Antibodies, binding fragments, and methods of use

Abstract

The present disclosure relates to anti-SSEA4 antibodies and bindings fragments thereof comprising specific complementarity determining regions capable of high affinity binding to SSEA4 molecules and SSEA4-associated expressing tumor cells, such as breast cancer, pancreatic cancer, and renal cancer cells. The anti-SSEA4 antibodies and binding fragments induce ADCC or CDC effects in the targeted tumor cells and inhibit and/or reduce the cancer/tumor proliferation. The present disclosure also provides anti-SSEA4 antibodies and binding fragments thereof as a pharmaceutical composition for treating cancer. In addition, the anti-SSEA4 antibodies and binding fragments are useful in the diagnosis of cancers.

What the claim is: 
     
         1 . An isolated monoclonal antibody or an antigen-binding fragment thereof comprising:
 (i) H-CDR1 selected from SEQ ID Nos. 10, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150 and 170, or 80% or more conserved sequence homologs of (i);   (ii) H-CDR2 selected from SEQ ID Nos. 11, 41, 51, 61, 71, 81, 101, 121, 131, 141, 151, and 171, or 80% or more conserved sequence homologs of (ii);   (iii) H-CDR3 selected from SEQ ID Nos: 12, 42, 52, 62, 72, 82, 102, 122, 132, 142, 152 and 172, or 80% or more conserved sequence homologs of (iii);   (iv) L-CDR1 selected from SEQ ID Nos. 15, 45, 55, 65, 75, 85, 105, 125, 135, 145, 155 and 175, or 80% or more conserved sequence homologs of (iv);   (v) L-CDR2 selected from SEQ ID Nos. 16, 46, 56, 66, 76, 86, 106, 126, 136, 146, 156 and 176, or 80% or more conserved sequence homologs of (v); and   (vi) L-CDR3 selected from SEQ ID Nos: 17, 47, 57, 67, 77, 87, 107, 127, 137, 147, 157, and 177, or 80% or more conserved sequence homologs of (vi); respectively.   
     
     
         2 . An isolated monoclonal antibody or an antigen-binding fragment thereof comprising:
 (i) H-CDR1 selected from SEQ ID Nos. 10, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150 and 170, or conserved sequence homologs of (i) containing less than 5 amino acid substitutions;   (ii) H-CDR2 selected from SEQ ID Nos. 11, 41, 51, 61, 71, 81, 101, 121, 131, 141, 151, and 171, or conserved sequence homologs of (ii) containing less than 5 amino acid substitutions;   (iii) H-CDR3 selected from SEQ ID Nos: 12, 42, 52, 62, 72, 82, 102, 122, 132, 142, 152 and 172, or conserved sequence homologs of (iii) containing less than 5 amino acid substitutions;   (iv) L-CDR1 selected from SEQ ID Nos. 15, 45, 55, 65, 75, 85, 105, 125, 135, 145, 155 and 175, or conserved sequence homologs of (iv) containing less than 5 amino acid substitutions;   (v) L-CDR2 selected from SEQ ID Nos. 16, 46, 56, 66, 76, 86, 106, 126, 136, 146, 156 and 176, or conserved sequence homologs of (v) containing less than 5 amino acid substitutions; and   (vi) L-CDR3 selected from SEQ ID Nos: 17, 47, 57, 67, 77, 87, 107, 127, 137, 147, 157, and 177, or conserved sequence homologs of (vi) containing less than 5 amino acid substitutions; respectively.   
     
     
         3 . The isolated monoclonal antibody or an antigen-binding fragment thereof of  claim 1  or  2 , further comprising amino acid substitution on the CDR selected from one or more of A100R, N31S, T62A on the heavy chain and/or S52Y on the light chain. 
     
     
         4 . The isolated monoclonal antibody or an antigen-binding fragment thereof of  claim 1  or  2 , further comprising amino acid substitution on the CDR selected from one or more of V50A, G53A, S35T on the heavy chain and/or one or more of V301/A, G91A, Y94F on the light chain. 
     
     
         5 . An isolated monoclonal antibody or an antigen-binding fragment thereof, comprising:
 (i) a heavy chain variable domain selected from SEQ ID Nos. 13, 23, 33, 43, 53, 63, 73, 83, 103, 123, 133, 143, 153, and 173 or 80% or more conserved sequence homologs thereof; and   (ii) a light chain variable domain selected from SEQ ID Nos. 18, 28, 38, 48, 58, 68, 78, 88, 108, 128, 138, 148, 158, and 178 or 80% or more conserved sequence homologs thereof .   
     
