# US 10,501,559 B2 · T cell receptor-like antibody agents specific for EBV latent membrane protein 2A peptide presented by human HLA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,559 B2 - Document ID: US10501559B2 - Status: Granted - Kind: B2 - Application number: 15/735,133 - Filed: 2016-06-09 - Published: 2018-09-13 - Granted: 2019-12-10 - Earliest claimed: 2015-06-09 - Assignees: Memorial Sloan Kettering Cancer Center; Eureka Therapeutics, Inc. - Applicants: Memorial Sloan Kettering Cancer Center; Eureka Therapeutics, Inc. - Inventors: Nai-Kong V. Cheung; Mahiuddin Ahmed; Andres Lopez-Albaitero; Cheng Liu; Su Yan; Jingyi Xiang; Hong Liu - Web page: https://patentgrep.com/patent/US10501559B2 ## CPC - A61P 31/22: for herpes viruses - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - A61P 35/00: Antineoplastic agents - C07K 16/085: Orthoherpesviridae (F), e.g. pseudorabies virus or Epstein-Barr virus - C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex} - G01N 33/6854: {Immunoglobulins} - A61K 2039/505: {comprising antibodies} - A61K 2039/572: {cytotoxic response} - C07K 2317/21: from primates, e.g. man - C07K 2317/31: multispecific - C07K 2317/32: specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor - C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity - C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues - C07K 2317/40: characterized by post-translational modification - C07K 2317/41: Glycosylation, sialylation, or fucosylation - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - C07K 2317/565: Complementarity determining region [CDR] - C07K 2317/622: Single chain antibody (scFv) - C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC] - C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Described herein are antibodies, fragments thereof and multi-specific binding agents that bind an Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) peptide presented by HLA class I molecules, in particular, HLA-A02. Also provided herein are methods of using the same or compositions thereof for the detection, prevention and/or therapeutic treatment of diseases characterized by expression of an EBV-LMP2 peptide presented by HLA-A02, in particular, Burkit's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinoma. ## Claims ### Claim 1 A human antibody agent that binds an EBV-LMP2/HLA peptide complex comprising (a) heavy chain CDR1 of SEQ ID NO:31, heavy chain CDR2 of SEQ ID NO:33 and heavy chain CDR3 of SEQ ID NO:35; and light chain CDR1 of SEQ ID NO:73, light chain CDR2 of SEQ ID NO:75 and light chain CDR3 of SEQ ID NO:77; (b) heavy chain CDR1 of SEQ ID NO:37, heavy chain CDR2 of SEQ ID NO:39 and heavy chain CDR3 of SEQ ID NO:41; and light chain CDR1 of SEQ ID NO:79, light chain CDR2 of SEQ ID NO:81 and light chain CDR3 of SEQ ID NO:83; (c) heavy chain CDR1 of SEQ ID NO:43, heavy chain CDR2 of SEQ ID NO:45 and heavy chain CDR3 of SEQ ID NO:47; and light chain CDR1 of SEQ ID NO:85, light chain CDR2 of SEQ ID NO:87 and light chain CDR3 of SEQ ID NO:89; (d) heavy chain CDR1 of SEQ ID NO:49, heavy chain CDR2 of SEQ ID NO:51 and heavy chain CDR3 of SEQ ID NO:53; and light chain CDR1 of SEQ ID NO:91, light chain CDR2 of SEQ ID NO:93 and light chain CDR3 of SEQ ID NO:95; (e) heavy chain CDR1 of SEQ ID NO:55, heavy chain CDR2 of SEQ ID NO:57 and heavy chain CDR3 of SEQ ID NO:59; and light chain CDR1 of SEQ ID NO:97, light chain CDR2 of SEQ ID NO:99 and light chain CDR3 of SEQ ID NO:101; (f) heavy chain CDR1 of SEQ ID NO:61, heavy chain