Patent Application
20220144956
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 5, 2021, is named 067461-5277-WO_SL.txt and is 2,853,990 bytes in size.
One mechanism by which tumors evade immune surveillance is by producing the immunosuppressive cytokine TGFß which directly inhibits the expression of cytolytic proteins such as IFNγ which are necessary for T cell-mediated tumor cytotoxicity. Additionally, TGFß is pro-fibrotic and promotes the expansion of fibroblasts. Cancer-associated fibroblasts (CAFs) have been reported to promote tumor survival and proliferation (Orimo et al., 2006; Xing et al., 2011), for example by providing growth factors for angiogenesis and by further encouraging an immunosuppressive environment, and have been associated with poor prognosis (Underwood et al, 2015).
Accordingly, a number of therapies have been developed to block the TGFß/TGFßR axis such as anti-TGFßRII antibodies. However, such therapies have varied in success. For example, an anti-TGFßRII mAb resulted in uncontrolled cytokine release syndrome. Therefore, there is a need and potential for effective therapy targeting the TGFß/TGFßR axis with enhanced safety profiles.
Provided herein are novel antibodies that bind TGFßRII. In some embodiments, the anti-TGFßRII antibodies bispecific heterodimeric antibodies. In some embodiments, the anti-TGFßRII antibodies are anti-TGFßRII×anti-CD5 bispecific antibodies. In some embodiments, the anti-TGFßRII antibodies are anti-TGFßRII×anti-PD-1 bispecific antibodies. Also provided herein are methods of making and using such antibodies. The antibodies provided advantageously block TGFß activity in a broad population of cells (e.g., active and unactivated hematopoietic cells), and find use wherein such blockage of TGFß activity is desirable, for example, for the treatment of cancers.
In a first aspect, provided herein is a heterodimeric antibody comprising:
a) a first monomer comprising: i) a scFv comprising a first variable heavy domain, an scFv linker and a first variable light domain; and ii) a first Fc domain, wherein the scFv is covalently attached to the N-terminus of the first Fc domain using a domain linker; b) a second monomer comprising, from N-terminus to C-terminus, a VH1-CH1-hinge-CH2-CH3, wherein VH is a first variable heavy domain and CH2-CH3 is a second Fc domain; and c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a variable light domain and CL is a constant light domain. The VH1 and the VL1 together form a first antigen binding domain (ABD). The scFv comprises a second VH domain (VH2), a scFv linker, and a second VL domain (VL2), wherein the VH2 and the VL2 together form a second ABD. Further, one of the first ABD and second ABD is a TGFßRII binding domain and the other of the first ABD and second ABD is a CD5 binding domain. The TGFßRII binding domain comprises a variable heavy domain selected from the group consisting of: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NO:1863, SEQ ID NO:1871, SEQ ID NO:1875, SEQ ID NOs:1323-1605, SEQ ID NO:525, SEQ ID NO:533, SEQ ID NO:541, SEQ ID NO:549, SEQ ID NO:557, SEQ ID NO:562, SEQ ID NO:564, SEQ ID NO:572, SEQ ID NO:990, SEQ ID NO:994, SEQ ID NO:998, SEQ ID NO:1002, SEQ ID NO:1006, SEQ ID