Antibodies to cd40 with enhanced agonist activity

Abstract

Provided herein are agonistic antibodies, or antigen binding portions thereof, that bind to human CD40. Such antibodies optionally comprise Fc regions with enhanced specificity for FcγRIIb. The invention also provides methods of treatment of cancer or chronic infection by administering the antibodies of the invention to a subject in need thereof.

We claim: 
     
         1 . An isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40 and competes for binding to human CD40 in a cross-blocking assay with one or more of antibodies selected from the group consisting of 12D6 (SEQ ID NOs: 3 and 4), 5F11 (SEQ ID NOs: 23 and 24), 8E8 (SEQ ID NOs: 40 and 41), 5G7 (SEQ ID NOs: 52 and 53), and 19G3 (SEQ ID NOs: 58 and 59). 
     
     
         2 . The isolated antibody or antigen binding portion thereof of  claim 1 , wherein the competition in a cross-blocking assay comprises the ability to reduce binding of the selected antibody to human CD40 in a competition ELISA by at least 20% when used at an equal molar concentration with the selected antibody. 
     
     
         3 . An isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40 at:
 a. an epitope comprising or consisting of the sequence EPPTACREKQYLINS (residues 21-35 of SEQ ID NO: 1) (antibodies 12D6, 5G7 and 19G3); or   b. an epitope comprising or consisting of the sequence ECLPCGESE (residues 58-66 of SEQ ID NO: 1) (antibody 5F11).   
     
     
         4 . An isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40 comprising:
 a) heavy chain CDR sequences derived at least in part from murine V region germline VH1-39_01 and J region germline IGHJ4 and
 light chain CDR sequences derived at least in part from murine V region germline VK1-110_01 and J region germline IGKJ1 (12D6); 
   b) heavy chain CDR sequences derived at least in part from murine V region germline VH1-4_02 and J region germline IGHJ3 and
 light chain CDR sequences derived at least in part from murine V region germline VK3-5_01 and J region germline IGKJ5 (5F11); 
   c) heavy chain CDR sequences derived at least in part from murine V region germline VH1-80_01 and J region germline IGHJ2 and
 light chain CDR sequences derived at least in part from murine V region germline VK1-110_01 and J region germline IGKJ2 (8E8); 
   d) heavy chain CDR sequences derived at least in part from murine V region germline VH1-18_01 and J region germline IGHJ4 and
 light chain CDR sequences derived at least in part from murine V region germline VK10-96_01 and J region germline IGKJ2 (5G7); or 
   e) heavy chain CDR sequences derived at least in part from murine V region germline VH5-9-4_01 and J region germline IGHJ3 and
 light chain CDR sequences derived at least in part from murine V region germline VK1-117_01 and J region germline IGKJ2 (19G3). 
   
     
     
         5 . The isolated antibody or antigen binding portion thereof of  claim 1  comprising a heavy chain and a light chain, wherein the heavy chain comprises CDRH1, CDRH2 and CDRH3 sequences and the light chain comprises CDRL1, CDRL2 and CDRL3 sequences selected from the group consisting of:
 a) the CDRs of antibody 12D6-03 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-108, respectively, of SEQ ID NO:5 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:6; 
 b) the CDRs of antibody 12D6-22 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-108, respectively, of SEQ ID NO:7 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:9; 
 c) the CDRs of antibody 12D6-23 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-108, respectively, of SEQ ID NO:10 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:11; 
 d) the CDRs of antibody 12D6-24 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-108, respectively, of SEQ ID NO:12 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:9; 
 e) the CDRs of antibody 5F11-17 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-106, respectively, of SEQ ID NO:25 and CDRL1, CDRL2 and CDRL3 comprise residues 24-38, 54-60 and 93-101, respectively, of SEQ ID NO:26; 
 f) the CDRs of antibody 5F11-23 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-106, respectively, of SEQ ID NO:27 and CDRL1, CDRL2 and CDRL3 comprise residues 24-38, 54-60 and 93-101, respectively, of SEQ ID NO:28; 
 g) the CDRs of antibody 5F11-45 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-106, respectively, of SEQ ID NO:29 and CDRL1, CDRL2 and CDRL3 comprise residues 24-38, 54-60 and 93-101, respectively, of SEQ ID NO:30; 
 h) the CDRs of antibody 8E8-56 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID NO:42 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:43; 
 i) the CDRs of antibody 8E8-62 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID NO:44 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:45; 
 j) the CDRs of antibody 8E8-67 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID NO:46 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:47; 
 k) the CDRs of antibody 8E8-70 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID NO:48 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:49; 
 l) the CDRs of antibody 8E8-71 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-111, respectively, of SEQ ID NO:50 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:51; 
 m) the CDRs of antibody 5G7-22 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-102, respectively, of SEQ ID NO:54 and CDRL1, CDRL2 and CDRL3 comprise residues 24-34, 50-56 and 89-97, respectively, of SEQ ID NO:55; 
 n) the CDRs of antibody 5G7-25 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-102, respectively, of SEQ ID NO:56 and CDRL1, CDRL2 and CDRL3 comprise residues 24-34, 50-56 and 89-97, respectively, of SEQ ID NO:57; 
 o) the CDRs of antibody 19G3-11 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-101, respectively, of SEQ ID NO:60 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:62; and 
 p) the CDRs of antibody 19G3-22 wherein CDRH1, CDRH2 and CDRH3 comprise residues 31-35, 50-66 and 99-101, respectively, of SEQ ID NO:63 and CDRL1, CDRL2 and CDRL3 comprise residues 24-39, 55-61 and 94-102, respectively, of SEQ ID NO:64. 
 