CDR2 of SEQ ID NO:63 and heavy chain CDR3 of SEQ ID NO:65; and light chain CDR1 of SEQ ID NO:103, light chain CDR2 of SEQ ID NO:105 and light chain CDR3 of SEQ ID NO:107; or (g) heavy chain CDR1 of SEQ ID NO:67, heavy chain CDR2 of SEQ ID NO:69 and heavy chain CDR3 of SEQ ID NO:71; and light chain CDR1 of SEQ ID NO:109, light chain CDR2 of SEQ ID NO:111 and light chain CDR3 of SEQ ID NO:113. ### Claim 2 (depends on 1) The human antibody agent of claim 1, wherein the human antibody agent has a KD of about 2.0 to about 170 nM. ### Claim 3 (depends on 1) The human antibody agent of claim 1, wherein the EBV-LMP2 peptide has an amino acid sequence that is or comprises CLGGLLTMV (SEQ ID NO:1). ### Claim 4 (depends on 3) The human antibody agent of claim 3, wherein the human antibody agent interacts directly with amino acid residues at one or more positions selected from the group consisting of position 1, 2, 3, 4, 5, 6, 7, 8, and combinations thereof, in the EBV-LMP2 peptide. ### Claim 5 (depends on 1) The human antibody agent of claim 1, wherein the human antibody agent interacts directly with amino acid residues at one or more positions selected from the group consisting of position 1, position 5, position 8, and combinations thereof, in the EBV-LMP2 peptide. ### Claim 6 (depends on 1) The human antibody agent of claim 1, wherein the human antibody agent comprises (a) a heavy chain variable region sequence as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, and 29, and (b) a light chain variable region sequence as set forth in any one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, and 27. ### Claim 7 (depends on 1) The human antibody agent of claim 1, wherein the human antibody agent comprises (a) the light chain variable region of SEQ ID NO:3 and the heavy chain variable region of SEQ ID NO:5; (b) the light chain variable region of SEQ ID NO:7 and the heavy chain variable region of SEQ ID NO:9; (c) the light chain variable region of SEQ ID NO:11 and the heavy chain variable region of SEQ ID NO:13; (d) the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:17; (e) the light chain variable region of SEQ ID NO:19 and the heavy chain variable region of SEQ ID NO:21; (f) the light chain variable region of SEQ ID NO:23 and the heavy chain variable region of SEQ ID NO:25; or (g) the light chain variable region of SEQ ID NO:27 and the heavy chain variable region of SEQ ID NO:29. ### Claim 8 (depends on 1) The human antibody agent of claim 1, wherein the human antibody agent is a human monoclonal antibody that is defucosylated and/or glycosylated with terminal mannose, N-acetylglucose or glucose. ### Claim 9 (depends on 1) The human antibody agent of claim 1, which human antibody agent comprises one or more amino acid substitutions that increase affinity to the EBV-LMP2/HLA peptide complex, wherein the one or more amino acid substitutions is selected from a light chain I48V substitution, a light chain K66R substitution, a heavy chain V5E substitution, a heavy chain E10D substitution, a heavy chain G26E substitution, and a heavy chain V78A substitution. ### Claim 10 (depends on 9) The human antibody agent of claim 9, which human antibody agent that comprises one or more amino acid substitutions is characterized by (a) a KA of at least about 1.6 higher affinity than affinity to a reference peptide/HLA complex, and/or (b) no cross-reactivity to a reference peptide/HLA complex. ### Claim 11 (depends on 1) The human antibody agent of claim 1, wherein the human antibody