NO:1010, SEQ ID NO:1014, SEQ ID NO:1018, SEQ ID NO:1022, SEQ ID NO: 1026, SEQ ID NO: 1030, SEQ ID NO:1034, SEQ ID NO:1038, SEQ ID NO:1042, SEQ ID NO:1046, SEQ ID NO:1050, SEQ ID NO:1054, SEQ ID NO:1058, SEQ ID NO:1062, SEQ ID NO:1066, SEQ ID NO:1070, SEQ ID NO:1074, SEQ ID NO: 1078, SEQ ID NO: 1082, SEQ ID NO:1086, SEQ ID NO:1090, SEQ ID NO:1094, SEQ ID NO:1098, NO:1102, SEQ ID NO:1106, SEQ ID NO:1110, SEQ ID NO:1114, SEQ ID NO:1118, SEQ ID NO:1122, SEQ ID NO: 1126, SEQ ID NO: 1130, SEQ ID NO:1134, SEQ ID NO:1138, SEQ ID NO:1142, SEQ ID NO:1146, SEQ ID NO:1150, SEQ ID NO:1154, SEQ ID NO:1158, SEQ ID NO:1162, SEQ ID NO:1166, SEQ ID NO:1170, SEQ ID NO:1174, SEQ ID NO: 1178, SEQ ID NO: 1182, SEQ ID NO:1186, SEQ ID NO:1190, SEQ ID NO:1194, SEQ ID NO:1198, NO:1202, SEQ ID NO:1206, SEQ ID NO:1210, SEQ ID NO:1214, SEQ ID NO:1218, SEQ ID NO:1222, SEQ ID NO: 1226, SEQ ID NO: 1230, SEQ ID NO:1234, SEQ ID NO:1238, SEQ ID NO:1242, SEQ ID NO:1246, SEQ ID NO:1250, SEQ ID NO:1254, SEQ ID NO:1258, SEQ ID NO:1262, SEQ ID NO:1266, SEQ ID NO:1270, SEQ ID NO:1274, SEQ ID NO: 1278, SEQ ID NO: 1282, SEQ ID NO:1286, SEQ ID NO:1290, SEQ ID NO:1294, SEQ ID NO:1298, SEQ ID NO:1302, SEQ ID NO:1306, and SEQ ID NO:1310, and a variable light domain selected from the group consisting of: SEQ ID NO:1867, SEQ ID NO:1879, and SEQ ID NOs:1606-1703, SEQ ID NO:529, SEQ ID NO:537, SEQ ID NO:545, SEQ ID NO:553, SEQ ID NO:561, SEQ ID NO:563, SEQ ID NO:568, SEQ ID NO:576, SEQ ID NO:1314, SEQ ID NO:1315, and SEQ ID NO:1319. The CD5 binding domain comprises a variable heavy domain selected from the group consisting of: SEQ ID NO:2187, SEQ ID NO:2147, SEQ ID NO:2155, VH: SEQ ID NO:2163, SEQ ID NO:2171, SEQ ID NO:2179, SEQ ID NO:2183, SEQ ID NO:s1704-1754, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO: 113, and SEQ ID NO:2137, and a variable light domain selected from the group consisting of: SEQ ID NO:2175, SEQ ID NO:2151, SEQ ID NO:2159, SEQ ID NO:2167, SEQ ID NO:2191, SEQ ID NOs:1755-1757, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, and SEQ ID NO:2141.
In some embodiments, the first ABD is a CD5 binding domain and the second ABD is a TGFßRII binding domain. In exemplary embodiments, the TGFßRII binding domain comprises: a) a variable heavy domain selected from the group consisting of: SEQ ID NOs: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, 1859, 1863, 1871, 1875, and 1323-1605, and b) a variable light domain selected from the group consisting of: SEQ ID NOs:1867, 1879, and 1606-1703.
In some embodiments, the TGFßRII binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:2389 and 1867, and SEQ ID NOs:2393 and 1867, respectively.
In certain embodiments, the CD5 binding domain comprises: a) a variable heavy domain selected from the group consisting of: SEQ ID NO:2187, SEQ ID NO:2163, SEQ ID NO:2171, SEQ ID NO:2179, SEQ ID NO:2183, SEQ ID NO:s1704-1754, SEQ ID NO:2147 and SEQ ID NO:2155, and b) a variable light domain selected from the group consisting of: SEQ ID NO:2175, SEQ ID NO:2167, SEQ ID NO:2191, SEQ ID NOs:1755-1757, SEQ ID NO:2151 and SEQ ID NO:2159. In some embodiments, the CD5 binding domain comprises a variable heavy domain of SEQ ID NO:2187 and a variable light domain of SEQ ID NO:2175.