     
         6 . An isolated monoclonal antibody or an antigen-binding fragment thereof, comprising:
 (i) a heavy chain variable domain selected from SEQ ID Nos. 13, 23, 33, 43, 53, 63, 73, 83, 103, 123, 133, 143, 153, and 173, or a conserved sequence homolog thereof containing less than 10 amino acid substitutions; and   (ii) a light chain variable domain selected from SEQ ID Nos. 18, 28, 38, 48, 58, 68, 78, 88, 108, 128, 138, 148, 158, and 178, or a conserved sequence homolog thereof containing less than 10 amino acid substitutions.   
     
     
         7 . An isolated monoclonal antibody or an antigen-binding fragment thereof comprising:
 (i) a heavy chain variable domain selected from SEQ ID Nos. 13, 23, 33, 43, 53, 63, 73, 83, 103, 123, 133, 143, 153, and 173 or 80% or more conserved sequence homologs thereof further comprising H-CDR1 selected from SEQ ID Nos. 10, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150 and 170; H-CDR2 selected from SEQ ID Nos. 11, 41, 51, 61, 71, 81, 101, 121, 131, 141, 151, and 171; H-CDR3 selected from SEQ ID Nos: 12, 42, 52, 62, 72, 82, 102, 122, 132, 142, 152 and 172; respectively, and   (ii) a light chain variable domain selected from SEQ ID Nos. 18, 28, 38, 48, 58, 68, 78, 88, 108, 128, 138, 148, 158, and 178 or 80% or more conserved sequence homologs thereof further comprising L-CDR1 selected from SEQ ID Nos. 15, 45, 55, 65, 75, 85, 105, 125, 135, 145, 155 and 175; L-CDR2 selected from SEQ ID Nos. 16, 46, 56, 66, 76, 86, 106, 126, 136, 146, 156 and 176; and L-CDR3 selected from SEQ ID Nos: 17, 47, 57, 67, 77, 87, 107, 127, 137, 147, 157, and 177; respectively.   
     
     
         8 . An isolated monoclonal antibody or an antigen-binding fragment thereof comprising:
 (i) a heavy chain variable domain selected from SEQ ID Nos. 13, 23, 33, 43, 53, 63, 73, 83, 103, 123, 133, 143, 153, and 173 or a conserved sequence homologs thereof containing less than 10 amino acid substitutions further comprising H-CDR1 selected from SEQ ID Nos. 10, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150 and 170; H-CDR2 selected from SEQ ID Nos. 11, 41, 51, 61, 71, 81, 101, 121, 131, 141, 151, and 171; H-CDR3 selected from SEQ ID Nos: 12, 42, 52, 62, 72, 82, 102, 122, 132, 142, 152 and 172; respectively, and   (ii) a light chain variable domain selected from SEQ ID Nos. 18, 28, 38, 48, 58, 68, 78, 88, 108, 128, 138, 148, 158, and 178 or a conserved sequence homologs thereof containing less than 10 amino acid substitutions further comprising L-CDR1 selected from SEQ ID Nos. 15, 45, 55, 65, 75, 85, 105, 125, 135, 145, 155 and 175; L-CDR2 selected from SEQ ID Nos. 16, 46, 56, 66, 76, 86, 106, 126, 136, 146, 156 and 176; and L-CDR3 selected from SEQ ID Nos: 17, 47, 57, 67, 77, 87, 107, 127, 137, 147, 157, and 177; respectively.   
     
     
         9 . The isolated monoclonal antibody or an antigen-binding fragment thereof, further comprising:
 (i) a heavy chain variable domain selected from SEQ ID Nos. 13, 23, 33, 43, 53, 63, 73, 83, 103, 123, 133, 143, 153, and 173; and   (ii) a light chain variable domain selected from SEQ ID Nos. 18, 28, 38, 48, 58, 68, 78, 88, 108, 128, 138, 148, 158, and 178 or a conserved sequence homologs thereof containing less than 10 amino acid substitutions further comprising L-CDR1 selected from SEQ ID Nos. 15, 45, 55, 65, 75, 85, 105, 125, 135, 145, 155 and 175; L-CDR2 selected from SEQ ID Nos. 16, 46, 56, 66, 76, 86, 106, 126, 136, 146, 156 and 176; and L-CDR3 selected from SEQ ID Nos: 17, 47, 57, 67, 77, 87, 107, 127, 137, 147, 157, and 177; respectively.   
     
     
         10 . The isolated monoclonal antibody or an antigen-binding fragment thereof, further comprising:
 (i) a heavy chain variable domain selected from SEQ ID Nos. 13, 23, 33, 43, 53, 63, 73, 83, 103, 123, 133, 143, 153, and 173 or a conserved sequence homologs thereof containing less than 10 amino acid substitutions further comprising H-CDR1 selected from SEQ ID Nos. 10, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150 and 170; H-CDR2 selected from SEQ ID Nos. 11, 41, 51, 61, 71, 81, 101, 121, 131, 141, 151, and 171; H-CDR3 selected from SEQ ID Nos: 12, 42, 52, 62, 72, 82, 102, 122, 132, 142, 152 and 172; respectively, and   (ii) a light chain variable domain selected from SEQ ID Nos. 18, 28, 38, 48, 58, 68, 78, 88, 108, 128, 138, 148, 158, and 178.   
     