     
     
         6 . The antibody of  claim 5  comprising heavy and light chain variable domain sequences selected from the groups consisting of:
 a) the heavy and light chain variable regions of antibody 12D6-03 comprising residues 1-119 and 1-112 of SEQ ID NO:5 and SEQ ID NO:6, respectively; 
 b) the heavy and light chain variable regions of antibody 12D6-22 comprising residues 1-119 and 1-112 of SEQ ID NO:7 and SEQ ID NO:9, respectively; 
 c) the heavy and light chain variable regions of antibody 12D6-23 comprising residues 1-119 and 1-112 of SEQ ID NO:10 and SEQ ID NO:11, respectively; 
 d) the heavy and light chain variable regions of antibody 12D6-24 comprising residues 1-119 and 1-112 of SEQ ID NO:12 and SEQ ID NO:9, respectively; 
 e) the heavy and light chain variable regions of antibody 5F11-17 comprising residues 1-117 and 1-111 of SEQ ID NO:25 and SEQ ID NO:26, respectively; 
 f) the heavy and light chain variable regions of antibody 5F11-23 comprising residues 1-117 and 1-111 of SEQ ID NO:27 and SEQ ID NO:28, respectively; 
 g) the heavy and light chain variable regions of antibody 5F11-45 comprising residues 1-117 and 1-111 of SEQ ID NO:29 and SEQ ID NO:30, respectively; 
 h) the heavy and light chain variable regions of antibody 8E8-56 comprising residues 1-122 and 1-112 of SEQ ID NO:42 and SEQ ID NO:43, respectively; 
 i) the heavy and light chain variable regions of antibody 8E8-62 comprising residues 1-122 and 1-112 of SEQ ID NO:44 and SEQ ID NO:45, respectively; 
 j) the heavy and light chain variable regions of antibody 8E8-67 comprising residues 1-122 and 1-112 of SEQ ID NO:46 and SEQ ID NO:47, respectively; 
 k) the heavy and light chain variable regions of antibody 8E8-70 comprising residues 1-122 and 1-112 of SEQ ID NO:45 and SEQ ID NO:49, respectively; 
 l) the heavy and light chain variable regions of antibody 8E8-71 comprising residues 1-122 and 1-112 of SEQ ID NO:50 and SEQ ID NO:51, respectively; 
 m) the heavy and light chain variable regions of antibody 5G7-22 comprising residues 1-113 and 1-107 of SEQ ID NO:54 and SEQ ID NO:55, respectively; 
 n) the heavy and light chain variable regions of antibody 5G7-25 comprising residues 1-113 and 1-107 of SEQ ID NO:56 and SEQ ID NO:57, respectively; 
 o) the heavy and light chain variable regions of antibody 19G3-11 comprising residues 1-112 and 1-112 of SEQ ID NO:60 and SEQ ID NO:62, respectively; and 
 p) the heavy and light chain variable regions of antibody 19G3-22 comprising residues 1-112 and 1-112 of SEQ ID NO:63 and SEQ ID NO:64, respectively. 
 
     
     
         7 . The antibody of  claim 1  wherein the antibody further comprises an Fc region modified to enhance specificity of binding to FcγRIIb. 
     
     
         8 . The antibody of  claim 7  exhibiting an A/I ratio of less than 5. 
     
     
         9 . The antibody of  claim 8  exhibiting an A/I ratio of less than 1. 
     
     
         10 . The antibody of  claim 7  wherein the modified Fc region is in a heavy chain constant region selected from the group consisting of SE (SEQ ID NO: 66), SELF (SEQ ID NO: 67), P238D (SEQ ID NO: 68), V4 (SEQ ID NO: 69), V4 D270E (SEQ ID NO: 70), V7 (SEQ ID NO: 71), V8 (SEQ ID NO: 72), V9 (SEQ ID NO: 73), V9 D270E (SEQ ID NO: 74), V11 (SEQ ID NO: 75), and V12 (SEQ ID NO: 76). 
     