In some embodiments, the TGFßRII binding domain comprises a variable heavy domain selected from the group consisting of: SEQ ID NOs: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, 1859, 1863, 1871, 1875, and 1323-1605, and a variable light domain selected from the group consisting of: SEQ ID NOs:1867, 1879, and 1606-1703, and the CD5 binding domain comprises a variable heavy domain selected from the group consisting of: SEQ ID NO:2187, SEQ ID NO:2163, SEQ ID NO:2171, SEQ ID NO:2179, SEQ ID NO:2183, SEQ ID NO:s1704-1754, SEQ ID NO:2147 and SEQ ID NO:2155, and a variable light domain selected from the group consisting of: SEQ ID NO:2175, SEQ ID NO:2167, SEQ ID NO:2191, SEQ ID NOs:1755-1757, SEQ ID NO:2151 and SEQ ID NO:2159. In some embodiments, the TGFßRII binding domain comprises a variable heavy domain and variable light domain are selected from the group consisting of: SEQ ID NOs:2389 and 1867, and SEQ ID NOs:2393 and 1867, and the CD5 binding domain comprises a variable heavy domain of SEQ ID NO:2187 and a variable light domain of SEQ ID NO:2175.
In some embodiments, the scFv comprises, from N- to C-terminal, VL2-scFv linker-VH2. In certain embodiments, the scFv comprises, from N- to C-terminal, VH2-scFv linker-VL2.
In some embodiments, the first Fc domain and second Fc domain are each variant Fc domains. In some embodiments, the first and second Fc domains comprise a set of heterodimerization skew variants selected from the following heterodimerization variants: S364K/E357Q:L368D/K370S; S364K:L368D/K370S; S364K:L368E/K370S; D401K: T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering. In exemplary embodiments, the first and second Fc domains comprise heterodimerization skew variants S364K/E357Q:L368D/K370S.
In some embodiments, the first and second Fc domains each comprise one or more ablation variants. In exemplary embodiments, the one or more ablation variants are E233P/L234V/L235A/G236del/S267K, wherein numbering is according to EU numbering.
In some embodiments, the one of the first or second monomer further comprises a pI variant. In exemplary embodiments the CH1-hinge-CH2-CH3 of the second monomer comprises pI variants N208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EU numbering.
In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises amino acid variants L368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K, and the first Fc domain comprises amino acid variants S364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering is according to EU numbering.
In exemplary embodiments, the first and second variant Fc domains each comprise amino acid variants 428L/434S.
In another aspect, provided herein is a heterodimeric antibody comprising: a) a first monomer comprising: i) a scFv comprising a first variable heavy domain, an scFv linker and a first variable light domain; and ii) a first Fc domain, wherein the scFv is covalently attached to the N-terminus of the first Fc domain using a domain linker; b) a second monomer comprising, from N-terminus to C-terminus, a VH1-CH1-hinge-CH2-CH3, wherein VH is a first variable heavy domain and CH2-CH3 is a second Fc domain; and c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a variable light domain and CL is a constant light domain. The VH1 and the VL1 together form a first antigen binding domain (ABD). The scFv comprises a second VH domain (VH2), a scFv linker, and a second VL domain (VL2), wherein the VH2 and the VL2 together form a second ABD. One of the first ABD and second ABD is a TGFßRII binding domain and the other of the first ABD and second ABD is a PD-1 binding domain. The TGFßRII binding domain comprises a variable heavy domain selected from the group consisting of: SEQ ID NOs: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, 1859, 1863, 1871, 1875, and 1323-1605, and a variable light domain selected from the group consisting of: SEQ ID NOs:1867, 1879, and 1606-1703, and the PD-1 binding domain comprises a variable heavy domain selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, and a variable light domain selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479.
In some embodiments, the first ABD is a PD-1 binding domain and the second ABD is a TGFßRII binding domain.
In certain embodiments, the PD-1 binding domain comprises: a) a variable heavy domain selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, and b) a variable light domain selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, and SEQ ID NO:521. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In some embodiments, the scFv comprises, from N- to C-terminal, VL2-scFv linker-VH2. In certain embodiments, the scFv comprises, from N- to C-terminal, VH2-scFv linker-VL2.
In some embodiments, the first Fc domain and second Fc domain are each variant Fc domains. In some embodiments, the first and second Fc domains comprise a set of heterodimerization skew variants selected from the following heterodimerization variants: S364K/E357Q:L368D/K370S; S364K:L368D/K370S; S364K:L368E/K370S; D401K: T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering. In exemplary embodiments, the first and second Fc domains comprise heterodimerization skew variants S364K/E357Q: L368D/K370S.