agent is a human monoclonal antibody or a fragment thereof. ### Claim 12 (depends on 11) The human antibody agent of claim 11, wherein the human monoclonal antibody is an IgG1. ### Claim 13 (depends on 11) The human antibody agent of claim 11, wherein the human monoclonal antibody fragment is an scFv. ### Claim 14 A bispecific antibody comprising a first antigen-binding site that binds an EBV-LMP2/HLA peptide complex and a second antigen-binding site, wherein the first antigen-binding site comprises (a) heavy chain CDR1 of SEQ ID NO:31, heavy chain CDR2 of SEQ ID NO:33 and heavy chain CDR3 of SEQ ID NO:35; and light chain CDR1 of SEQ ID NO:73, light chain CDR2 of SEQ ID NO:75 and light chain CDR3 of SEQ ID NO:77; (b) heavy chain CDR1 of SEQ ID NO:37, heavy chain CDR2 of SEQ ID NO:39 and heavy chain CDR3 of SEQ ID NO:41; and light chain CDR1 of SEQ ID NO:79, light chain CDR2 of SEQ ID NO:81 and light chain CDR3 of SEQ ID NO:83; (c) heavy chain CDR1 of SEQ ID NO:43, heavy chain CDR2 of SEQ ID NO:45 and heavy chain CDR3 of SEQ ID NO:47; and light chain CDR1 of SEQ ID NO:85, light chain CDR2 of SEQ ID NO:87 and light chain CDR3 of SEQ ID NO:89; (d) heavy chain CDR1 of SEQ ID NO:49, heavy chain CDR2 of SEQ ID NO:51 and heavy chain CDR3 of SEQ ID NO:53; and light chain CDR1 of SEQ ID NO:91, light chain CDR2 of SEQ ID NO:93 and light chain CDR3 of SEQ ID NO:95; (e) heavy chain CDR1 of SEQ ID NO:55, heavy chain CDR2 of SEQ ID NO:57 and heavy chain CDR3 of SEQ ID NO:59; and light chain CDR1 of SEQ ID NO:97, light chain CDR2 of SEQ ID NO:99 and light chain CDR3 of SEQ ID NO:101; (f) heavy chain CDR1 of SEQ ID NO:61, heavy chain CDR2 of SEQ ID NO:63 and heavy chain CDR3 of SEQ ID NO:65; and light chain CDR1 of SEQ ID NO:103, light chain CDR2 of SEQ ID NO:105 and light chain CDR3 of SEQ ID NO:107; or (g) heavy chain CDR1 of SEQ ID NO:67, heavy chain CDR2 of SEQ ID NO:69 and heavy chain CDR3 of SEQ ID NO:71, and light chain CDR1 of SEQ ID NO:109, light chain CDR2 of SEQ ID NO:111 and light chain CDR3 of SEQ ID NO:113. ### Claim 15 (depends on 14) The bispecific antibody of claim 14, wherein the bispecific antibody is characterized by an EC50 of about 0.2 to about 135 pM in T2 cells pulsed with EBV-LMP2 peptide CLGGLLTMV (SEQ ID NO:1). ### Claim 16 (depends on 14) The bispecific antibody of claim 14, wherein the first and/or second antigen-binding sites are selected from the group consisting of an immunoglobulin molecule, scFv, scFab, Fab, Fv or a combination thereof. ### Claim 17 (depends on 14) The bispecific antibody of claim 14, wherein the first and second antigen-binding sites are configured such that they form a single polypeptide chain. ### Claim 18 (depends on 17) The bispecific antibody of claim 17, wherein the first and second antigen-binding sites are each scFvs. ### Claim 19 (depends on 14) The bispecific antibody of claim 14, wherein the second antigen-binding site binds an immunological cell selected from the group consisting of a T cell, B cell, NK cell, dendritic cell, monocyte, macrophage, neutrophil, mesenchymal stem cell and neural stem cell. ### Claim 20 (depends on 19) The bispecific antibody of claim 19, wherein the second antigen-binding site binds CD3 on T cells. ### Claim 21 (depends on 14) The bispecific antibody of claim 14, comprising a first scFv that binds to EBV-LMP2 peptide CLGGLLTMV (SEQ ID NO:1) presented by an HLA-A02 molecule, and a second scFv that binds CD3 on T cells, wherein the bispecific antibody is characterized by an EC50 of about 