG2C, contains a positively charged residue in its transmembrane region by which it interacts with an adaptor molecule containing an ITAM. In some embodiments, the adaptor molecule comprises DNAX-activating protein of 12 kDa (DAP- 12). In some embodiments, the antibody or antigen binding fragment, does not prevent interaction of the positively charged residue within NKG2C transmembrane region with an adaptor molecule containing an ITAM. In some embodiments, the antibody or antigen binding fragment does not prevent activation of ITAM mediated by at least one of: NKG2C, NKG2E and NKG2H. In some embodiments, the antibody or antigen binding fragment does not prevent activation of NK cell or CD8+T cell expressing NKG2C or CD94 / NKG2C dimer. In some embodiments, the antibody or antigen binding fragment, does not inhibit the activation of a kinase by ITAM, mediated by the NKG2C receptor or CD94 / NKG2C dimer. In some embodiments, the kinase comprises at least one of: Src homology domain containing kinase Syk, Zeta-chain- associated protein kinase 70 (Zap70), and any combination thereof.

[0081] In some embodiments, the antibody or antigen binding fragment does not inhibit a cytotoxicity effect mediated by activated NK cell or by CD8+T cell via NKG2C or CD94 / NKG2C dimer. In some embodiments, a cytotoxicity effect of NK cell or CD8+T cell comprisesdegranulation (e.g., the release of lytic granule contents onto the surface of a target cell). In some embodiments, extemalization of CD107a (lysosome-associated membrane protein 1) is a marker of degranulation of NK cell or CD8+T cell. In some embodiments, activation of NK cell or CD8+T cell comprises cytotoxicity. In some embodiments, activation of NK cell or CD 8+ T cell comprises expression of a pro- inflammatory cytokine and / or chemokine. In some embodiments, the antibody or antigen binding fragment reduces NK or CD8+T cell activation mediated by NKG2C, by no more than 5%, 10%, 20% or 30%. Each possibility presents a separate embodiment of the invention.

[0082] In some embodiments, the antibody or antigen binding fragment of the invention enhances activation of NK cell, CD8+T cell, or any combination thereof. In some embodiments, the NK or CD8+T cell expresses NKG2A or CD94 / NKG2A dimer. In some embodiments, the NK or CD8+T cell expresses NKG2C or CD94 / NKG2C dimer. In some embodiments, activation comprises cytotoxicity. In some embodiments, cytotoxicity comprises degranulation. In some embodiments, cytotoxicity comprises expression of CD107a. In some embodiments, activation comprises expression of a pro- inflammatory cytokine and / or a chemokine. In some embodiments, a pro- inflammatory cytokine comprises interferon gamma (IFN-y), tumor necrosis factor alpha (TNF-a) or any combination thereof. In some embodiments, the antibody or antigen binding fragment enhances activation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 500%. Each possibility represents a separate embodiment of the invention.

[0083] In some embodiments, the heavy chain comprises SEQ ID NO: 7. In some embodiments, the heavy chain consists of SEQ ID NO: 7. In some embodiments, the heavy chain variable region comprises SEQ ID NO: 7. In some embodiments, the heavy chain variable region consists of SEQ ID NO: 7. In some embodiments, the heavy chain variable region comprises at least 80% homology to SEQ ID NO: 7. In some embodiments, homology is identity. In some embodiments, at least 80% is at least 80, 85, 90, 92, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 80% is at least 85%.

[0084] In some embodiments, the light chain comprises SEQ ID NO: 8. In some embodiments, the light chain consists of SEQ ID NO: 8. In some embodiments, the light chain variable region comprises SEQ ID NO: 8. In some embodiments, the light chain variable region consists of SEQ ID NO: 8. In some embodiments, the light chain variable region comprises at least 80% homology to SEQ ID NO: 8. In some embodiments, homology is identity. In some embodiments, at least 80% is at least 80, 85, 90, 92, 95, 97, 99 or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 80% is at least 85%.

[0085] In some embodiments, there is provided an antibody or antigen binding fragment, comprising a functional analog thereof, having a heavy chain comprising at least 80% homology to SEQ ID NO:7, and a light chain having at least 80% homology to SEQ ID NO: 8. In some embodiments, at least 80% comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, homology thereto. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody or antigen binding fragment, comprises a functional analog thereof, having a heavy chain comprising one of: 80-99%, 85-99%, 90-99%, or 95-99% homology to SEQ ID NO:7, and a light chain comprising one of: 80-99%, 85-99%, 90-99%, or 95- 99% homology to SEQ ID NO: 8. Each possibility represents a separate embodiment of the invention.

[0086] The term “analog” as used herein, refers to a antibody or antigen binding fragment that is similar, but not identical, to the antibody or antigen binding fragment of the invention that still is capable in binding the complex comprising HLA-E and SEQ ID NO: 9, wherein X is I or F. In some embodiments, the analog is still able to block NKG2A binding to HLA-E. In some embodiments, the analog does not block binding of NKG2C to HLA-E. An analog may have deletions, additions or mutations that result in nucleotide sequence that is different than the nucleotide sequence of the monoclonal antibody or antigen binding fragment of the invention. It should be understood, that all analogs of the monoclonal antibody or antigen binding fragment of the invention would still be capable of binding the complex comprising HLA-E and SEQ ID NO: 9. Further, an analog may be analogous to a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 50 consecutive nucleotides of SEQ ID NO: 7 and at least 50 consecutive nucleotides of SEQ ID NO: 8.

[0087] In some embodiments, the antibody or antigen binding fragment comprises a signal peptide. In some embodiments, the heavy chain comprises a signal peptide. In some embodiments, the light chain comprises a signal peptide. In some embodiments, the heavy chain signal peptide comprises MGWSRIFLFLLSIIAGVHC (SEQ ID NO: 25). In some embodiments, the heavy chain signal peptide consists of SEQ ID NO: 25. In some embodiments, the light chain signal peptide comprises MESQIQAFVFVFLWLSGVDG (SEQ ID NO: 26). In some embodiments, the light chain signal peptide consists of SEQ ID NO: 26).Bispecific antibodies

[0088] According to another aspect there is provided a bispecific antibody comprising: (i) a first antigen binding fragment comprising an antigen binding fragment disclosed herein; and (ii) a second antigen binding fragment.

[0089] In some embodiments, the antibody or antigen binding fragment is a bispecific antibody. In some embodiments, the second antigen binding fragment binds to a protein expressed by an immune cell. In some embodiments, the second antigen binding fragment is specific to a protein expressed by an immune cell. In some embodiments, the protein is a surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a signaling protein. In some embodiments, the second antigen binding fragment binds to an immune cell protein. In some embodiments, the second antigen binding fragment binds to an immune cell surface protein. In some embodiments, the second antigen binding fragment binds an immune cell receptor. In some embodiments, the second antigen binding fragment activates the immune cell upon binding. In some embodiments, a signal mediated by the receptor is an activating signal. In some embodiments, the second antigen binding fragment is an activating antigen binding fragment. In some embodiments, an antigen binding fragment is a single chain antibody (scAb). In some embodiments, the antigen binding fragment is a single chain variable fragment (scFv). In some embodiments, the antigen binding fragment is a single domain antibody (sdAb). In some embodiments, the sdAb is a camelid or shark antibody. In some embodiments, the sdAb is a VHH.

[0090] In some embodiments, the immune cell is a cytotoxic immune cell. In some embodiments, the immune cell is selected from a T cell, an NK cell, a neutrophil, a monocyte and a macrophage. In some embodiments, the immune cell is selected from: a T cell, and an NK cell. In some embodiments, a T cell is selected from a CD4 T cell and a CD8 T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell comprises an NK cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the receptor comprises an activating receptor expressed by the NK cell.

[0091] As used herein, the term “bispecific antibody” refers to an artificial protein that can simultaneously bind to two different antigens or two different epitopes on the same antigen. In some embodiments, the bispecific antibody is designed to recruit and / or activate an immune cell. In some embodiments, the bispecific antibody activates a signaling ligand or receptor. In some embodiments, the bispecific antibody enables approximation between a cancer cell and an immune cell. In someembodiments, the bispecific antibody enables approximation between an HLA-E expressing cancer cell and an immune cell.

[0092] Bispecific antibodies are well known in the art, and include the IgG-like format and the non- IgG-like format. An IgG-like bispecific antibody retains the traditional monoclonal antibody structure of two Fab arms and one Fc region, except that the two Fab sites bind different antigens. In some embodiments, the bispecific antibody is a trifunctional antibody. In some embodiments, the bispecific antibody is a multi- specific antibody. Methods for producing bispecific antibodies are known in the art (e.g., the quadroma method or the hybrid hybridoma method). Non-IgG-like bispecific antibodies include proteins that lack the Fc region, including chemically linked Fabs, bivalent or trivalent single-chain variable fragments (ScFvs), and fusion proteins mimicking the Fabs of two antibodies. Examples of non-IgG-like bispecific antibodies include bi-specific T-cell engagers (BiTEs), which uses the G4S linker to connect a ScFv with an antigen binding fragment that binds CD3 and a ScFv with tumor-specificity, tetravalent antiparallel structure (TandAbs) and VH only (Bi-Nanobody).Single chain variable fragments

[0093] According to another aspect there is provided a single chain variable fragment (scFv) comprising a variable region from a heavy chain and a variable region from a light chain separated by a linker, wherein said variable region from a heavy chain comprises CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1 (NYYVH), CDR-H2 comprising the amino acid sequence as set forth in SEQ ID NO: 2 (WIYSGNFFTKFNENFKG), and CDR-H3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 (YGNYYFDY), and wherein said variable region from a light chain comprises CDR-L1 comprising the amino acid sequence as set forth in SEQ ID NO: 4 (KASQDVSTAVA), CDR-L2 comprising the amino acid sequence as set forth in SEQ ID NO: 5 (WASTRHT), and CDR-L3 comprising the amino acid sequence as set forth in SEQ ID NO: 6 (QQHYSTPWT).

[0094] In some embodiments, the antibody or antigen binding fragment is a scFv. In some embodiments, the antigen binding fragment of the invention is a scFv of the invention. In some embodiments, the scFv comprises an antigen binding fragment of the invention. In some embodiments, the scFv comprises a heavy chain variable region of the invention. In some embodiments, the scFv comprises a light chain variable region of the invention. scFvs are well known in the art and methods of converting a full antibody into a scFv are well known as well.

[0095] In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a GS linker. In some embodiments, the linker is a GGGGS (SEQ ID NO: 32) linker. In some embodiments, the linker comprises or consists of (GGGGS)n, where n is an integer. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each possibility represents a separate embodiment of the invention. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, the linker comprises or consists of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 35).

[0096] In some embodiments, the variable region from a heavy chain is N-terminal to the variable region from a light chain. In some embodiments, the variable region from a heavy chain is C- terminal to the variable region from a light chain. In some embodiments, the variable region from a light chain is N-terminal to the variable region from a heavy chain. In some embodiments, the variable region from a light chain is C-terminal to the variable region from a heavy chain. In some embodiments, the variable region from a heavy chain is the N-terminal region of the scFv. In some embodiments, the variable region from a light chain is the N-terminal region of the scFv.

[0097] In some embodiments, the scFv comprises a structure of variable region from a heavy chainlinker-variable region from a light chain. In some embodiments, the scFv consists of a structure of variable region from a heavy chain-linker-variable region from a light chain. In some embodiments, the scFv comprises a structure of variable region from a light chain-linker-variable region from a heavy chain. In some embodiments, the scFv consists of a structure of variable region from a light chain-linker- variable region from a heavy chain.