     
         11 . The antibody of  claim 5  comprising heavy and light chain sequences selected from the groups consisting of:
 a) the heavy and light chains of antibody 12D6-24-P238D comprising the sequences of SEQ ID NO:13 and SEQ ID NO:9, respectively; 
 b) the heavy and light chains of antibody 12D6-24-SE comprising the sequences of SEQ ID NO:14 and SEQ ID NO:9, respectively; 
 c) the heavy and light chains of antibody 12D6-24-SELF comprising the sequences of SEQ ID NO:15 and SEQ ID NO:9, respectively; 
 d) the heavy and light chains of antibody 12D6-24-V4 comprising the sequences of SEQ ID NO:16 and SEQ ID NO:9, respectively; 
 e) the heavy and light chains of antibody 12D6-24-V4 D270E comprising the sequences of SEQ ID NO:17 and SEQ ID NO:9, respectively; 
 f) the heavy and light chains of antibody 12D6-24-V8 comprising the sequences of SEQ ID NO:18 and SEQ ID NO:9, respectively; 
 g the heavy and light chains of antibody 12D6-24-V9 comprising the sequences of SEQ ID NO:19 and SEQ ID NO:9, respectively; 
 h) the heavy and light chains of antibody 12D6-24-V9 D270E comprising the sequences of SEQ ID NO:20 and SEQ ID NO:9, respectively; 
 i) the heavy and light chains of antibody 12D6-24-V11 comprising the sequences of SEQ ID NO:21 and SEQ ID NO:9, respectively; 
 j) the heavy and light chains of antibody 12D6-24-V12 comprising the sequences of SEQ ID NO:22 and SEQ ID NO:9, respectively; 
 k) the heavy and light chains of antibody 5F11-45-SE comprising the sequences of SEQ ID NO:31 and SEQ ID NO:30, respectively; 
 l) the heavy and light chains of antibody 5F11-45-SELF comprising the sequences of SEQ ID NO:32 and SEQ ID NO:30, respectively; 
 m) the heavy and light chains of antibody 5F11-45-V4 comprising the sequences of SEQ ID NO:33 and SEQ ID NO:30, respectively; 
 n) the heavy and light chains of antibody 5F11-45-V4 D270E comprising the sequences of SEQ ID NO:34 and SEQ ID NO:30, respectively; 
 o) the heavy and light chains of antibody 5F11-45 V8 comprising the sequences of SEQ ID NO:35 and SEQ ID NO:30, respectively; 
 p) the heavy and light chains of antibody 5F11-45-V9 comprising the sequences of SEQ ID NO:36 and SEQ ID NO:30, respectively; 
 q) the heavy and light chains of antibody 5F11-45-V9 D270E comprising the sequences of SEQ ID NO:37 and SEQ ID NO:30, respectively; 
 r) the heavy and light chains of antibody 5F11-45-V11 comprising the sequences of SEQ ID NO:38 and SEQ ID NO:30, respectively; and 
 s) the heavy and light chains of antibody 5F11-45-V12 comprising the sequences of SEQ ID NO:39 and SEQ ID NO:30, respectively. 
 
     
     
         12 . A nucleic acid encoding the heavy and/or light chain variable region of the antibody, or antigen binding portion thereof, of  claim 1 . 
     
     
         13 . An expression vector comprising the nucleic acid molecule of  claim 12 . 
     
     
         14 . A cell transformed with an expression vector of  claim 13 . 
     
     
         15 . A method of preparing an anti-human CD40 antibody, or antigen binding portion thereof, comprising:
 a) expressing the antibody, or antigen binding portion thereof, in the cell of  claim 14 ; and   b) isolating the antibody, or antigen binding portion thereof, from the cell.   
     
     
         16 . A pharmaceutical composition comprising:
 a) the antibody, or antigen binding portion thereof, of  claim 1 ; and   b) a carrier.   
     
     
         17 . A method of stimulating an immune response in a subject comprising administering to the subject the pharmaceutical composition of  claim 16 . 
     
     
         18 . The method of  claim 17 , wherein the subject has a tumor and an immune response against the tumor is stimulated. 
     
     
         19 . The method of  claim 17 , wherein the subject has a chronic viral infection and an immune response against the viral infection is stimulated. 
     
     
         20 . A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of  claim 16 . 
     
     
         21 . The method of  claim 20 , wherein the cancer is selected from the group consisting of: bladder cancer, breast 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 virus-related cancer. 
     
     
         22 . A method of treating a chronic viral infection comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of  claim 16 .