0.2 to about 135 pM in T2 cells pulsed with the EBV-LMP2 peptide. ### Claim 22 (depends on 21) The bispecific antibody of claim 21, wherein the first scFv comprises (a) the light chain variable region of SEQ ID NO:3 and the heavy chain variable region of SEQ ID NO:5; (b) the light chain variable region of SEQ ID NO:7 and the heavy chain variable region of SEQ ID NO:9; (c) the light chain variable region of SEQ ID NO:11 and the heavy chain variable region of SEQ ID NO:13; (d) the light chain variable region of SEQ ID NO:15 and the heavy chain variable region of SEQ ID NO:17; (e) the light chain variable region of SEQ ID NO:19 and the heavy chain variable region of SEQ ID NO:21; (f) the light chain variable region of SEQ ID NO:23 and the heavy chain variable region of SEQ ID NO:25; or (g) the light chain variable region of SEQ ID NO:27 and the heavy chain variable region of SEQ ID NO:29. ### Claim 23 (depends on 21) The bispecific antibody of claim 21, wherein the HLA-A02 molecule is selected from HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05 and HLA-A*02:06. ### Claim 24 (depends on 21) The bispecific antibody of claim 21, wherein the first scFv of the bispecific antibody interacts directly with at least position 5 of the EBV-LMP2 peptide. ### Claim 25 A human antibody agent that binds an EBV-LMP2/HLA peptide complex comprising (i) heavy chain CDR1 of SEQ ID NO:43, heavy chain CDR2 of SEQ ID NO:45 or a sequence that differs from SEQ ID NO:45 in an I51V substitution, and heavy chain CDR3 of SEQ ID NO:47; and (ii) light chain CDR1 of SEQ ID NO:85, light chain CDR2 of SEQ ID NO:87 or a sequence that differs from SEQ ID NO:87 in one or both of a S52G and P55H substitution, and light chain CDR3 of SEQ ID NO:89 or a sequence that differs from SEQ ID NO:89 in a N95I substitution. ### Claim 26 (depends on 25) The human antibody agent of claim 25, comprising (i) a light chain variable region sequence that comprises a sequence of SEQ ID NO: 11 or a sequence that differs from SEQ ID NO:11 in one or more amino acid substitutions selected from I48V, S52G, P55H, K66R and N95I; and (ii) a heavy chain variable region that comprises a sequence of SEQ ID NO: 13 or a sequence that differs from SEQ ID NO:13 in one or more amino acid substitutions selected from V5E, E10D, G26E, I51V and V78A. ### Claim 27 A bispecific antibody comprising a first antigen-binding site that binds an EBV-LMP2/HLA peptide complex and a second antigen-binding site, wherein the first antigen-binding site comprises (i) heavy chain CDR1 of SEQ ID NO:43, heavy chain CDR2 of SEQ ID NO:45 or a sequence that differs from SEQ ID NO:45 in an I51V substitution, and heavy chain CDR3 of SEQ ID NO:47; and (ii) light chain CDR1 of SEQ ID NO:85, light chain CDR2 of SEQ ID NO:87 or a sequence that differs from SEQ ID NO:87 in one or both of a S52G and P55H substitution, and light chain CDR3 of SEQ ID NO:89 or a sequence that differs from SEQ ID NO:89 in a N95I substitution. ### Claim 28 (depends on 27) The bispecific antibody of claim 27, wherein the first antigen-binding site comprises (i) a light chain variable region sequence that comprises a sequence of SEQ ID NO: 11 or a sequence that differs from SEQ ID NO:11 in one or more amino acid substitutions selected from I48V, S52G, P55H, K66R and N95I; and (ii) a heavy chain variable region that comprises a sequence of SEQ ID NO: 13 or a sequence that differs from SEQ ID NO:13 in one or more amino acid substitutions selected from V5E, E10D, G26E, I51V and V78A.