[0098] In some embodiments, the scFv further comprises a signal peptide (SP). Signal peptides are also known as signal sequences and target to the ER proteins that are to be excreted or expressed in a membrane. In some embodiments, the SP is an N-terminal SP. It is well known that SPs are often N-terminal as translation begins in the cytoplasm but upon recognition of the nascent SP chain the ribosome is brought to the ER where co-translational insertion into the ER occurs. In some embodiments, the scFv comprises a structure of SP-variable region from a heavy chain-linker- variable region from a light chain. In some embodiments, the scFv consists of a structure of SP- variable region from a heavy chain-linker-variable region from a light chain. In some embodiments, the scFv comprises a structure of SP-variable region from a light chain-linker-variable region from a heavy chain. In some embodiments, the scFv consists of a structure of SP-variable region from a light chain-linker-variable region from a heavy chain.

[0099] In some embodiments, the variable region from a heavy chain is N-terminal to the variable region from a light chain and the SP is a SP of an IgG heavy chain. In some embodiments, a SP of the IgG heavy chain comprises the amino acid sequence MGWSRIFLFLLSIIAGVHC (SEQ ID NO: 25). In some embodiments, the variable region from a light chain is N-terminal to the variable region from a heavy chain and the SP is a SP of an IgG light chain. In some embodiments, a SP of the IgG light chain comprises the amino acid sequence MESQIQAFVFVFLWLSGVDG (SEQ ID NO: 26). In some embodiments, the variable region from a light chain is N-terminal to the variable region from a heavy chain and the SP comprises the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 34). In some embodiments, the SP comprises a sequence SEQ ID NO: 25. In some embodiments, the SP comprises a sequence SEQ ID NO: 26. In some embodiments, the SP comprises a sequence SEQ ID NO: 34. In some embodiments, the SP consists of SEQ ID NO: 25. In some embodiments, the SP consists of SEQ ID NO: 26. In some embodiments, the SP consists of SEQ ID NO: 34.

[0100] In some embodiments, the scFV comprises the same specificity as antibody or antigen binding fragment of the invention. In some embodiments, the scFV comprises the same function as antibody or antigen binding fragment of the invention.Chimeric antigen receptors (CARs)

[0101] According to another aspect there is provided a chimeric antigen receptor (CAR) comprising: an antigen recognition domain comprising an antigen binding fragment of the invention.

[0102] According to another aspect, there is provided a CAR comprising: an scFv of the invention.

[0103] A CAR is a genetically engineered receptor that combines both antigen-binding and an immune cell activating function into a single receptor. In some embodiments, the CAR comprises an extracellular domain. In some embodiments, the extracellular domain comprises or consists of an antigen recognition domain. In some embodiments, the antigen recognition domain comprises or consists of a scFv of the invention. In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the CAR comprises an intracellular domain. In some embodiments, the intracellular domain is an activation domain. In some embodiments, an activation domain is an immune cell activation domain. In some embodiments, the activation domain is a T cell signaling domain. In some embodiments, a CAR comprises an extracellular hinge domain.

[0104] In some embodiments, the antigen recognition domain of the CAR comprises a single-chain variable fragment (scFv). In some embodiments, the scFv is the scFv of the invention. In someembodiments the antigen recognition domain of the CAR comprises three CDR-H and three CDR- L, wherein: CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the antigen recognition domain comprises SEQ ID NO: 7 and SEQ ID NO: 8. In some embodiments, the antigen recognition domain comprises a functional analog of SEQ ID NO: 7 and SEQ ID NO: 8. In some embodiments, the heavy chain variable region and the light chain variable region are separated by a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a GS linker, n some embodiments, the linker is a flexible linker. In some embodiments, the linker is a GGS linker. In some embodiments, the linker is a GGGS (SEQ ID NO: 31) linker. In some embodiments, the linker is a GGGGS (SEQ ID NO: 32) linker. In some embodiments, the linker comprises (GS)n where n is an integer. In some embodiments, the linker comprises (GGS)n where n is an integer. In some embodiments, the linker comprises (GGGS)n where n is an integer. In some embodiments, the linker comprises (GGGGS )n where n is an integer. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is SEQ ID NO: 35. In some embodiments, the light chain variable region is N-terminal to the heavy chain variable region. In some embodiments, the heavy chain variable region is C-terminal to the light chain variable region. In some embodiments, the scFv comprises or consists of the structure light chain variable-linker- heavy chain variable region. In some embodiments, the scFv comprises or consists of the structure SP-light chain variable-linker-heavy chain variable region.

[0105] As known in the art, the first generation of CAR comprised an immunoreceptor tyrosinebased activation motif (IT AM) present in the cytoplasmic domain of CD3-zeta. The second generation comprised additional costimulatory domain, e.g., 4-1BB (CD137), CD28, CD27 or 0X40 (CD134), within the backbone of the first generation, and more than one costimulatory domain was added in the third CAR generation. In some embodiments, the intracellular domain comprises at least one IT AM domain. In some embodiments, the intracellular domain comprises 1, 2, 3 or 4 ITAM domains. Each possibility represents a separate embodiment of the invention. In some embodiments, the intracellular domain comprises a CD3-zeta intracellular signaling domain. In some embodiments, the intracellular domain is selected from at least one of the following intracellular domains: CD3Z, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD- 1, ICOS, lymphocytefunction-associated antigen- 1 (LFA- 1), CD2, CD 7, LIGHT, NKG2C, and B7- H3. In some embodiments, the intracellular domain comprises and at least one costimulatory domain selected from: a 4- IBB domain, a CD28 domain, a CD27 domain, an 0X40 domain, or any combination thereof. In some embodiments, the intracellular domain comprises CD3 zeta chain and a CD28 costimulatory domain or a CD137 (4-1BB) costimulatory domain.

[0106] In some embodiments, the transmembrane domain of the CAR is a single-pass transmembrane domain. In some embodiments, the transmembrane domain comprises a CD3 transmembrane domain. In some embodiments, the CD3 transmembrane domain comprises the amino acid sequence LCYLLDGILFIYGVIITALYL (SEQ ID NO: 29). In some embodiments, the intracellular domain of the CAR comprises a CD3 zeta chain. In some embodiments, the CD3 zeta chain comprises the amino acid sequenceRAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEG VYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR (SEQ ID NO: 30). In some embodiments, the CAR disclosed herein comprises SEQ ID NO: 29, SEQ ID NO: 30, or both.

[0107] In some embodiments, the CAR is a multi-antigen CAR. In some embodiments, the CAR comprises a first antigen recognition domain comprising an antigen binding domain of the invention and a second antigen recognition domain. In some embodiments, the second antigen recognition domain comprises a second antigen binding domain.Cells

[0108] According to another aspect there is provided a cell comprising a monoclonal antibody or antigen binding fragment of the invention.

[0109] According to another aspect, there is provided a cell comprising a bispecific antibody of the invention.

[0110] According to another aspect, there is provided a cell comprising a scFv of the invention.

[0111] According to another aspect, there is provided a cell comprising a CAR of the invention.

[0112] In some embodiments, any one of the monoclonal antibody or antigen binding fragment, the bispecific antibody disclosed herein, or the CAR, are anchored to the plasma membrane. In some embodiments, the antibody or fragment thereof is attached to the plasma membrane of the cell. In some embodiments, anchored is attached. In some embodiments, attached is in the plasma membrane. In some embodiments, attached is bound to the plasma membrane. In someembodiments, bound is covalently bound. In some embodiments, attached is adsorbed to the plasma membrane.

[0113] In some embodiments, the cell comprises a first antibody or antigen binding fragment of the invention, and further comprises a second antibody or antigen binding fragment. In some embodiments, the second antibody or antigen binding fragment binds to a protein expressed by an immune cell. In some embodiments, the second antibody or antigen binding fragment is specific to a protein expressed by an immune cell. In some embodiments, the protein is a surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a signaling protein. In some embodiments, the second antibody or antigen binding fragment binds to an immune cell protein. In some embodiments, the second antibody or antigen binding fragment binds to an immune cell surface protein. In some embodiments, the second antibody or antigen binding fragment binds an immune cell receptor. In some embodiments, the second antibody or antigen binding fragment is specific to a receptor expressed by an immune cell. In some embodiments, the second antibody or antigen binding fragment activates the immune cell.

[0114] In some embodiments, the cell comprises the antibody or antigen binding fragment in its plasma membrane. In some embodiments, the cell secretes the antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment is in the plasma membrane of the cell.

[0115] In some embodiments, the cell comprises within its plasma membrane a monoclonal antibody or an antigen binding fragment that binds HLA-E and SEQ ID NO: 9, wherein X is I or F. In some embodiments, the monoclonal antibody or antigen binding fragment binds HLA-E and SEQ ID NO: 10. In some embodiments, the cell comprises a monoclonal antibody or antigen binding fragment, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3, CDR-L 1 comprises the amino acid sequence as set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the cell comprises a monoclonal antibody or antigen binding fragment, comprising a heavy chain as set forth in SEQ ID NO: 7 and a light chain as set forth in SEQ ID NO: 8. In some embodiments, the antibody or an antigen binding fragment is expressed on the cell surface. In some embodiments, the antibody or an antigen binding fragment is anchored to the cell plasma membrane. In some embodiments, the cell expresses a monoclonal antibody or an antigen binding fragment,comprising a functional analog having a heavy chain comprising at least 80% homology to SEQ ID NO:7, and a light chain having at least 80% homology to SEQ ID NO: 8.

[0116] In some embodiments, the cell comprises two antibodies or antigen binding fragments, that are specific to two different ligands. In some embodiments, the antibodies or the antigen binding fragments are anchored to the cell plasma membrane. In some embodiments, the cell expresses a monoclonal antibody or antigen binding fragment, as disclosed herein, and a monoclonal antibody or antigen binding fragment that binds and activates a receptor involved in NK and / or CD8+T cell cytotoxicity. In some embodiments, a monoclonal antibody or antigen binding fragment that binds and activates a receptor involved in NK and / or CD8+T cell cytotoxicity comprises a receptor agonist. In some embodiments the receptor agonist enhances a NK and / or CD8+T cell cytotoxic effect.

[0117] Examples for agonists, or activating antibodies, that activate NK cytotoxicity are known in the art, as described in Koch J and Tesar M. Recombinant Antibodies to Arm Cytotoxic Lymphocytes in Cancer Immunotherapy. Transfus Med Hemother. 2017; 44:337-350, hereby incorporated by reference in its entirety.

[0118] In some embodiments, the CAR is inserted into the plasma membrane. In some embodiments, the CAR is anchored by its intracellular domain. In some embodiments, the CAR is anchored in the plasma membrane such that the antigen recognition domain is extracellular and the intracellular domain is intracellular. In some embodiments, the cell is a CAR cell. In some embodiments, the cell is a CAR T cell. In some embodiments, the cell is a CD4+CAR T cell. In some embodiments, the cell is a CD8+CAR T cell. In some embodiments, the cell is a CAR-NK cell.

[0119] As used herein, the terms "CAR-T cell” and “CAR-NK cell” refer to an engineered receptor which has specificity for at least one protein of interest (for example HLA-E) and is grafted onto an immune effector cell (a T cell or NK cell). CAR-T and CAR-NK cells target and are cytotoxic to the protein for which the receptor binds. In some embodiments, the CAR-T cell has the specificity of a monoclonal antibody of the invention grafted onto a T-cell. In some embodiments, the CAR- NK cell has the specificity of a monoclonal antibody of the invention grafted onto a NK-cell. In some embodiments, the T cell is selected from a cytotoxic T lymphocyte and a regulatory T cell.