In some embodiments, the first and second Fc domains each comprise one or more ablation variants. In exemplary embodiments, the one or more ablation variants are E233P/L234V/L235A/G236del/S267K, wherein numbering is according to EU numbering.
In some embodiments, the one of the first or second monomer further comprises a pI variant. In exemplary embodiments the CH1-hinge-CH2-CH3 of the second monomer comprises pI variants N208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EU numbering.
In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises amino acid variants L368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K, and the first Fc domain comprises amino acid variants S364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering is according to EU numbering.
In exemplary embodiments, the first and second variant Fc domains each comprise amino acid variants 428L/434S.
In another aspect, provided herein is a composition comprising a TGFßRII binding domain comprising: a) a variable heavy domain with an amino acid sequence selected from the group consisting of: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NOs:1859, 1863, 1323-1605; and b) variable light domain with an amino acid sequence selected from the group consisting of: SEQ ID NOs:1867, and 1606-1703. In some embodiments, the composition is an antibody comprising: a) a heavy chain comprising the VH-CH1-hinge-CH2-CH3; and b) a light chain comprising the VL-CL.
In another aspect, provided herein is a composition comprising a CD5 binding domain comprising: a) a variable heavy domain with an amino acid sequence selected from the group consisting of: SEQ ID NO:2155 and SEQ ID NO:2147; and b) variable light domain with an amino acid sequence selected from the group consisting of: SEQ ID NO:2159 and SEQ ID NO:2151. In some embodiments, the composition is an antibody comprising: a) a heavy chain comprising the VH-CH1-hinge-CH2-CH3; and b) a light chain comprising the VL-CL.
In yet another aspect, provided herein is a composition comprising a CD5 binding domain comprising: a) a variable heavy domain with an amino acid sequence selected from the group consisting of: SEQ ID NO:2187, SEQ ID NO:2163, SEQ ID NO:2171, SEQ ID NO:2179, SEQ ID NO:2183, and SEQ ID NO:s1704-1754; and b) variable light domain with an amino acid sequence selected from the group consisting of: SEQ ID NO:2175, SEQ ID NO:2167, SEQ ID NO:2191, and SEQ ID NOs:1755-1757. In some embodiments, the composition is an antibody comprising: a) a heavy chain comprising the VH-CH1-hinge-CH2-CH3; and b) a light chain comprising the VL-CL.b) a light chain comprising the VL-CL.
In another aspect, provided herein is a heterodimeric, bispecific antibody comprising: a) a means for binding TGFßRII attached to a first monomer; b) a means for binding CD5 (or PD-1) attached to a second monomer; and c) a means for heterodimerization of the first monomer and second monomer.
In one aspect, provided herein is a heterodimeric, bispecific antibody comprising: a) a means for binding TGFßRII attached to a first monomer; and b) a means for binding CD5 attached to a second monomer; wherein the first monomer comprises a first variant Fc domain, the second monomer comprises a second variant Fc domain, and the first variant Fc domain and second variant Fc domain comprises heterodimerization skew variants S364K/E357Q:L368D/K370S.
In one aspect, provided herein is a heterodimeric, bispecific antibody comprising: a) a means for binding TGFßRII attached to a first monomer; and b) a means for binding PD-1 attached to a second monomer; wherein the first monomer comprises a first variant Fc domain, the second monomer comprises a second variant Fc domain, and the first variant Fc domain and second variant Fc domain comprises heterodimerization skew variants S364K/E357Q:L368D/K370S.