     
         11 . An isolated monoclonal antibody or an antigen-binding fragment thereof, comprising the respective corresponding V H , V L  and respective H-CDRs and L-CDRS as set forth in each variant in Tables 2A-2D. 
     
     
         12 . The isolated antibody or antigen-binding fragment of any one of  claims 1 - 11 , wherein the antibody or antigen-binding fragment is:
 a) a chimeric antibody or a fragment thereof; or   b) a humanized antibody or fragment thereof; or   c) a human antibody or fragment thereof; or   d) an antigen-binding fragment selected from the group consisting of Fab, Fab′, Fv, scFv, dsFv, F(ab) 2 , Fd and a diabody.   
     
     
         13 . The isolated antibody or antigen-binding fragment of any one of  claims 1 - 12 , wherein the antibody is IgG. 
     
     
         14 . The isolated antibody or antigen-binding fragment thereof of any one of  claims 1 - 13  wherein the antibody binding or the binding fragment target is carbohydrate antigen SSEA4 having the structure Neu5Acα 2→3Galβ 1→3GalNAcβ 1→3Galα 1→4Galβ 1→4G1cβ 1. 
     
     
         15 . The isolated antibody or antigen-binding fragment of any one of  claims 1 - 14 , wherein the antibody has CDC and/or ADCC inducing activity upon binding to the target cells. 
     
     
         16 . A pharmaceutical composition, comprising the isolated antibody or antigen-binding fragment thereof of any one of  claims 1 - 14  and a pharmaceutical acceptable carrier. 
     
     
         17 . A pharmaceutical composition of  claim 16 , further comprising one or more therapeutic agent. 
     
     
         18 . A pharmaceutical composition of  claim 17 , wherein the therapeutic agent is selected from therapeutic antibodies, chemotherapeutic agents, or cytokines. 
     
     
         19 . An immunoconjugate comprising the antibody of any one of  claims 1  to  14  and a cytotoxic agent. 
     
     
         20 . The immunoconjugate of  claim 19 , having the formula AB-(L-D)p, wherein:
 (a) AB is the antibody of anyone of  claims 1 - 14 ;   (b) L is a linker;   (c) D is a suitable cytotoxic drug, and (d) p ranges from 1 to 8.   
     
     
         21 . The immunoconjugate (ADC) of  claim 20 , wherein the drug is MMAE or MMAF. 
     
     
         22 . The immunoconjugate of  claim 20 , wherein the linker is cleavable linker. 
     
     
         23 . The ADC of  claim 22 , wherein the cleavable linker is an alkoxyamine-cleavable linker. 
     
     
         24 . A pharmaceutical formulation comprising the immunoconjugate of claims and a pharmaceutically acceptable carrier. 
     
     
         25 . The pharmaceutical formulation of  claim 24 , further comprising an additional therapeutic agent. 
     
     
         26 . Isolated nucleic acid (cDNA) encoding the antibody of any one of  claims 1 - 14 . 
     
     
         27 . A host cell comprising the nucleic acid of  claim 26 . 
     
     
         28 . A method of producing an antibody comprising culturing the host cell of  claim 27  so that the antibody is produced. 
     
     
         29 . An antibody produced by steps comprising:
 (a) providing a nucleic acid encoding 3 VH domain CDRs having sequences of: L-CDR1 selected from SEQ ID Nos. 15, 45, 55, 65, 75, 85, 105, 125, 135, 145, 155 and 175; and L-CDR2 selected from SEQ ID Nos. 16, 46, 56, 66, 76, 86, 106, 126, 136, 146, 156 and 176, and L-CDR3 selected from SEQ ID Nos: 17, 47, 57, 67, 77, 87, 107, 127, 137, 147, 157, and 177, or conserved sequence homologs of each respective L-CDRs having 5 or less conserved amino acid substitutions;   (b) combining a repertoire of nucleic acids encoding 3 VH domain CDRs having the sequences of H-CDR1 selected from SEQ ID Nos. 10, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150 and 170; H-CDR2 selected from SEQ ID Nos. 11, 41, 51, 61, 71, 81, 101, 121, 131, 141, 151, and 171, H-CDR3 selected from SEQ ID Nos: 12, 42, 52, 62, 72, 82, 102, 122, 132, 142, 152 and 172, or conserved sequence homologs of each respective H-CDRs having 5 or less conserved amino acid substitutions;   with the nucleic acid encoding the 3 VL domain CDRs, so as to provide a product repertoire of nucleic acids encoding the 3 VL domain CDRs and the repertoire of 3 VH domain CDRs   (c) expressing the nucleic acids of the product repertoire;   (d) selecting an antigen-binding fragment comprising a variable domain that specifically binds to SSEA4 and that is expressed from the nucleic acids of the product repertoire; and   (e) producing an antibody comprising the antigen-binding fragment.   
     