[0120] Construction of CAR cells is well known in the art. In one non-limiting example, a monoclonal antibody to a cancer protein can be made and then a vector coding for the antibody will be constructed. The vector will also comprise a costimulatory signal region. In some embodiments,the costimulatory signal region comprises the intracellular domain of a known T cell or NK cell stimulatory molecule. In some embodiments, the intracellular domain is selected from at least one of the following: CD3Z, CD27, CD28, 4- IBB, 0X40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD 7, LIGHT, NKG2C, B7- H3, and a ligand that specifically binds with CD83. In some embodiments, the vector also comprises a CD3Z signaling domain. This vector is then transfected, for example by lentiviral infection, into a T-cell.

[0121] In some embodiments, the cell is a multi-antigen targeted CAR cell. As used herein, the term “multi-antigen targeted CAR cell” encompasses a cell comprising at least two different antigen binding domains. In some embodiments, the two different antigen binding domains target at least two different ligands, or receptors. Several types of multi-antigen-targeted CAR cells are known in the art. In some embodiments, the multi-antigen targeted CAR cell is a dual CAR cell. In some embodiments, a dual CAR cell comprises two distinct CARs, a first CAR that is a CAR of the invention and a second CAR. In some embodiments, the second CAR comprises an antigen recognition domain comprising a second antigen binding domain. In some embodiments, the multiantigen targeted CAR cell is a tandem CAR cell. In some embodiments, a tandem CAR cell comprises two distinct antigen -binding domains in a single CAR. In some embodiments, the multiantigen targeted CAR cell is a trivalent CAR cell.Pharmaceutical compositions

[0122] The present invention also contemplates pharmaceutical formulations for medical use, which comprise as the active agent of at least one antibody or antigen binding fragment, which recognizes HLA-E[pHLA G], for the manufacture of a therapeutic composition for the treatment, diagnosis or prophylaxis of the conditions variously described herein.

[0123] By another aspect, there is provided a composition comprising an antibody or antigen binding fragment of the invention.

[0124] By another aspect, there is provided a composition comprising a bispecific antibody of the invention .

[0125] By another aspect, there is provided a composition comprising a scFv of the invention.

[0126] By another aspect, there is provided a composition comprising a CAR of the invention .

[0127] By another aspect, there is provided a composition comprising a cell of the invention.

[0128] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

[0129] In some embodiments, the pharmaceutical composition comprises an antibody or antigen binding fragment, or a cell, comprising an antigen binding fragment that binds HLA-E and SEQ ID NO: 9, wherein X is I or F. In some embodiments, the monoclonal antibody or antigen binding fragment, or the cell, within the pharmaceutical composition binds HLA-E and SEQ ID NO: 10. In some embodiments, the pharmaceutical comprises a monoclonal antibody or antigen binding fragment, or a cell, comprising the amino acid sequences as set forth in SEQ ID NOs: 1-6. In some embodiments, the pharmaceutical composition comprises SEQ ID NO: 7 and SEQ ID NO: 8, or a functional analog thereto.

[0130] In such pharmaceutical and medicament formulations, the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

[0131] Typically, the molecules of the present invention comprising the antigen binding portion of an antibody will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of active ingredient (molecule comprising the antigen binding portion of an antibody) or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a poly anhydride copolymer of a stearic acid dimer and sebaric acid. The rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.

[0132] The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, intralesionally, intratumorally or parenterally. Ordinarily, intravenous (i.v.), intraarticular, topical or parenteral administration will be preferred.

[0133] It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician.

[0134] Although an appropriate dosage of a molecule (an antibody or a fragment thereof) of the invention varies depending on the administration route, type of molecule (polypeptide, polynucleotide, organic molecule etc.) age, body weight, sex, or conditions of the patient, and should be determined by the physician in the end, in the case of oral administration, the daily dosage can generally be between about 0.01 mg to about 500 mg, preferably about 0.01 mg to about 50 mg, more preferably about 0.1 mg to about 10 mg, per kg body weight. In the case of parenteral administration, the daily dosage can generally be between about 0.001 mg to about 100 mg, preferably about 0.001 mg to about 10 mg, more preferably about 0.01 mg to about 1 mg, per kg body weight. The daily dosage can be administered, for example in regimens typical of 1-4 individual administration daily. Other preferred methods of administration include intraarticular administration of about 0.01 mg to about 100 mg per kg body weight. Various considerations in arriving at an effective amount are described, e.g., in Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

[0135] Suitable dosing regimens of combination chemotherapies are known in the art and described in, for example, Saltz et al. Proc ASCO 1999, 18, 233a and Douillard et al., Lancet 2000, 355, 1041- 7.

[0136] The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

[0137] As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicleforming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0138] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0139] In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for administration to a human.

[0140] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, intravenous, intramuscular, intratumoral or intraperitoneal.

[0141] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.Methods and use thereof

[0142] According to another aspect, there is provided a method of blocking binding of NKG2A to HLA-E, the method comprising contacting HLA-E with one of: an antibody or antigen binding fragment of the invention, a bispecific antibody of the invention, a scFv of the invention, a CAR of the invention, a cell of the invention, a composition of the invention, or any combination thereof, thereby blocking binding.

[0143] According to another aspect, there is provided a method of blocking binding of NKG2A to a complex comprising HLA-E and a peptide comprising SEQ ID NO: 9, wherein X is I or F, the method comprising contacting the complex with one of: an antibody or antigen binding fragment of the invention, a bispecific antibody of the invention, a scFv of the invention, a cell of the invention, a composition of the invention, or any combination thereof, thereby blocking binding.

[0144] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in culture.

[0145] As used herein, the term “contacting” refers to at least one of: incubating, mixing, centrifuging or any combination thereof, allowing the binding between the monoclonal antibody or antigen binding fragment of the invention, and the complex comprising HLA-E and SEQ ID NO: 9, wherein X is I or F. In some embodiments, contacting is in the presence of a NK cell. In some embodiments, contacting is for 10 - 120 minutes in RT, or 37° C.

[0146] In some embodiments, the HLA-E is in complex with a peptide. In some embodiments, the binding is to a complex comprising HLA-E and a peptide. In some embodiments, the peptide comprises SEQ ID NO: 9. In some embodiments, the peptide consists of SEQ ID NO: 9. In some embodiments, the peptide comprises SEQ ID NO: 10. In some embodiments, the peptide consists of SEQ ID NO: 10.

[0147] In some embodiments, blocking comprises increasing activation of NK cell or CD8+T cell. In some embodiments, the cell expresses NKG2A or CD94 / NKG2A dimer. In some embodiments, the cell expresses NKG2C or CD94 / NKG2C dimer. In some embodiments, increased activation is of NK cell and is compared to a control NK cell. In some embodiments, increased activation is of CD8+T cell and is compared to a control CD8+T cell.

[0148] In some embodiments, contacting in in vivo. In some embodiments, contacting comprises administering a therapeutic effective amount of the pharmaceutical composition of the invention, comprising, the monoclonal antibody or antigen binding fragment, the cell, or any combination thereof, to a subject in need thereof.

[0149] In some embodiments, contacting of at least one of: (a) the monoclonal antibody or antigen binding fragment of the invention, (b) the cell of the invention (c) the pharmaceutical composition of the invention, and (d) any combination thereof, with a cell that expresses the complex comprising HLA-E and SEQ ID NO: 9, wherein X is I or F, increases activation and / or cytotoxicity of at least one cell type selected from: NK, CD8+cytotoxic T cell, and any combination thereof. In some embodiments increased activation of NK cell, CD8+T cell, or any combination thereof leads toincreased death of a target cell. In some embodiments, the target cell comprises a cancer cell. In some embodiments, the target cell expresses HLA-E. In some embodiments, the target cell expresses HLA-E and SEQ ID NO: 9, wherein X is I or F. In some embodiments, the target cell expresses HLA-E and SEQ ID NO: 10. In some embodiments, a target cell death comprises apoptosis. In some embodiments, the apoptosis of a target cell is by exocytosis. In some embodiments, apoptosis of a target cell by NK cell is by the release of the membrane-disrupting protein perforin and / or serine protease granzyme. In some embodiments, a NK cell expressing FasL and / or tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) interacts with a target cell expressing death receptors (Fas / CD95), leading to caspase -dependent apoptosis. In some embodiments, the antibody or antigen binding fragment, or the cell disclosed herein, increases a cell target death by at least one of the abovementioned mechanisms.

[0150] Methods for examination of NK or CD8+T cell activity and / or cytotoxicity are known in the art. NK-cell effector function can be monitored by one of: degranulation assay, cytotoxicity assay, chromium release assay, colorimetric lactic dehydrogenase (LDH) measurement-based NK cell- mediated cytotoxicity assay, and calcein acetoxymethyl (AM) staining-based microscopic method. In some embodiments, at least one of: the monoclonal antibody or the antigen binding fragment thereof, the cell, the composition, and any combination thereof, increases NK activity and / or cytotoxicity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, at least 1000%. Each possibility presents a different embodiment of the invention. In some embodiments, increased activity and / or cytotoxicity comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, cell death of the target cell. Each possibility presents a different embodiment of the invention. In some embodiments, increased activity and / or cytotoxicity comprises at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, cell death of HLA-E expressing cancer cell. Each possibility presents a different embodiment of the invention. In some embodiments, increased activity and / or cytotoxicity is compared to a control NK cell, or a control CD8+T cell.

[0151] As used herein, a “control NK cell” refers to a NK cell that was not exposed to the antibody, the antigen binding fragment or the cell disclosed herein. A “control CD8+T cell” refers to a CD8+T cell that was not exposed to the antibody, the antigen binding fragment or the cell disclosed herein. In some embodiments, a control NK or a control CD8+T cell refers to a NK or CD8+T cell, respectively, from a subject in need thereof, that was not treated by the method disclosed herein.

[0152] In some embodiments, increased activity and / or cytotoxicity is compared to a control target cell. As used herein, a “control target cell” refers to a cell that was not exposed to the antibody, the antigen binding fragment or the cell disclosed herein. In some embodiments, a control target cell refers to a cancer cell that was not contacted with the antibody, the antigen binding fragment or the cell disclosed herein. In some embodiments, a control cancer cell is obtained from a subject in need thereof, that was not treated by the method disclosed herein.

[0153] In some embodiments, the antibody or antigen binding fragment thereof does not inhibit activation of NKG2C in at least one cell selected from: NK cell, CD8+T cell, and any combination thereof. In some embodiments, there is no inhibited activation of NKG2C in a NK cell. In some embodiments, the NK cell expresses at least one molecule selected from: NKG2A, NKG2C, and any combination thereof. In some embodiments, the antibody or antigen binding fragment thereof does not inhibit activation of NKG2C in a NK cell expressing NKG2C. In some embodiments, the antibody or antigen binding fragment thereof does not inhibit activation of NKG2C in a NK cell expressing NKG2C and NKG2A. In some embodiments, the antibody or antigen binding fragment thereof reduces NK cell activation by no more than 30%, compared to a control NK cell. In some embodiments, reduced NK activation is by no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, or no more than 30%, compared to a control NK cell. Each possibility represents a separate embodiment of the invention.

[0154] According to some embodiments, there is provided a method of treating an HLA expressing cancer. In some embodiments, HLA is HLA-E. In some embodiments, HLA is a non-canonical HLA. In some embodiments, the HLA is a canonical HLA.

[0155] According to another aspect, there is provided a method of treating an HLA-E associated disease or disorder, in a subject in need thereof, the method comprising administering to the subject an antibody or antigen binding fragment thereof of the invention, a bispecific antibody of the invention, a cell of the invention, or a pharmaceutical composition of the invention, thereby treating an HLA-E associated disease or disorder.

[0156] According to another aspect, the antibody or antigen binding fragment thereof of the invention, the bispecific antibody of the invention, the cell of the invention, or the pharmaceutical composition of the invention is for use in treating an HLA-E associated disease or disorder. In some embodiments, for use in treating is for use in producing a medicament for treating.