In another aspect, provided herein is heterodimeric, bispecific antibody comprising: a) a means for binding TGFßRII attached to a first monomer; b) a CD5 binding domain attached to a second monomer, wherein the CD5 binding domain comprises: i) a variable heavy domain selected from the group consisting of: SEQ ID NO:2187, SEQ ID NO:2147, SEQ ID NO:2155, VH: SEQ ID NO:2163, SEQ ID NO:2171, SEQ ID NO:2179, SEQ ID NO:2183, SEQ ID NO:s1704-1754, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO: 113, and SEQ ID NO:2137, and ii) a variable light domain selected from the group consisting of: SEQ ID NO:2175, SEQ ID NO:2151, SEQ ID NO:2159, SEQ ID NO:2167, SEQ ID NO:2191, SEQ ID NOs:1755-1757, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, and SEQ ID NO:2141; and c) a means for heterodimerization of the first monomer and second monomer. In some embodiments, the variable heavy domain is SEQ ID NO:2187 and the variable light domain is SEQ ID NO:2175.
In one aspect, provided herein is a heterodimeric, bispecific antibody comprising: a) a means for binding CD5 attached to a first monomer; b) a TGFßRII binding domain attached to a second monomer, wherein the TGFßRII binding domain comprises: i) a variable heavy domain selected from the group consisting of: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NO:1863, SEQ ID NO:1871, SEQ ID NO:1875, SEQ ID NOs:1323-1605, SEQ ID NO:525, SEQ ID NO:533, SEQ ID NO:541, SEQ ID NO:549, SEQ ID NO:557, SEQ ID NO:562, SEQ ID NO:564, SEQ ID NO:572, SEQ ID NO:990, SEQ ID NO:994, SEQ ID NO:998, SEQ ID NO:1002, SEQ ID NO:1006, SEQ ID NO:1010, SEQ ID NO:1014, SEQ ID NO:1018, SEQ ID NO:1022, SEQ ID NO: 1026, SEQ ID NO: 1030, SEQ ID NO:1034, SEQ ID NO:1038, SEQ ID NO:1042, SEQ ID NO:1046, SEQ ID NO:1050, SEQ ID NO:1054, SEQ ID NO:1058, SEQ ID NO:1062, SEQ ID NO:1066, SEQ ID NO:1070, SEQ ID NO:1074, SEQ ID NO: 1078, SEQ ID NO: 1082, SEQ ID NO:1086, SEQ ID NO:1090, SEQ ID NO:1094, SEQ ID NO:1098, NO:1102, SEQ ID NO:1106, SEQ ID NO:1110, SEQ ID NO:1114, SEQ ID NO:1118, SEQ ID NO:1122, SEQ ID NO: 1126, SEQ ID NO: 1130, SEQ ID NO:1134, SEQ ID NO:1138, SEQ ID NO:1142, SEQ ID NO:1146, SEQ ID NO:1150, SEQ ID NO:1154, SEQ ID NO:1158, SEQ ID NO:1162, SEQ ID NO:1166, SEQ ID NO:1170, SEQ ID NO:1174, SEQ ID NO: 1178, SEQ ID NO: 1182, SEQ ID NO:1186, SEQ ID NO:1190, SEQ ID NO:1194, SEQ ID NO:1198, NO:1202, SEQ ID NO:1206, SEQ ID NO:1210, SEQ ID NO:1214, SEQ ID NO:1218, SEQ ID NO:1222, SEQ ID NO: 1226, SEQ ID NO: 1230, SEQ ID NO:1234, SEQ ID NO:1238, SEQ ID NO:1242, SEQ ID NO:1246, SEQ ID NO:1250, SEQ ID NO:1254, SEQ ID NO:1258, SEQ ID NO:1262, SEQ ID NO:1266, SEQ ID NO:1270, SEQ ID NO:1274, SEQ ID NO: 1278, SEQ ID NO: 1282, SEQ ID NO:1286, SEQ ID NO:1290, SEQ ID NO:1294, SEQ ID NO:1298, SEQ ID NO:1302, SEQ ID NO:1306, and SEQ ID NO:1310, and ii) a variable light domain selected from the group consisting of: SEQ ID NO:1867, SEQ ID NO:1879, and SEQ ID NOs:1606-1703, SEQ ID NO:529, SEQ ID NO:537, SEQ ID NO:545, SEQ ID NO:553, SEQ ID NO:561, SEQ ID NO:563, SEQ ID NO:568, SEQ ID NO:576, SEQ ID NO:1314, SEQ ID NO:1315, and SEQ ID NO:1319; and c) a means for heterodimerization of the first monomer and second monomer. In some embodiments, the variable heavy domain and variable light domain are selected from the group consisting of: SEQ ID NOs:2389 and 1867, and SEQ ID NOs:2393 and 1867, respectively.
