Anti-lap antibody variants and uses thereof

Abstract

Provided herein are anti-LAP antibodies (e.g., recombinant humanized, chimeric, and human anti-LAP antibodies) or antigen binding fragments thereof which have therapeutically beneficial properties, such as binding specifically to LAP-TGFβ1 on cells but not to LAP-TGFβ1 in extracellular matrix, as well as compositions including the same. Also provided are uses of these antibodies or antigen binding fragments in therapeutic applications, such as in the treatment of cancer, and diagnostic applications.

1 . An isolated antibody or antigen binding fragment thereof which specifically binds to LAP comprising:
 (a) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and 112, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;   (b) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and 112, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;   (c) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 16, 26, and 18, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively;   (d) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively;   (e) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively;   (f) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 68, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively; or   (g) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and 68, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively.   
     
     
         2 - 42 . (canceled) 
     
     
         43 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof, a bispecific molecule comprising the antibody or antigen binding fragment thereof or an immunoconjugate comprising the antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof specifically binds to LAP and comprises:
 (a) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 120, and 112, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;   (b) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and 112, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;   (c) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 16, 26, and 18, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively;   (d) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively;   (e) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and 56, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively:   (f) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 55, and 68, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively; or   (g) a heavy chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 54, 66, and 68, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 57, 58, and 59, respectively.   
     
     
         44 . The method of  claim 43 , wherein the cancer is characterized by abnormal TGFβ activity. 
     
     
         45 . The method of  claim 43 , wherein the cancer is associated with infiltration of CD4+ regulatory T cells, CD8+ regulatory T cells, regulatory B cells, myeloid-derived suppressor cells, tumor-associated macrophages, cancer-associated fibroblasts, and/or innate lymphoid cells. 
     
     
         46 . The method of  claim 43 , wherein the cancer is selected from the group consisting of: breast cancer, bladder cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and myelodysplastic syndromes. 
     
     
         47 . The method of  claim 43 , further comprising administering one or more additional therapies. 
     
     
         48 . The method of  claim 47 , wherein the one or more additional therapies is selected from radiation therapy, chemotherapy, an immune checkpoint inhibitor, immunosuppressive therapy, immunostimulatory therapy, cell therapy, and a therapeutic agent. 
     
     
         49 . The method of  claim 48 , wherein the immune checkpoint inhibitor is an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, or an anti-TIM3 antibody. 
     
     
         50 - 54 . (canceled) 
     
     
         55 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof comprises heavy and light chain variable region sequences which are at least 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 218 and 154, respectively, (b) SEQ ID NOs: 133 and 154, respectively, (c) SEQ ID NOs: 42 and 52, respectively; (d) SEQ ID NOs: 101 and 104, respectively; and (e) SEQ ID NOs: 98 and 104, respectively. 
     
     
         56 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:218 with 1-5, 5-10, 10-15, 15-20, or 20-25 amino acid substitutions; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154 or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154 with 1-5, 5-10, 10-15, 15-20, or 20-25 amino acid substitutions. 
     
     
         57 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof comprises heavy and light chain variable region sequences selected from the group consisting of: (a) SEQ ID NOs: 218 and 154, respectively, (b) SEQ ID NOs: 133 and 154, (c) SEQ ID NOs: 42 and 52, respectively; (d) SEQ ID NOs: 101 and 104, respectively; and (e) SEQ ID NOs: 98 and 104, respectively. 
     
     
         58 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof comprises heavy and light chain sequences which are at least 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequences selected from the group consisting of: (a) SEQ ID NOs: 219 and 155, respectively, (b) SEQ ID NOs: 220 and 155, respectively, (c) SEQ ID NOs: 134 and 155, respectively; (d) SEQ ID NOs: 135 and 155, respectively, (e) SEQ ID NOs: 43 and 53, respectively; (f) SEQ ID NOs: 45 and 53, respectively; (g) SEQ ID NOs: 102 and 105, respectively; (h) SEQ ID NOs: 103 and 105, respectively; (i) SEQ ID NOs: 99 and 105, respectively; and (j) SEQ ID NOs: 100 and 105, respectively. 
     
     
         59 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof of  claim 1  which comprises heavy and light chain sequences selected from the group consisting of: (a) SEQ ID NOs: 219 and 155, respectively, (b) SEQ ID NOs: 220 and 155, respectively, (c) SEQ ID NOs: 134 and 155, respectively; (d) SEQ ID NOs: 135 and 155, respectively, (e) SEQ ID NOs: 43 and 53, respectively; (f) SEQ ID NOs: 45 and 53, respectively; (g) SEQ ID NOs: 102 and 105, respectively; (h) SEQ ID NOs: 103 and 105, respectively; (i) SEQ ID NOs: 99 and 105, respectively; and (j) SEQ ID NOs: 100 and 105, respectively. 
     
     
         60 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof comprises a heavy chain comprising amino acid sequences as set forth in SEQ ID NO: 218 and SEQ ID NO: 245 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 155. 
     
     
         61 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to human LAP, cyno LAP, rat LAP, and/or murine LAP. 
     
     
         62 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof inhibits TGFβ1 activation. 
     
     
         63 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to human LAP with a K D  of 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less. 
     
     
         64 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to human LAP in the absence of an anchor protein. 
     
     
         65 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to immunosuppressive cells. 
     
     
         66 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to LAP complexed with an anchor protein on immunosuppressive cells, but does not bind to the anchor protein or to an epitope composed of residues of both LAP and the anchor protein. 
     
     
         67 . The method of  claim 66 , wherein the anchor protein is GARP or LRRC33. 
     
     
         68 . The method of  claim 65 , wherein the immunosuppressive cells are regulatory T cells, M2 macrophages, cancer cells expressing LAP, and/or myeloid-derived suppressor cells. 
     
     
         69 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to both GARP-positive immunosuppressive cells and GARP-negative immunosuppressive cells. 
     
     
         70 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof does not bind to LAP on extracellular matrix. 
     
     
         71 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof does not bind to LAP complexed with LTBP1, LTBP3 and/or LTBP4. 
     
     
         72 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof comprises an IgG constant region or variant thereof. 
     
     
         73 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof is a chimeric, human or humanized antibody. 
     
     
         74 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof does not bind to human LAP comprising a Y74T mutation or binds to human LAP comprising K27C and Y75C mutations. 
     
     
         75 . The method of  claim 43 , wherein the antibody or antigen binding fragment thereof binds to one or more residues of residues 31-40, 274-280, and 340-343 of human LAP-TGFβ1 (SEQ ID NO: 1), or binds to one or more residues of residues 31-43, 272-283, and 340-344 of human LAP-TGFβ1 (SEQ ID NO: 1). 
     
     
         76 . The method of  claim 43 , wherein the bispecific molecule is linked to a molecule having a second binding region and the second binding region binds to a tumor-associated antigen. 
     
     
         77 . The method of  claim 77 , wherein the second binding region binds to CD4, CD8, CD45, CD56, CD14, CD16, CD19, CD11b, CD25, CD20, CD22, CD30, CD38, CD 114, CD23, CD73, CD163, CD206, CD203, CD200R, or CD39. 
     
     
         78 . The method of  claim 43 , wherein the immunoconjugate is linked to a detectable moiety, a binding moiety, a labeling moiety, or a biologically active moiety.