[0157] As used herein, an “HLA-E associated disease or disorder” refers to at least one of: (a) a disease in which HLA-E is expressed on the cell surface of a cell, that does not normally expressthe HLA-E in a control subject, (b) a disease in which HLA-E expression is elevated, and (c) any combination thereof. In some embodiments, an HLA-E associated disease comprises a disease with elevated levels of cell surface HLA-E in a subject’s cell or tissue. In some embodiments, an HLA- E associated disease comprises a disease with elevated levels of soluble HLA-E. In some embodiments, the soluble HLA-E is in a bodily fluid. In some embodiments, the bodily fluid is blood. In some embodiments, the elevated soluble HLA-E levels are observed in the serum of the subject in need thereof. In some embodiments, elevated levels of cell surface or soluble HLA-E are above a predetermined threshold. In some embodiments, elevated levels of cell surface or soluble HLA-E are compared to control subject. As used herein a “control subject” refers to a subject that lacks the pathology that leads to induced levels of HLA-E. In some embodiments, a control subject comprises a healthy individual.

[0158] In some embodiments, the method further comprises a preceding step before “administering”, comprising detection of at least one of: (i) HLA-E expression on the surface of cell, a tissue, or any combination thereof, of a subject in need thereof, (ii) circulating soluble HLA- E expression in the subject’s serum; and, (iii) any combination thereof. Methods for detection of HLA-E, soluble or cell surface, are well known in the art and include, PCR, Q-PCR, northern blot, immunoblot, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), immunostaining, and FACS. In some embodiments, mRNA expression is confirmed. In some embodiments, protein expression is confirmed. In some embodiments, the method comprises FACS analysis of the subject’s tissue or cell to confirm surface expression. In some embodiments, the method comprises ELISA analysis of the subject’s serum to confirm soluble HLA-E levels.

[0159] In some embodiments, the subject is afflicted with a disease that was found to be correlated with expression of a specific allele comprising HLA-E*01:01 or HLA-E*01:03, as described in Kanevskiy L et al. Dimorphism of HLA-E and its Disease Association. Int I Mol Sci. 2019; 20:5496, hereby incorporated by reference in its entirety.

[0160] In some embodiments, an HLA-E associated disease or disorder comprises a viral infection. In some embodiments, the subject in need thereof is afflicted with a viral infection. In some embodiments a viral infection comprises cytomegalovirus (CMV), hepatitis C virus, Epstein-Barr virus, hepatitis B virus, or human immunodeficiency virus 1 (HIV-1).

[0161] In some embodiments, an HLA-E associated disease or disorder is HLA-E expressing cancer. In some embodiments, HLA-E expressing cancer is HLA-E positive cancer. In some embodiments, HLA-E expressing is HLA-E[pHLA-G]expressing. In some embodiments, HLA-Eexpressing is HLA-E[pHLA-C]expressing. According to some embodiments, there is provided a method of treating an HLA-E expressing cancer in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition, comprising the monoclonal antibody or the antigen binding fragment of the invention, the cell of the invention, or any combination thereof, thereby treating an HLA-E expressing cancer.

[0162] In some embodiments, there is provided a method of treating an HLA-E[pHLA G]expressing cancer in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition, comprising the monoclonal antibody or the antigen binding fragment of the invention, the cell of the invention, or any combination thereof, thereby treating an HLA- glpHLA-G]expressing cancer. In some embodiments, the complex comprising HLA-E and SEQ ID NO: 10, induces immune suppression in a subject afflicted with cancer.

[0163] In some embodiments, there is provided a method of treating an HLA-E[pHLA C]expressing cancer in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition, comprising the monoclonal antibody or the antigen binding fragment of the invention, the cell of the invention, or any combination thereof, thereby treating an HLA- E[pHLA-c] expressing cancer. In some embodiments, the complex comprising HLA-E and SEQ ID NO: 11, induces immune suppression in a subject afflicted with cancer. It will be understood by a skilled artisan that as the antibody or the invention also binds to HLA-E in complex with the HLA- C leader peptide (SEQ ID NO: 11) an HLA-E[pHLA C]expressing cancer can also be treated. As such, all embodiments referring to an HLA-Elpl ll AAlexpressing cancer will also be understood to recite separate embodiments, referring to an HLA-E[pHLA-C]expressing cancer.

[0164] In some embodiments, the method comprises administering to a subject in need thereof an agent that inhibits HLA-E[pHLA Gbased immune suppression. In some embodiments, the agent that inhibits HLA-E[pHLA G]based immune suppression binds to HLA-E[pHLA G]. In some embodiments, the agent binds the HLA-E[pHLA G]extracellular domain. In some embodiments, the agent is an HLA- glpHLA G]antagOnist ]nsome embodiments, the agent is an HLA-E[pHLA-G]blocking antibody. In some embodiments, the agent inhibits HLA-E[pHLA G]interaction with NKG2A. In some embodiments, the agent comprises the antibody or the antigen binding fragment of the invention .

[0165] In some embodiments, the method further comprises confirming expression of the complex comprising HLA-E[pHLA G]in the subject is above a predetermined threshold and administering to the subject an agent that inhibits HLA-E[pHLA-G]- based immune suppression, comprising the monoclonal antibody or the antigen binding fragment of the invention, the cell of the invention, orany combination thereof, thereby treating a cancer in a subject. In some embodiments, HLA-E[pHLA-G]comprises soluble HLA-E[pHLA-G]. In some embodiments, HLA-E[pHLA G]comprises cell surface HLA-E[pHLA G]. In some embodiments, confirming comprises detection of cell surface HLA-E[pHLA"G]in a cell or a tissue of a subject in need thereof. In some embodiments, confirming comprises detection of circulating soluble HLA-E[pHLA G]. In some embodiments, soluble HLA-E[pHLA G]levels are detected in the subject’s serum. In some embodiments, elevated levels of cell surface or soluble HLA-E[pHLA G]are above a predetermined threshold. In some embodiments, elevated levels of cell surface or soluble HLA-E[pHLA G]are compared to control subject. As used herein a “control subject” refers to a subject that lacks the pathology that leads to induced levels of HEA-E[pHLA-G]. In some embodiments, a control subject comprises a healthy individual.

[0166] In some embodiments, the subject in need thereof is afflicted with cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises carcinoma. In some embodiments, the cancer is selected from: hematologic cancer, gynecologic cancer, breast cancer, non-small cell lung carcinoma (NSCLC), liver cancer, pancreas cancer, kidney cancer, melanoma, prostate cancer, head and neck cancer, stomach cancer, rectal cancer, colorectal cancer, and any combination thereof. In some embodiments, the cancer comprises a hematological cancer. In some embodiments, the cancer comprises a hematopoietic cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is acute myeloid leukemia (AML).

[0167] In some embodiments, the cancer comprises head and neck cancer. In some embodiments, head and neck cancer comprises a malignancy developed in at least one tissue selected from: lip and oral cavity (mouth), larynx (throat), salivary glands, nose, sinuses, skin of the face, and any combination thereof. In some embodiments, the cancer comprises a squamous cell carcinoma. In some embodiments, the cancer comprises lymphoepithelioma. In some embodiments the cancer comprises a human papillomavirus (HPV) positive cancer. In some embodiments, the cancer comprises at least one cancer type selected from: an oral cancer, paranasal sinus or nasal cavity cancer, sinonasal undifferentiated carcinoma, nasopharynx cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, and any combination thereof.

[0168] In some embodiments, the cancer is a hematologic cancer. In some embodiments, hematologic cancer is selected from leukemia, lymphoma and multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is multiple myeloma. In some embodiments, leukemia comprises acutemyeloid leukemia (AML). In some embodiments, the method disclosed herein is for treating AML, multiple myeloma, or both, in a subject in need thereof.

[0169] In some embodiments, treating comprises increasing immune surveillance. In some embodiments, treating comprises increasing an immune response. In some embodiments, treating comprises decreasing tumor burden. In some embodiments, treating comprises reducing cancer metastasis. In some embodiments, treating comprises increasing cytotoxicity against the cancer. In some embodiments, treating comprises increasing inflammatory response against the cancer. In some embodiments, treating comprises increased phagocytosis of the cancer .

[0170] In some embodiments, treating a subject afflicted with cancer, with the pharmaceutical composition comprising the monoclonal antibody, or antigen binding fragment of the invention, a bispecific antibody of the invention, a scFv of the invention, a CAR of the invention, or the cell of the invention, comprises inducing activation of a NK cell or a CD8+cytotoxic T cell. In some embodiments, inducing activation is of NKG2A expressing NK cell or CD8+T cell. In some embodiments, inducing activation is by reducing internalization of NKG2C on NK cell or CD8+T cell expressing NKG2C. In some embodiments, treating is by (i) inducing activation of NK cell expressing NKG2A and (ii) not inhibiting the activation of a NK cell expressing NKG2C.

[0171] In some embodiments, an antibody or antigen binding fragment of the invention, bispecific antibody of the invention, scFV of the invention, CAR of the invention, or the cell of the invention, is for use in shifting a tumor microenvironment from immunosuppressive to immuno -stimulatory. In some embodiments, said shifting the tumor microenvironment comprises one or more of: inducing / enhancing an anti-tumor T-cell response, increasing T-cell proliferation, and increasing NK cell activity. In some embodiments, antibody or antigen binding fragment of the invention is for use in increasing a T-cell response against a cancer cell. In some embodiments, the T cell response comprises increased pro-inflammatory cytokine secretion. In some embodiments, T cell response comprises increased cytotoxicity. In some embodiments, the T cell response comprises increased T cell proliferation. In some embodiments, antibody or antigen binding fragment of the invention is for use in increasing NK cell cytotoxicity against a cancer cell. In some embodiments, a tumor is a tumor microenvironment (TME) .

[0172] In some embodiments, the antibody or antigen binding fragment, bispecific antibody, scFv, CAR or cell induces in a subject at least 1, 2, or 3 anti-cancer effects. Each possibility represents a separate embodiment of the invention. In some embodiments, the antibody or antigen binding fragment, bispecific antibody, scFv, CAR or cell induces in a subject at least 2 effects. In someembodiments, the antibody or antigen binding fragment, bispecific antibody, scFv, CAR or cell induces in a subject at least 3 effects. In some embodiments, the effects are selected from: increased NK cell cytotoxicity, increased T cell cytotoxicity, increased T cell proliferation, increased secretion of pro-inflammatory cytokine, and any combination thereof. In some embodiments, cytotoxicity is cytotoxicity against a cancer. In some embodiments, the antibody or antigen binding fragment induces in a subject an anti-cancer effect on at least one cell type selected from: T cells, NK cells, dendritic cells and macrophages. In some embodiments, the antibody or antigen binding fragment induces in a subject an anti-cancer effect on T cells, NK cells, and any combination thereof. In some embodiments, the antibody or antigen binding fragment, bispecific antibody, scFv, CAR or cell induces the effect as a monotherapy. In some embodiments, the antibody or antigen binding fragment induces the effect without combination.

[0173] In some embodiments, the antibody or antigen binding fragment, bispecific antibody, scFv, CAR, cell or composition of the invention is for use in combination with another agent. In some embodiments, the use in combination with another agent is for treating an HLA-E expressing cancer. In some embodiments, the agent is an opsonizing agent. In some embodiments, the agent is at least one of: an anti- programmed cell death protein 1 (anti-PD-1) agent, anti-PD-Ll agent, and anti- cytotoxic T lymphocyte-associated antigen (CTLA-4) agent. In some embodiments, the antibody or antigen binding fragment, bispecific antibody, scFv, CAR, cell or composition of the invention is for use in combination with anti-PD-l / PD-Ll based therapy, and / or ant-CTLA-4 based therapy.