In another aspect, provided herein is a heterodimeric, bispecific antibody comprising: a) a means for binding TGFßRII attached to a first monomer; b) a PD-1 binding domain attached to a second monomer, wherein the PD-1 binding domain comprises: i) a variable heavy domain selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, and ii) a variable light domain selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479; and c) a means for heterodimerization of the first monomer and second monomer. In some embodiments, the variable heavy domain and the variable light domain are selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In another aspect, provided herein is a bispecific antibody comprising: a) a means for binding PD-1 attached to a first monomer; b) a TGFßRII binding domain attached to a second monomer, wherein the TGFßRII binding domain comprises: i) a variable heavy domain selected from the group consisting of: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NO:1863, SEQ ID NO:1871, SEQ ID NO:1875, SEQ ID NOs:1323-1605, SEQ ID NO:525, SEQ ID NO:533, SEQ ID NO:541, SEQ ID NO:549, SEQ ID NO:557, SEQ ID NO:562, SEQ ID NO:564, SEQ ID NO:572, SEQ ID NO:990, SEQ ID NO:994, SEQ ID NO:998, SEQ ID NO:1002, SEQ ID NO:1006, SEQ ID NO:1010, SEQ ID NO:1014, SEQ ID NO:1018, SEQ ID NO:1022, SEQ ID NO: 1026, SEQ ID NO: 1030, SEQ ID NO:1034, SEQ ID NO:1038, SEQ ID NO:1042, SEQ ID NO:1046, SEQ ID NO:1050, SEQ ID NO:1054, SEQ ID NO:1058, SEQ ID NO:1062, SEQ ID NO:1066, SEQ ID NO:1070, SEQ ID NO:1074, SEQ ID NO: 1078, SEQ ID NO: 1082, SEQ ID NO:1086, SEQ ID NO:1090, SEQ ID NO:1094, SEQ ID NO:1098, NO:1102, SEQ ID NO:1106, SEQ ID NO:1110, SEQ ID NO:1114, SEQ ID NO:1118, SEQ ID NO:1122, SEQ ID NO: 1126, SEQ ID NO: 1130, SEQ ID NO:1134, SEQ ID NO:1138, SEQ ID NO:1142, SEQ ID NO:1146, SEQ ID NO:1150, SEQ ID NO:1154, SEQ ID NO:1158, SEQ ID NO:1162, SEQ ID NO:1166, SEQ ID NO:1170, SEQ ID NO:1174, SEQ ID NO: 1178, SEQ ID NO: 1182, SEQ ID NO:1186, SEQ ID NO:1190, SEQ ID NO:1194, SEQ ID NO:1198, NO:1202, SEQ ID NO:1206, SEQ ID NO:1210, SEQ ID NO:1214, SEQ ID NO:1218, SEQ ID NO:1222, SEQ ID NO: 1226, SEQ ID NO: 1230, SEQ ID NO:1234, SEQ ID NO:1238, SEQ ID NO:1242, SEQ ID NO:1246, SEQ ID NO:1250, SEQ ID NO:1254, SEQ ID NO:1258, SEQ ID NO:1262, SEQ ID NO:1266, SEQ ID NO:1270, SEQ ID NO:1274, SEQ ID NO: 1278, SEQ ID NO: 1282, SEQ ID NO:1286, SEQ ID NO:1290, SEQ ID NO:1294, SEQ ID NO:1298, SEQ ID NO:1302, SEQ ID NO:1306, and SEQ ID NO:1310, and ii) a variable light domain selected from the group consisting of: SEQ ID NO:1867, SEQ ID NO:1879, and SEQ ID NOs:1606-1703, SEQ ID NO:529, SEQ ID NO:537, SEQ ID NO:545, SEQ ID NO:553, SEQ ID NO:561, SEQ ID NO:563, SEQ ID NO:568, SEQ ID NO:576, SEQ ID NO:1314, SEQ ID NO:1315, and SEQ ID NO:1319; and c) a means for heterodimerization of the first monomer and second monomer. In some embodiments, the variable heavy domain and variable light domain are selected from the group consisting of: SEQ ID NOs:2389 and 1867, and SEQ ID NOs:2393 and 1867, respectively.