     
         30 . A method of treating a subject having a SSEA4-positive cancer, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of any one of  claims 16 ,  17 ,  18 ,  24 , and  25 . 
     
     
         31 . The method of  claim 30 , wherein the SSEA4-positive cancer is selected from brain, lung, breast, oral, esophageal, stomach, liver, bile duct, pancreatic, colon, kidney, cervical, ovarian, and prostate cancer. 
     
     
         32 . The method of  claim 30 , further comprising administering one or more additional therapeutic modality or agent in combination to the individual. 
     
     
         33 . The method of  claim 32 , wherein the combined treatment modality is selected from therapeutic antibodies, cell therapies, radiation, cytokines, or chemotherapeutic agents. 
     
     
         34 . A method of inhibiting proliferation of a SSEA4-positive cell, the method. comprising exposing the cell to the pharmaceutical formulations of any one of  claims 16 ,  17 ,  18 ,  24 , and  25  under conditions permissive for binding of the antibodies/fragments/ADCs to SSEA4 on the surface of the cell expressing carbohydrate andgen, thereby inhibiting proliferation of the cell. 
     
     
         35 . A method of treating a subject having a SSEA4-positive cancer, wherein the SSEA4-positive cancer is resistant to a first therapeutic agent, the method comprising administering to the individual an effective amount of the pharmaceutical formulation of any one of claims  claims 16 ,  17 ,  18 ,  24 , and  25 . 
     
     
         36 . The method of  claim 35 , wherein the SSEA4-positive cancer is brain, lung, breast, oral, esophageal, stomach, liver, bile duct, pancreatic, colon, kidney, cervical, ovarian, and/or prostate cancer. 
     
     
         37 . The method of  claim 35 , wherein the first therapeutic agent comprises a first antibody/binding fragment/ADC that binds an antigen other than SSEA4, and/or radiation, and/or chemotherapeutic agent. 
     
     
         38 . A method of detecting SSEA4 in a biological sample comprising contacting the biological sample with the anti-SSEA4 and body of any one of  claims 1 - 14  under conditions permissive for binding of the anti-SSEA4 antibody to a naturally occurring SSEA.4, and detecting whether a complex. is formed between the anti-SSEA4 antibody and a naturally occurring SSEA4 in the biological sample. 
     
     
         39 . The method of  claim 38 , wherein the biological sample is a cancer sample. 
     
     
         40 . A method for detecting a SSEA4-positive cancer comprising (i) administering a labeled anti-SSEA4 antibody to a subject having or suspected of having a carbohydrate antigen expressing tumor, wherein the labeled anti-SSEA4 antibody comprises the anti-SSEA4 antibody of any one of  claims 1 - 14 , and (ii) detecting the labeled anti-SSEA4 antibody in the subject, wherein detection of the labeled anti-SSEA4 antibody indicates a SSEA4-positive cancer in the subject. 
     
     
         41 . A method for detecting a SSEA4-positive cancer comprising (i) contacting a labeled anti-SSEA4 antibody with a sample from a subject having or suspected of haying a carbohydrate antigen expressing tumor, wherein the labeled anti-SSEA4 antibody comprises the anti-SSEA4 antibody of any one of  claims 1 - 14 , and (ii) detecting the labeled anti SSEA4 antibody in the sample, wherein detection of the labeled anti-SSEA4 antibody indicates a SSEA4-positive cancer in the sample. 
     
     
         42 . The isolated antibody of any one of  claims 1 - 14 , wherein the antibody specifically binds to SSEA4 with an affinity constant less than 10 −7  M. 
     
     
         43 . The isolated antibody of any one of  claims 1 - 14 , wherein the antibody is IgG 1 , IgG 2 , IgG 3 , or IgG 4 . 
     
     
         44 . The isolated antibody of any one of  claims 1 - 14 , wherein the antibody is IgG 1λ  or IgG 1κ . 
     
     
         45 . The monoclonal antibody or antigen-binding portion thereof of any one of  claims 1 - 14 , wherein the monoclonal antibody or antigen-binding portion thereof binds to SSEA4 with a K D  of 1×10 −7 M or less, and wherein the K D  is measured by surface plasmon resonance (Biacore) analysis. 
     
     
         46 . The isolated anti-SSEA4 antibody or binding fragment thereof of  claim 45  whererin the binding affinity is <50 nM.