[0174] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[0175] As used herein the term “treatment” refers to clinical intervention in an attempt to alter the course of disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of the disease, alleviation of symptoms, reducing a pathological consequence of the disease, reducing the rate of disease progression, amelioration of the disease state, remission or improved prognosis. The term “treatment” may also encompass ex vivo procedures affecting cells or tissues in culture.

[0176] In some embodiments, administering: an antibody or antigen binding fragment of the invention, or a cell of the invention, comprises administering a pharmaceutical composition comprising the antibody or antigen binding fragment of the invention, or the cell of the invention. In some embodiments, a therapeutically effective amount of antibody, antigen binding fragment or a cell is administered. In some embodiments, the pharmaceutical composition further comprises a carrier, excipient or adjuvant. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0177] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. The term “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition.Nucleic acids

[0178] By another aspect, there is provided, a nucleic acid sequence encoding an antibody or the antigen binding fragment of the invention.

[0179] By another aspect, there is provided, a nucleic acid sequence encoding a bispecific antibody of the invention.

[0180] By another aspect, there is provided, a nucleic acid sequence encoding a scFv of the invention.

[0181] By another aspect, there is provided a nucleic acid sequence encoding a CAR of the invention.

[0182] In some embodiments, the nucleic acid sequence is a nucleic acid molecule. In one embodiment, an antibody or antigen binding fragment as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 75% identity to a DNA sequence as described herein. In one embodiment, an antibody or antigen binding fragment as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 80% identity to a DNA sequence as described herein. In one embodiment, an antibody as described herein, or antigen binding fragment is encoded by a DNA molecule comprising a DNA sequence having at least 85% identity to a DNA sequence as described herein. In one embodiment, an antibody or antigen binding fragment as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 90% identity to a DNA sequence as described herein. In one embodiment, an antibody orantigen binding fragment as described herein is encoded by a DNA molecule comprising a DNA sequence having at least 95% identity to a DNA sequence as described herein.

[0183] In some embodiments, there is a nucleic acid molecule encoding an antibody or antigen binding fragment that binds the complex comprising HLA-E[pHLA-G]. In some embodiments, there is a nucleic acid molecule encoding an antibody or antigen binding fragment that binds the complex comprising HLA-E and SEQ ID NO: 9, wherein X is I or F.

[0184] In some embodiments, the nucleic acid is within an expression vector. In some embodiments, the expression vector comprises a plasmid or a virus.

[0185] The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and / or translation of said gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or translation of RNA into a precursor or mature protein (polypeptide).

[0186] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly- Adenine sequence.

[0187] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno- associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.

[0188] In some embodiments, the nucleic acid molecule comprises nucleotide sequences of three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 16 (AACTACTATGTACAC), CDR-H2 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 17 (TGGATTT ATTCTGGAAATTTTTTT ACT AAGTTC A ATGAGAACTTC AAGGGC) , CDR-H3 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 18 (TATGGCAACTACTACTTTGACTAC), CDR-L1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 19 (AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC), CDR-L2 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 20 (TGGGCATCCACCCGGCACACT), and CDR-L3 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 21 (CAGCAACATTATAGCACTCCGTGGACG).

[0189] In some embodiments, the heavy chain signal peptide is encoded by a sequence comprising ATGGGATGGAGCCGGATCTTTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGC (SEQ ID NO: 27). In some embodiments, the heavy chain signal peptide is encoded by a sequence consisting of SEQ ID NO: 27. In some embodiments, the light chain signal peptide is encoded by a sequence comprisingATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGG A (SEQ ID NO: 28). In some embodiments, the light chain signal peptide is encoded by a sequence consisting of SEQ ID NO: 28).

[0190] In some embodiments, the heavy chain variable region is encoded by a nucleic acid sequence comprising ATGGGATGGAGCCGGATCTTTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGCC AGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGA TATCCTGCAAGGCTTCTGGCTACACCTTCACAAACTACTATGTACACTGGGTGAAGCA GAGGCCTGGGCAGGGACTTGAGTGGATTGGATGGATTTATTCTGGAAATTTTTTTACT AAGTTCAATGAGAACTTCAAGGGCAAGGCCACACTGACTGCAGACAAGTCCTCCAGC ACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTACTTCTGTG CATCATATGGCAACTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTC CTCA (SEQ ID NO: 23). In some embodiments, the heavy chain variable region is encoded by a nucleic acid sequence consisting of SEQ ID NO: 23. In some embodiments, the light chain variable region is encoded by a nucleic acid sequence comprising ATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGG AGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGT CAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACA AAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGG AGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGC AGTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGT GGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 24). In some embodiments, the light chain variable region is encoded by a nucleic acid sequence consisting of SEQ ID NO: 24.General definitions

[0191] The term "monoclonal antibody" or “mAb” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variantsthat may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed antibodies to be used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581-597 (1991), for example.

[0192] The mAb of the present invention may be of any immunoglobulin class including IgG, IgM, IgE or IgA. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristine -primed Balb / c mice to produce ascites fluid containing high concentrations of the desired mAbs. mAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

[0193] "Antibody fragments" or “antigen-binding fragment” are used synonymously and comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al, Protein Eng. 8(10): 1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to, Db- Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)-scFv); and Bi-specific T-cell engagers (BiTEs).

[0194] An "antigen" is a molecule or a portion of a molecule capable of eliciting antibody formation and being bound by an antibody. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

[0195] The term "antigenic determinant" or "epitope" according to the invention refers to the region of an antigen molecule that specifically reacts with particular antibody. Peptide sequences derived from an epitope can be used, alone or in conjunction with a carrier moiety, applying methods known in the art, to immunize animals and to produce additional polyclonal or monoclonal antibodies. Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt. cines.fr / ) (IMGT® / V-Quest) to identify variable region segments, including CDRs. See, e.g., Brochet, X. et al, Nucl. Acids Res. J6:W503-508 (2008).

[0196] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0197] The basic unit of the naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains, linked together by both noncovalent associations and by disulfide bonds. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Five human antibody classes (IgG, IgA, IgM, IgD and IgE) exist, and within these classes, various subclasses, are recognized based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0198] The amino terminal regions of the heavy and light chains are more diverse in sequence than the carboxy terminal regions, and hence are termed the variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. A heavy variable (VH) domain and a light variable (VL) domain together form a single antigen-binding site, thus, the basic immunoglobulin unit has two antigen-binding sites. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 824592-4596).

[0199] The carboxy terminal portion of the heavy and light chains form the constant domains i.e., CHI, CH2, CH3, CL. While there is much less diversity in these domains, there are differences from one animal species to another, and further, within the same individual there are several different isotypes of antibody, each having a different function.

[0200] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984)). In addition, complementarity determining region (CDR) grafting may be performed to alter certain properties of the antibody molecule including affinity or specificity. A non-limiting example of CDR grafting is disclosed in US patent 5,225,539.

[0201] Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Antibodies which have variable region framework residues substantially from human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a mouse antibody (termed a donor antibody) are also referred to as humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human / murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (for example PCT patent applications WO 86 / 01533, WO 97 / 02671, WO 90 / 07861, WO 92 / 22653 and US patents 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539). As used herein, the term “humanized antibody” refers to an antibody comprising a framework region from a human antibody and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. Parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. In some cases, however, specific amino acid residues, for example in the framework regions, may be modified, so as to optimize performance of the humanized antibody. Importantly, the humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. For further details, see e.g. U.S. Pat. No. 5,225,539 assigned to Medical Research Council, UK. The terms “a framework region from an acceptor human immunoglobulin” and “a framework region derived from an acceptor human immunoglobulin”, and similar grammatical expressions are used interchangeably herein to refer to a framework region or portion thereof that has the same amino acid sequence of the acceptor human immunoglobulin.

[0202] The term “framework region” or “FR” refers to the amino acid residues in the variable domain of an antibody, which are other than the hypervariable region amino acid residues as herein defined. The term “hypervariable region” as used herein refers to the amino acid residues in the variable domain of an antibody, which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR”. The CDRs are primarily responsible for binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see, Kabat et al.). In some embodiments, CDRs are determined using the KABAT system. In some embodiments, CDRs are determined using the Clothia system. In some embodiments, the Clothia system is the enhanced Clothia system (Martin system) .

[0203] Papain digestion of antibodies produces two identical antigen -binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0204] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three surface of the VH- VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0205] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0206] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0207] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies:IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, delta, e, gamma, and micro, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0208] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0209] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0210] The monoclonal antibodies of the invention may be prepared using methods well known in the art. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pg. 77-96 in “Monoclonal antibodies and cancer therapy”, Alan R. Liss, Inc. (1985).

[0211] Besides the conventional method of raising antibodies in vivo, antibodies can be generated in vitro using phage display technology. Such a production of recombinant antibodies is much faster compared to conventional antibody production, and they can be generated against an enormous number of antigens. Furthermore, when using the conventional method, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation (i.e., increasing the affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, large numbers of different antibodies against a specific antigen can be generated in one selection procedure. To generate recombinant monoclonal antibodies one can use various methods all based on display libraries to generate a large pool of antibodies with different antigen recognition sites. Such a library can be made in several ways: One can generate a synthetic repertoire by cloning synthetic CDR3 regions in a pool of heavy chaingermline genes and thus generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected. One can use the lymphocyte pool of humans as starting material for the construction of an antibody library. It is possible to construct naive repertoires of human IgM antibodies and thus create a human library of large diversity. This method has been widely used successfully to select a large number of antibodies against different antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are provided in the well-known reference text Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0212] Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human complementarity determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0213] In some embodiments, antibodies as described herein are neutralizing antibodies. "Neutralization", as discussed here, is defined as the reduction in protein function by antibodies of the invention. In one embodiment, "neutralization", as discussed here, is binding of antibodies to the surface of immune cells, preferably to immature and mature myeloid linage derived cells, T cells and NK cells, thereby blocking the propagation of inhibitory signals inside these cells and conferring a less suppressive phenotype and function.

[0214] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0215] Polynucleotides encoding polypeptides may be obtained from any source including, but not limited to, a cDNA library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level. Accordingly, polynucleotides encoding a polypeptide can be conveniently obtained from a cDNA library prepared from human tissue. The polypeptide -encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0216] For example, the polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH) but also a heavy chain constant region (CH), which typically will comprise three constant domains: CHI, CH2 and CH3; and a "hinge" region. In some situations, the presence of a constant region is desirable.

[0217] Other polypeptides which may be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and CHI and CK or CL domain has been excised. As minibodies are smaller than conventional antibodies they should achieve better tissue penetration in clinical / diagnostic use, but being bivalent they should retain higher binding affinity than monovalent antibody fragments, such as dAbs. Accordingly, unless the context dictates otherwise, the term "antibody" as used herein encompasses not only whole antibody molecules, but also antigen-binding antibody fragments of the type discussed above. Each framework region present in the encoded polypeptide may comprise at least one amino acid substitution relative to the corresponding human acceptor framework. Thus, for example, the framework regions may comprise, in total, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions relative to the acceptor framework regions. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0218] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0219] As used herein the term “subject” refers to an individual, or a patient, which is a vertebrate, e.g., a mammal, including especially a human. In some embodiments, the subject is a human. In some embodiments, the subject is a mammal. In some embodiments, the subject suffers from cancer.

[0220] The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the appended claims.EXAMPLES

[0221] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: Alaboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I- III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); "Monoclonal Antibodies: Methods and Protocols". Vincent Ossipow, Nicolas Fischer. Humana Press (2014); "Monoclonal Antibodies: Methods and Protocols". Maher Albitar. Springer Science & Business Media (2007), all of which are incorporated by reference. Other general references are provided throughout this document.Materials and Methods

[0222] Cell lines and culture conditions: 721.221 WT (ATCC CVCL_6263), 721.221 transfectants expressing HLA-E and HLA-G molecules, RPMI 8266 (ATCC CRM-CCL-155), U266 (ATCC TIB-196), and U937 (ATCC CRL-1593.2) Cells were cultured in RPMI 1640 (Gibco, Life Technologies) medium and SP2 / 0-Agl4 (ATCC no. CRL-1581) was cultured in DMEM (mention the company name). All media were supplemented with 10% FBS (Gibco, 12657-029), 1% L- Glutamine, 1% Pen-Strep, 1% MEM-Eagle, 1% Sodium Pyruvate, and 1% HEPES IM (Biological Industries).