In addition, provided herein are nucleic acid compositions that include nucleic acids encoding components of the subject heterodimeric antibodies (e.g., first and second monomers and light chains) and antigen binding domains provided herein, expression vectors that include the nucleic acid compositions and host cells that include the nucleic acid compositions or expression vectors. Also provided herein is a method of making the subject bispecific heterodimeric antibody by culturing a host cell described herein under conditions wherein the bispecific heterodimeric antibody is produced and recovering the antibody.
In another aspect, provided herein is a method of treating a patient with cancer comprising administering a bispecific heterodimeric as provided herein.
Included within each of these backbones are sequences that are 90, 95, 98 and 99% identical (as defined herein) to the recited sequences, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions (as compared to the “parent” of the Figure, which, as will be appreciated by those in the art, already contain a number of amino acid modifications as compared to the parental human IgG1 (or IgG2 or IgG4, depending on the backbone). That is, the recited backbones may contain additional amino acid modifications (generally amino acid substitutions) in addition or as an alternative to the skew, pI and ablation variants contained within the backbones of this Figure.
In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.
By “ablation” herein is meant a decrease or removal of activity. Thus for example, “ablating FcγR binding” means the Fc region amino acid variant has less than 50% starting binding as compared to an Fc region not containing the specific variant, with more than 70-80-90-95-98% loss of activity being preferred, and in general, with the activity being below the level of detectable binding in a Biacore, SPR or BLI assay. Of particular use in the ablation of FcγR binding are those shown in
By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is correlated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to an increase in ADCC activity.
By “ADCP” or antibody dependent cell-mediated phagocytosis as used herein is meant the cell-mediated reaction wherein nonspecific phagocytic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
As used herein, term “antibody” is used generally. Antibodies described herein can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, described herein.
Traditional immunoglobulin (Ig) antibodies are “Y” shaped tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light chain” monomer (typically having a molecular weight of about 25 kDa) and one “heavy chain” monomer (typically having a molecular weight of about 50-70 kDa).
Other useful antibody formats include those outlined herein and depicted in
Antibody heavy chains typically include a variable heavy (VH) domain, which includes vhCDR1-3, and an Fc domain, which includes a CH2-CH3 monomer. In some embodiments, antibody heavy chains include a hinge and CH1 domain. Traditional antibody heavy chains are monomers that are organized, from N- to C-terminus: VH-CH1-hinge-CH2-CH3. The CH1-hinge-CH2-CH3 is collectively referred to as the heavy chain “constant domain” or “constant region” of the antibody, of which there are five different categories or “isotypes”: IgA, IgD, IgG, IgE and IgM. Thus, “isotype” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It should be understood that therapeutic antibodies can also comprise hybrids of isotypes and/or subclasses. For example, as shown in US Publication 2009/0163699, incorporated by reference, the antibodies described herein include the use of human IgG1/G2 hybrids.
In some embodiments, the antibodies provided herein include IgG isotype constant domains, which has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By “immunoglobulin (Ig) domain” herein is meant a region of an immunoglobulin having a distinct tertiary structure. Of interest in the antibodies described herein are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, “CH” domains in the context of IgG are as follows: “CH1” refers to positions 118-220 according to the EU index as in Kabat. “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat. As shown herein and described below, the pI variants can be in one or more of the CH regions, as well as the hinge region, discussed below.
It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). The sequences depicted herein use the 356D/358M allotype, however the other allotype is included herein. That is, any sequence inclusive of an IgG1 Fc domain included herein can have 356E/358L replacing the 356D/358M allotype. It should be understood that therapeutic antibodies can also comprise hybrids of isotypes and/or subclasses. For example, as shown in US Publication 2009/0163699, incorporated by reference, the present antibodies, in some embodiments, include IgG1/IgG2 hybrids.