[0223] Generation of Hybridoma: To produce HLA-E-specific mAb 4D7-B6, 4-week-old BALB / c mice were injected subcutaneously (SC) with 15pg\mouse of pHLA-E-mIgG2a-Fc carried with VMAPRTLFL peptide complex (8343.1) diluted in PBS 1:1 with Complete Freund Adjuvant (CFA). On days 14, 35, 52, and 101 post-injection, mice received the 2nd, 3rd, 4th, and 5th injections with incomplete Freund Adjuvant (IFA). On day 112, mice received an IFA injection booster with 15pg\mouse of 8343.1 complex diluted in PBS; while control mice were injected with adjuvant only. Blood was drawn from the tail of all mice 24 and 45 days post-first injection and was assayed for 8343.1 complex recognition using ELISA assay. 4 days after boost, mice with the higher antibody titer were sacrificed and the splenocytes were isolated. 1x108 splenocyte cells werecultured with 2x107 SP2 / 0-Agl4 myeloma cells, and the splenocytes were fused with mouse Sp2 / 0 using standard fusion methods. The cells were spread into 96-well plates, cultured in DMEM with 20% FBS, and selected with HAT supplement (Sigma). After 14 days of HAT selection, hybridoma supernatants were screened for HLA-E peptide recognition using an ELISA assay. Positive hybridomas were selected, subcloned to ensure monoclonality, and compared to the recognition of the 8341.1 complex. The 4D7-B6 hybridoma was cultured in HT supplement (Sigma) selection medium and further in FreeStyle 293 Expression Medium (Gibco, Life Technologies) for production and purification of the antibody. Antibody purification was performed using a HiTrap Protein G HP Columns and FPLC System (GE Healthcare).

[0224] ELISA assay for mAb 4D7 antigen binding: A 96F-well plate (Costar™) was coated overnight at 4°C with 2ug / ml of pHLA-E-m!gG2a-Fc proteins that carried different peptides; VMAPRTLIL (8341.1), VMAPRTLFL (8343.1), QMRPVSRVL (8345.1) in Na2HPO4 buffer of pH=9. After washing with PBS-T (PBS + 0.05% TWEEN-20 (Sigma)) the plate was blocked with 1% BSA in PBS-T for 1.5 hours at 37°C followed by washing with PBS-T and incubated for a further 1 hour at 37°C with 2ug / ml of 4D7 mAb or purified mouse IgGlK isotype control antibody (BioLegend 401402). Detection was done after washing by Peroxidase AffiniPure Goat Anti-Mouse IgG - light chain specific with a final dilution of 1:500 (Jackson Immunoresearch). Following washing, TMB (BioFX) was added, and the absorbance was measured at 650nm (Thermo Electron Multiskan Spectrum).

[0225] Flow cytometry for surface HLA-E detection: Target cells were detached with Versene, diluted 1:5000 (Gibco, Life Technologies), and washed with PBS. The cells were counted, seeded in a 96-well U-bottom plate (50,000 cells / well), and incubated for 1.5 hours on ice with a final concentration of 2ug / ml of 4D7 mAb or purified mouse IgGlK isotype control antibody (BioLegend). The samples were then washed and stained with APC Goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) for 30 min on ice. Cells were washed twice and suspended with DAPI 1 g / mL followed by FACS analysis using a Beckman Coulter Cytoflex flow cytometer.

[0226] HLA-E Peptide Stripping: 0.5*106 cells / well were seeded in a 96-well plate and washed with PBS. Pellet was suspended in 25ul of ice-cold citric acid in Na2HPO4 buffer of pH=5 (made by mixing an equal volume of 0.263 M citric acid and 0.123 M Na2HPO4). The cells were incubated with the buffer for 1.5 min on ice followed by washing twice with 225ul / well of RPMI (0% FBS). Peptides were added at a final concentration of 20ug / ml diluted in RPMI (0% FBS) and 10% DMSO for 1 hour at 37°C and 5% CO2. Cells were stained with 4D7 to test the recognition of the mAb against the set of peptides.

[0227] Molecular docking studies: The structure for HLA-EVMAPRTLFL (SEQ ID NO: 10) complex was retrieved from protein data bank (PDB: 3CDG). Structures of both 4D7 and HLA- SEQ ID NO: 10 were prepared, and any structural inconsistencies were corrected using Schrodinger's protein preparation module. 4D7 was docked to HLA-SEQ ID NO: 10 complex using ClusPro2 web server ( / / cluspro.bu.edu / home.php) using Antibody mode. The top poses were selected based on Cluster size and corresponding energy was further calculated by: E— 0.5 OEiep- — 0.20Eatt+600Eelec+0.25EDARS •

[0228] Kinetic analysis of the antibody by surface plasmon resonance: The binding of 4D7 to recombinant HLA-E[pHLA G]protein (8343.1) was measured using a ProteOn XPR36 instrument (Bio-Rad Laboratories, CA, USA). 8343.1 was immobilized on a GLC chip (Bio-Rad) using standard amino coupling procedures: activating the carboxymethyl groups on the chip’ s surface with 20 mM ED AC and 5 mM S-NHS run at 30ul / min for 300 sec. Next, 0.49ug of 8343.1 was resuspended in Acetate buffer pH4.5 and run at 30ul / sec for 300sec. 0.36 ug of recombinant HLA- E[PHSP60] prO(Cjn(8345.1) was also resuspended in buffer acetate pH4.5 and was run on another channel as a negative control. Deactivation of empty carboxyl residues was done with IM EA at 30ul / min for 300 sec. All running conditions were done using PBST (PBS xl + 0.005% Tween 20) and at 25°C.

[0229] 4D7 was run at 5 different concentrations (78, 39, 19.5, 9.75, 4.875, and 0 um) at 40ul / min for 613 sec followed by a dissociation step of 600 sec. The 8345.1 channel sensograms and the 4D7 zero concentration were subtracted as background. Data was analyzed on the ProteOn manager software version 3.01 using the equilibrium model for measuring affinity.

[0230] Isolation and culture of primary human NK cells: Primary human NK cells were isolated from the peripheral blood of healthy donors via RosetteSep Human NK Cell Enrichment Cocktail (EasySep-19615, STEMCELL-negative selection isolation kit). After isolation, purified NK cells were cultured in serum-free medium, Xeno-free (CellGenix GMP SCGM) supplemented with 10% heat-inactivated human AB plasma from healthy donors (SIGMA, male AB, H-4522, Israel, Jerusalem), 1% L-glutamine, 1% Pen-Strep, 1% sodium pyruvate, 1% MEM-Eagle, 1% HEPES IM, and 300 lU / mL recombinant human IL-2 (PeproTech).

[0231] NK Cell Functional Assay: Target cells were detached with Versene 1:5000 and washed with PBS. The cells were counted, seeded in a 96-well U-bottom plate (150,000 cells / well), and incubated for 1.5 hours at 37°C and 5% CO2 with 4D7 mAb or Purified mouse IgGlK Isotype control Antibody (BioLegend). The antibodies were diluted in RPMI with 10% FBS at a finalconcentration of lOug / ml. pNK cells were then counted and added to the plate (50,000 cells / well) along with anti-human CD107a FITC (Biogems, Rehovot, Israel), at a final concentration of 50 U / IL-2. After incubating the cells for 4 h at 37°C and 5% CO2, the cells were washed once and stained with anti-human CD107a FITC, NKG2A APC, NKG2C PE, CD56 APC / Cy7, CD16 PerCP / Cy5.5 (Biolegend) for 30 min on ice. Degranulation was assessed by FACS.EXAMPLE 1: Generation of anti- HLA-E[pHLA G]mAb, named 4D7

[0232] Mice were immunized to generate a hybridoma specific to the complex of HLA-E and the HLA-G derived leader peptide VMAPRTLFL, SEQ ID NO: 10 (HLA-E[pHLA G]). In order to enhance the chance of getting such a hybridoma, mice were immunized in two different consecutive sets of experiments. In each experimental set mice were immunized 4 times, with intervals of 3 weeks. The 1stimmunization was a subcutaneous administration of the recombinant peptide emulsified in Complete Freund's Adjuvant (CFA). The additional 3 immunizations comprised subcutaneous administration of the recombinant peptide emulsified in Incomplete Freund's Adjuvant (IFA). An additional boost of intravenous administration of the purified protein was performed 4 days prior to the splenocytes infusion.

[0233] In each experimental set, there were two mice groups: (i) a group immunized with the disulfide-trapped HLA-E / Bi-microglobulin single-chain trimers (SCT) conjugated with the HLA-G leader peptide (HLA-E[pHLA G], 8343.1); and, (ii) a group immunized with the disulfide-trapped HLA-E / Bi-microglobulin single-chain trimers (SCT) conjugated with the HSP60 leader peptide QMRPVSRVL (SEQ ID NO: 15) (8345.1).

[0234] Testing sera from immunized mice showed that following the 4thimmunization, the level of response to the antigen reached a plateau, therefore, the boost and splenocytes fusion with SP2 / 0 myeloma counterpart were performed right after. It should be noted that there were no observed differences in the response of mice immunized with HLA-E+VMAPRTLFL (SEQ ID NO: 10) (HLA-E[pHLA G]) to either the peptide HLA-E+VMAPRTLFL (SEQ ID NO: 10) (HLA-E[pHLA G]) or to the peptide HLA-E+QMRPVSRVL (SEQ ID NO: 15). This is a result of the presence of polyclonal antibodies in the sera and the fact that most clones recognize the HLA-E backbone, rather than the specific HLA-E+peptide complex. The rarity of plasma cells that produce mAb specific to the HLAE-leader peptide (HLA-E[pHLA-G]) complex was the basis for the inventor’s strategy to have two repeated experimental sets, with the aim to screen hundreds to thousands of fusion wells.

[0235] Following several fusion experiments, screening of growing hybridomas, cloning and recloning of hybridomas secreting plausible candidate mAbs, the inventors were able to stabilize ahybridoma, termed 4D7, that secretes a monoclonal antibody (4D7 mAb), that was found to specifically recognize the recombinant protein complex comprising HLA-E+VMAPRTLFL (SEQ ID NO: 10) (HLA-E[pHLA G]), as compared to HLA-E+QMRRPVSRVL (SEQ ID NO: 15) (Fig. 1A). The mAb 4D7, was found to also recognize the recombinant protein complex comprising HLA- E+VMAPRTLIL (SEQ ID NO: 11 ; from the leader peptide of HLA-C), that has a single amino acid (AA) change (F to I) within the peptide part of the complex.

[0236] The affinity of 4D7 to recombinant peptide-complexed HLAE was measured using SPR and the KD was calculated using an equilibrium model that was statistically validated with Chi-square divided by Rmax value of less than 10%. mAb 4D7 bound to recombinant HLA-E complexed with the SEQ ID NO: 10 peptide derived from the leader sequence of HLA-G (HLA-E[pHLA-G]) with a relatively high affinity (KD=1.17x10-9 mol / L; Fig. IB); no measurable binding affinity was detected for the interaction of 4D7 with recombinant HLA-E complexed with the SEQ ID NO: 15 peptide derived from HSP60 leader sequence (HLA-E[pHSP60]). To complement ELISA and SPR binding results with molecular docking analysis, the inventors sequenced the Fv of mAb 4D7 and generated the homology model for its VH-VL region using the Abodybuilder2 webserver which utilizes deep learning models to accurately predict the antibody's structure. The structure for HLA- gtpHLA-G]comp|ex wasretrieved from protein data bank (PDB: 3CDG, NKG2A and HLA-E[pHLA-G]complex). Structures of both mAb 4D7 and HLA-E[pHLA G]I HLA-E[pHSP60]I HLA-E without peptide were then prepared and corrected for inconsistencies (see Materials and Methods). 4D7 was docked to the different HLA-E complexes using ClusPro2 web server. The model of the 4D7 mAb docked to HLA-E[pHLA-G]is shown in Figure 1C while Figure ID shows the model of mAb 4D7 docked to HLA-E without a peptide. The model-observed shifting of 4D7 binding from the peptide groove (Fig. ID) which indicates irrelevant binding. Figure IE shows the center weighted scores for binding free energy of mAb 4D7 to the HLAE complexed with either the SEQ ID NO: 10 (HLA- E[pHLA-G])orSEQ j£) NO: 15 (HLA-E[pHSP60]) peptides as well as HLAE without complexed peptide. Significant energy differences were observed between the complexes which indicates that mAb 4D7 has a preferential binding towards HLAE complexed with the VMAPRTLFL (SEQ ID NO: 10) peptide.

[0237] To verify that 4D7 can bind to cell lines expressing membrane-associated HLA-E presenting endogenous peptides, HLA-E positive cancer cell lines were stained. Figure IF shows the mAb 4D7-based flow cytometry staining results for 5 cell lines. These cell lines include WT 721.221 that express null to very low levels of HLA-E and 4 other cell lines that express detectable levels of HLA-E including 2 multiple myeloma cell lines (RPMI 8226 & U266), one AML cell line (U937)and HLA-G-transfected 721.221 (721.221 HLA-G) that express endogenous HLA-E mostly loaded with the VMAPRTLFL (SEQ ID NO: 10) nonapeptide derived from the leader sequence of transfected HLA-G. Except for the HLAE-dull 721.221 cell line, all other cell lines were successfully stained with mAh 4D7.EXAMPLE 2: Characterization of mAh 4D7 binding to HLA-E loaded with single A A mutations of VMAPRTLFL (SEQ ID NO: 10)

[0238] To better characterize the effect of different single AA substitutions within the SEQ ID NO: 10 peptide on the binding of 4D7 mAb to HLA-E+peptide, the inventors employed 45 VMAPRTLFL (SEQ ID NO: 10) single AA mutated versions. In each version there was a substation of a single AA as compared to the original peptide. The consequential synthetic mutated peptides were loaded onto acid-washed 721.221-HLA-G cells, which are 721.221 cells transfected with HLA-G. After transfection, HLA-G is expressed, and its signal peptide cleaved. This nonapeptide, VMAPRTLFL (SEQ ID NO: 10), is known to be loaded on the endogenous HLA-E, which stabilizes it, and the complex HLA-E[pHLA G]is then expressed on the membrane of 721-HLA-G cells. Following the acid wash, endogenous peptides, including SEQ ID NO: 10, were stripped from the HLA-E, enabling the exogenously added peptides, either the original SEQ ID NO: 10 peptide or each of the 45 single AA-mutated peptides, to be loaded onto the HLA-E. Overall, the tested mutations included a single AA mutation in all nine positions of the peptide, but with emphasis on positions 1, 4, 5, and 8 from the N-terminal end of the peptide, considered to face out towards the epitope of NKG2A receptor. Based on these experimental results demonstrated in Figure 2, the inventors could identify some binding criteria regarding 4D7 recognition of HLA-E[pHLA-G]complex. The recognition of the HLA-E[pHLA G]complex by mAb 4D7 was significantly affected by substitutions in positions 1, 4 and 5 of the original SEQ ID NO: 10 peptide. It was found that 4 out of 7 mutations in positions 1, 4 out of 6 mutations in position 4, and 11 out of 13 mutations in position 5, caused a clear reduction in the binding of 4D7 mAb, as compared to binding with a commercial Ab to HLA-E. In contrast, only 1 out of 6 mutations in position 8 (F8D) reduced the binding of 4D7. Interestingly, some mutations at positions 2, 3, 6 and 9 also produced larger reductions. These findings demonstrate that the epitope for the 4D7 mAb, within the complex HLA- g[pHLA-G],C()|T1prjsesthe A A positioned at residues 1, 4 and 5 of the VMAPRTLFL (SEQ ID NO: 10) peptide. The other residues of the SEQ ID NO: 10 peptide (2, 3, 6, 7, 9) are associated with binding of the peptide to the HLA-E groove. Thus, interpretation of changes in recognition is more complex as reduced binding could be due to reduced binding affinity of the exogenously added peptides to HLA-E itself.EXAMPLE 3: The 4D7 mAb enhances the function of NKG2A-positive NK cells exposed to tumor cells

[0239] mAb 4D7 was isolated based on specificity to complex HLA-E[pHLA G]. Yet, based on the results shown in Figure 2, it should have even better affinity to a certain subset of HLA-E loaded peptides. Hence, it was asked was whether the subset of HLA-E loaded peptides that mAb 4D7 recognizes is correlated with the subgroup of HLA-E loaded peptides that are recognized by the NKG2A / CD94, which is a suppressor receptor expressed by NK and T cells. An additional question was whether mAb 4D7 recognizes the subset of peptides presented by HLA-E to the NKG2C / CD94 receptor, which is an activating receptor of NK cells.

[0240] To answer these questions, the inventors grew primary NK cells from healthy donors and tested the effect of the 4D7 mAb on the activation of NKG2A+and NKG2A" subpopulations. Figures 3A-B show results from primary NK cell culture from five donors that lacked NKG2C expression. Thus, the effect of 4D7 mAb was examined for either NK cells expressing (Fig. 3A) or not expressing (Fig. 3B) the NKG2A receptor. Six target cell lines were investigated. RPMI 8226 (human multiple myeloma cell line), U266 (human multiple myeloma cell line) and U937 (human acute myeloid leukemia cell line) are known to express detectable levels of HLA-E. The wildtype cell line 721.221 (b-lymphoblastoid cell line) expresses no to very low levels of surface HLA-E. However, transfection of 721.22 cells with either the HLA-E gene (721.221 HLA-E), or the HLA- G gene (721.221 HLA-G) induces HLA-E surface expression. Addition of the 4D7 mAb to the coculture significantly enhanced the activity of NKG2A-positive NK cells, in all the five examined cell lines expressing detectable levels of HLA-E (Fig. 3A, data for 721.221 HLA-E not shown). In the WT 721.221 coculture no enhanced NK activity was observed as these cells do not strongly express HLA-E. Co-culture of NKG2A-negative NK cells with the same target cells lines in the presence of 4D7 mAb did not enhance NK activity (Fig. 3B). The NKG2C receptor also recognizes peptide-loaded HLA-E complexes; albeit it recognizes subgroup of peptides with different affinity from NKG2A. Four NK donors that were NKG2A- and NKG2C+ were co-cultured with various cell lines and activity was measured. mAb 4D7 did not affect the activity of these cells; except that some reduction of activity was observed for the HLA-G-transfected 721.221 cells (Fig. 3C). These results demonstrate the ability of the 4D7 mAb to convert the HLA-E- mediated inhibition of NK cells to activation by inhibiting the binding of peptide-loaded HLA-E to NKG2A receptor.

[0241] The NKG2C / CD94 receptor also recognizes peptide- loaded HLA-E complexes; albeit it recognizes a subgroup of peptides with different affinity compared to NKG2A / CD94. NKG2C positive primary NK cells were acquired and cultured with 3 subsets of primary NK cells: NKG2A"NKG2C+, NKG2 A+NKG2C\ and NKG2A NKG2C". Similar results to those of the first donor were observed for the NKG2A+NKG2C" cells, although a mild, possible non-specific, reduction in activation upon treatment with the 4D7 antibody was observed in the 721 WT coculture (Fig. 4A). As these cells do not express HLA-E for the most part, and since the effect was a reduction rather than increase in activation, this result is likely an artifact or non-specific. Culture with the NKG2A" NKG2C" NK subsets, produced no enhancement of NK function when co-cultured with 721-HLAE in the presence of the 4D7 mAb, pointing to its specific activity via the NKG2A receptor (Fig. 4B). For the NKG2A’NKG2C+subset, which could not be studied for the 1stdonor, application of mAb 4D7 to the coculture had no effect on activation in 4 of the 5 HLA-E positive cell lines (Fig. 4C). Even in the HLA-G line which showed strong activation (likely due to interaction with NKG2C), the 4D7 antibody had no effect. A small reduction in activation was observed in the coculture with 721.HLA-E cells, however, this was similar to reduction observed in the WT coculture which as explained above is likely an artifact or non-specific. Therefore, it was demonstrated that the 4D7 mAb can efficiently bind and block the subset of HLA-E+peptide complexes that are recognized by the inhibitory receptor NKG2A / CD94, and yet, do so without blocking the subset of HLAE+peptide complexes that are recognized by the activating NK receptor NKG2C / CD94.

[0242] Finally, the activity of NKG2A+NKG2C-, NKG2A-NKG2C- and NKG2A-NKG2C+ NK cells in presence of mAb 4D7 was assessed for cells donated by six different MM patients (Fig. 5A- C). There was a clear enhancement of NK activity for NKG2A+NKG2C- NK cells when 4D7 was added to NK and HLA-E-positive target co-cultures (Fig. 5A). The NKG2C receptor also recognizes peptide-loaded HLA-E complexes; albeit it recognizes subgroup of peptides with different affinity from NKG2A. NKG2A-NKG2C+ activity was not affected by mAb 4D7 when co-cultured with autologous or allogenic primary MM targets as well as with two MM cell lines (Fig. 5B). Adding mAh 4D7 to a co-culture of target cells and NKG2A-NKG2C- (double negative) NK cells did not affect NK activity, though a small increase in activity was observed again the allogeneic primary MM cells (Fig. 5C).EXAMPLE 4: MAb 4D7 can bind and stain formalin-fixed paraffin-embedded (FFPE) cuts from patient derived xenografts (PDXs)

[0243] Figure 6 shows that the 4D7 mAb can recognize and stain primary tumor cells following in vivo growth of human cancer cells in immune incompetent NOD scid gamma (NSG) mice. mAb staining is shown both for HLA-E positive PDX (PDX1) and HLA-E negative PDX (PDX2).EXAMPLE 5: Generating Chimeric Antigen Receptors (CARs) from MAb 4D7

[0244] CARs were generated from Mab 4D7 according to standard protocols. The hinge, transmembrane domain and cytosolic regions of human CD28 were used along with mouse CD3 zeta (so the CAR could be tested in mouse cells). Two orientations for the single chain variable region (scFv) derived from 4D7 were investigated. In the first, the heavy chain variable region was placed N-terminal to the light chain variable region and in the second the light chain variable region was placed N-terminal. In both cases, the two variable regions were separated by a (G4S)4 (SEQ ID NO: 35) linker. A Myc tag was placed between the antigen binding domain and the transmembrane domain, making surface expression of the CARs detectable with an anti-Myc antibody. In order to express the CARs in BW cells, a signal peptide was included at the N-terminus of the construct. In the CAR with an N-terminal heavy chain variable region the signal peptide from the IgG heavy chain (SEQ ID NO: 25) was used. In the CAR with an N-terminal light chain variable region the signal peptide from the IgG light chain (SEQ ID NO: 26) was used. A third signal peptide, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 34) hereinafter referred to as the METD peptide, predicted to be compatible with the CAR, was also tested and was included in both the CAR with an N-terminal heavy chain variable region and the CAR with an N-terminal light chain variable region. This resulted in 4 constructs to be tested (Fig. 7A).

[0245] BW cells were transduced with the 4 CAR constructs and surface expression was measured using an anti-MyC antibody. This was done first with a mouse anti-Myc primary antibody (2 ug\ml), followed by washing and incubation with goat anti-mouse IgG APC labeled secondary antibody (2 ug\ml). Surface APC levels were measured by flow cytometry. Surprisingly, the METD peptide on a CAR with an N-terminal light chain variable region and CAR with a heavy chain signal peptide produced significantly more expression than the construct with the light chain signal peptide (Fig. 7B).

[0246] Next, it was checked if the scFv retained the binding ability of the parent antibody. To test this, a mouse IgGl fusion protein was created. This protein contains an N-terminal mouse IgGl constant region followed by HLA-E, a flexible linker and the peptide VMAPRTLFL (SEQ ID NO: 10). BW cells transduced with the 4 CAR constructs were incubated with the recombinant fusion protein, washed and then incubated with the same goat anti-mouse IgG APC antibody. Positive APC fluorescence is indicative of binding of the CAR to the HLA-E / peptide complex as the 4D7 antibody did. As can be seen in Figure 7C, the trend in fluorescence was the same as the previous experiment which only measured surface expression, indicating that the CARs that reached the cell surface were functionally capable of binding the target antigen. It was also apparent that the constructs with theMETD signal peptide were superior to the construct with the heavy chain signal peptide. This taken together with the results presented in Figure 7B indicate that the METD signal peptide are superior to both the heavy and light chain signal peptides. Further, the METD construct with the light chain variable region N-terminal to the heavy chain variable region consistently and surprisingly showed better expression than the METD construct with the reversed orientation.

[0247] Finally, the functionality of the CARs and their specificity was examined. Tissue culture wells were coated with 2 ug / ml of the recombinant IgG-HLA-E / peptide complex. Two complexes were tested: one that included SEQ ID NO: 10 and is expected to induce signaling by the CAR and one that included the peptide QMRPVSRVL (SEQ ID NO: 15) which is a negative control peptide and should not be bound by the CAR and not induce signaling. Only the two best expressing CARs (METD-light chain variable-linker-heavy chain and VH SP-heavy chain variable-linker-light chain variable) were tested. The CARs were transduced into BW cells and the cells were cultured either in wells containing the target complex or wells containing the control complex. IL-2 secretion into the media was measured by mouse IL-2 ELISA assay as a readout for CAR signaling. Both CARs induced IL-2 secretion in response to the target HLA-E / peptide complex, once again confirming that the scFv fragments in the CARs are functional (Fig. 7D). Surprisingly, while the METD-light chain variable-linker-heavy chain CAR produced only background IL-2 secretion in response to the control complex, the VH SP-heavy chain variable-linker-light chain variable CAR produced increased IL-2 in response to the negative control. This indicates that for some reason this configuration reduces the specificity of the antigen binding region and produces more promiscuous binding. Thus, the light chain variable region being N-terminal to the heavy chain variable region appears to be a superior configuration for the scFv and the CAR.

[0248] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A monoclonal antibody or antigen binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (NYYVH), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 (WIYSGNFFTKFNENFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 (YGNYYFDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 (KASQDVSTAVA), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 (WASTRHT), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 (QQHYSTPWT).

2. The antibody or antigen binding fragment of claim 1, comprising a heavy chain comprising the amino acid sequence:MGWSRIFLFLLSIIAGVHCQVQLQQSGPELVKPGASVRISCKASGYTFTNYYVH W VKQRPGQGLEWIGWIYS GNFFTKFNENFKGKATLT ADKS S S T A YMQLS S LTS EDSAVYFCASYGNYYFDYWGQGTTLTVSS (SEQ ID NO: 7).

3. The antibody or antigen binding fragment of claim 1 or 2, comprising a light chain comprising an amino acid sequence:MESQIQAFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAV AWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALY YCQQHYSTPWTFGGGTKLEIK (SEQ ID NO: 8).

4. The antibody or antigen binding fragment of any one of claims 1 to 3, wherein said antibody comprises an IgGl constant region.

5. The antibody or antigen binding fragment of any one of claims 1 to 4, wherein said antibody or antigen binding fragment is humanized.

6. The antibody or antigen binding fragment of any one of claims 1 to 5, wherein said antibody or antigen binding fragment thereof binds to a fragment of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is I or F; blocks interaction between NKG2A and HLA-E or both.

7. A monoclonal antibody, or antigen binding fragment, that binds an epitope comprising a fragment of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and a peptide comprising the sequence VMAPRTLXL (SEQ ID NO: 9), wherein X is I or F.

8. The antibody, or antigen binding fragment of claim 7, wherein said peptide and said HLA-E are complexed together.

9. The antibody, or antigen binding fragment of claim 7 or 8, wherein said SEQ ID NO: 9 is VMAPRTLFL (SEQ ID NO: 10).

10. A monoclonal antibody, or antigen binding fragment, that blocks interaction of Killer cell lectin-like receptor subfamily C, member 1 (NKG2A) with HLA-E.

11. The antibody, or antigen binding fragment of claim 10, that does not block interaction of NKG2C with HLA-E.

12. A bispecific antibody comprising: (i) the antigen binding fragment of any one of claims 1 to 11; and (ii) an antigen binding fragment specific to a receptor expressed by an immune cell.

13. A single chain variable fragment (scFv) comprising a variable region from a heavy chain and a variable region from a light chain separated by a peptide linker, wherein said variable region from a heavy chain comprises CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1 (NYYVH), CDR-H2 comprising the amino acid sequence as set forth in SEQ ID NO: 2 (WIYSGNFFTKFNENFKG), and CDR-H3 comprising the amino acid sequence as set forth in SEQ ID NO: 3 (YGNYYFDY), and wherein said variable region from a light chain comprises CDR-L1 comprising the amino acid sequence as set forth in SEQ ID NO: 4 (KASQDVSTAVA), CDR-L2 comprising the amino acid sequence as set forth in SEQ ID NO: 5 (WASTRHT), and CDR-L3 comprising the amino acid sequence as set forth in SEQ ID NO: 6 (QQHYSTPWT).

14. The scFv of claim 13, wherein said linker is a GGGGS (SEQ ID NO: 32) linker.

15. The scFv of claim 13 or 14, wherein said variable region from a heavy chain is N- terminal to said variable region from a light chain.

16. The scFv of claim 15, wherein said scFv further comprises a signal peptide of an IgG heavy chain, optionally wherein said signal peptide of an IgG heavy chain comprises the amino acid sequence MGWSRIFLFLLSIIAGVHC (SEQ ID NO: 25).

17. The scFv of claim 13 or 14, wherein said variable region from a light chain is N-terminal to said variable region from a heavy chain.

18. The scFv of claim 17, wherein said scFv further comprises a signal peptide of an IgG light chain, optionally wherein said signal peptide of an IgG light chain comprises the amino acid sequence MESQIQAFVFVFLWLSGVDG (SEQ ID NO: 26).

19. The scFv of claim 17, wherein said scFv further comprises a signal peptide comprising the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 34).

20. A chimeric antigen receptor (CAR) comprising an antigen recognition domain comprising the antigen binding fragment of any one of claims 1 to 11 or an scFv of any one of claims 13 to 19.

21. The CAR of claim 20, further comprising a transmembrane domain and an intracellular cell activation domain.

22. The CAR of claim 21, wherein said intracellular activation domain comprises CD3-zeta chain.

23. The CAR of claim 22, wherein said transmembrane domain comprises the amino acid sequence as set forth in SEQ ID NO: 29 and said CD3-zeta chain comprises the amino acid sequence as set forth in SEQ ID NO: 30.

24. A cell comprising any one of: (i) a monoclonal antibody or antigen binding fragment of any one of claims 1 to 11, (ii) the bispecific antibody of claim 12, (iii) a scFv of any one of claims 13 to 19, (iv) a CAR of any one of claims 20 to 23, and (v) any combination thereof.

25. The cell of claim 24, wherein any one of: said monoclonal antibody or antigen binding fragment, said bispecific antibody, and said CAR, are anchored to the plasma membrane of said cell.

26. The cell of claim 24 or 25, further comprising a monoclonal antibody or antigen binding fragment specific to a receptor expressed by an immune cell.

27. The cell of any one of claims 24 to 26, wherein said cell is a cytotoxic immune cell selected from an NK cell and a T cell.

28. A pharmaceutical composition comprising any one of: (i) an antibody or antigen binding fragment of any one of claims 1 to 11, (ii) the bispecific antibody of claim 12, (iii) a scFv of any one of claims 13 to 19, (iv) a CAR of any one of claims 20 to 23, (v) a cell of any one of claims 24 to 27, and (vi) any combination thereof, and a pharmaceutically acceptable carrier, excipient or adjuvant.

29. A method of blocking binding of Killer cell lectin-like receptor subfamily C, member 1 (NKG2A) to a complex comprising HLA-E and a peptide comprising SEQ ID NO: 9, the method comprising contacting said complex with any one of: (i) an antibody or antigen binding fragment of any one of claims 1 to 11, (ii) the bispecific antibody of claims 12, (iii) a scFv of any one of claims 13 to 19, (iv) a CAR of any one of claims 20 to 23, (v) a cell of any one of claims 24 to 27, (vi) the pharmaceutical composition of claim 28, and (vii) any combination thereof, thereby blocking binding.

30. The method of claim 29, wherein said blocking comprises increasing activation of an NK cell expressing said NKG2A.

31. The method of claim 29 or 30, wherein said antibody or antigen binding fragment thereof does not inhibit activation of NKG2C in an NK cell expressing said NKG2A.

32. A method of treating an HLA-E expressing disease in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition of claim 28, thereby treating an HLA-E expressing cancer.

33. The method of claim 32, wherein said HLA-E expressing disease comprises surface expression of HLA-E above a predetermined threshold or comprises circulating soluble HLA-E above a predetermined threshold.

34. The method of claim 32 or 33, wherein said HLA-E expressing disease is HLA-E expressing cancer.

35. The method of claim 34, wherein said cancer is selected from: hematologic cancer, gynecologic cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, skin cancer, prostate cancer, head and neck, stomach cancer, rectal cancer, and colorectal cancer.

36. The method of claim 35, wherein said cancer is hematologic cancer or head and neck cancer.

37. The method of claim 36, wherein said hematologic cancer is selected from acute myeloid leukemia (AML), and multiple myeloma.

38. The method of any one of claims 32 to 37, wherein said treating comprises increasing activation of an NK cell expressing NKG2A.

39. The method of any one of claims 32 to 38, wherein said treating does not inhibit activation of NKG2C in an NK cell expressing NKG2C.

40. A nucleic acid molecule encoding an antibody or antigen binding fragment of any one of claims 1 to 11, the bispecific antibody of claim 12, a scFv of any one of claims 13 to 19 or a CAR of any one of claims 20 to 23.

41. The nucleic acid molecule of claim 40, wherein said nucleic acid molecule is an expression vector.

42. The nucleic acid molecule of claim 40 or 41, comprising nucleotide sequences of three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:CDR-H1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 16 (AACTACTATGTACAC), CDR-H2 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 17(GGATTTATTCTGGAAATTTTTTTACTAAGTTCAATGAGAACTTCAAGGGC), CDR-H3 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 18 (TATGGCAACTACTACTTTGACTAC), CDR-L1 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 19(AAGGCCAGTCAGGATGTGAGTACTGCTGTAGCC), CDR-L2 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 20 (TGGGCATCCACCCGGCACACT), and CDR-L3 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 21(CAGCAACATTATAGCACTCCGTGGACG).

43. The nucleic acid molecule of any one of claims 40 to 42, comprising SEQ ID NO: 23 and SEQ ID NO: 24.

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