By “Fc” or “Fc region” or “Fc domain” as used herein is meant the polypeptide comprising the constant region of an antibody, in some instances, excluding all of the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and in some cases, optionally including all or part of the hinge. For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all or a portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, in some cases, the Fc domain includes, from N- to C-terminal, CH2-CH3 and hinge-CH2-CH3. In some embodiments, the Fc domain is that from IgG1, IgG2, IgG3 or IgG4, with IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3 finding particular use in many embodiments. Additionally, in the case of human IgG1 Fc domains, frequently the hinge includes a C220S amino acid substitution. Furthermore, in the case of human IgG4 Fc domains, frequently the hinge includes a S228P amino acid substitution. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues E216, C226, or A231 to its carboxyl-terminal, wherein the numbering is according to the EU index as in Kabat. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcγR or to the FcRn.
By “heavy chain constant region” herein is meant the CH1-hinge-CH2-CH3 portion of an antibody (or fragments thereof), excluding the variable heavy domain; in EU numbering of human IgG1 this is amino acids 118-447 By “heavy chain constant region fragment” herein is meant a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region.
Another type of Ig domain of the heavy chain is the hinge region. By “hinge” or “hinge region” or “antibody hinge region” or “hinge domain” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Thus for IgG the antibody hinge is herein defined to include positions 216 (E216 in IgG1) to 230 (p230 in IgG1), wherein the numbering is according to the EU index as in Kabat. In some cases, a “hinge fragment” is used, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain. As noted herein, pI variants can be made in the hinge region as well. Many of the antibodies herein have at least one cysteine at position 220 according to EU numbering (hinge region) replaced by a serine. Generally, this modification is on the “scFv monomer” side for most of the sequences depicted herein, although it can also be on the “Fab monomer” side, or both, to reduce disulfide formation. Specifically included within the sequences herein are one or both of these cysteines replaced (C220S).
As will be appreciated by those in the art, the exact numbering and placement of the heavy constant region domains can be different among different numbering systems. A useful comparison of heavy constant region numbering according to EU and Kabat is as below, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, entirely incorporated by reference.
The antibody light chain generally comprises two domains: the variable light domain (VL), which includes light chain CDRs vlCDR1-3, and a constant light chain region (often referred to as CL or Cκ). The antibody light chain is typically organized from N- to C-terminus: VL-CL.
By “antigen binding domain” or “ABD” herein is meant a set of Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds a target antigen (e.g., CD5 or TGFßRII) as discussed herein. As discussed herein, there are two types of ABDs that find use in the present invention, those that use a set of 6 CDRs and those that rely on a set of three, in the case of VHH ABDs as more fully discussed herein.
Many ABDs rely on a set of 6 CDRs, which are generally present as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 variable heavy CDRs and vlCDR1, vlCDR2 and vlCDR3 vhCDR3 variable light CDRs. The CDRs are present in the variable heavy domain (vhCDR1-3) and variable light domain (vlCDR1-3). The variable heavy domain and variable light domain from an Fv region.
The antibodies described herein provide a large number of different CDR sets. In this case, a “full CDR set” comprises the three variable light and three variable heavy CDRs, e.g., a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a larger variable light or variable heavy domain, respectfully. In addition, as more fully outlined herein, the variable heavy and variable light domains can be on separate polypeptide chains, when a heavy and light chain is used (for example when Fabs are used), or on a single polypeptide chain in the case of scFv sequences.
As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3) and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3). A useful comparison of CDR numbering is as below, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):
Throughout the present specification, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)).
In some embodiments, as outlined herein, a single domain antibody (sdAb, also referred to herein as “sdABD” or “VHH ABDs”) that contains only a single variable heavy domain (referred to herein as “VHH”) with three CDRs: VHHCDR1, VHHCDR2 and VHHCDR3.
Under either a VHH or standard VH and VL embodiment, the CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of the antigen binding domains and antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.
The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.
Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning.” As outlined below, the disclosure not only includes the enumerated antigen binding domains and antibodies herein, but those that compete for binding with the epitopes bound by the enumerated antigen binding domains.
In some embodiments, the six CDRs of the antigen binding domain are contributed by a variable heavy and a variable light domain. In a “Fab” format, the set of 6 CDRs are contributed by two different polypeptide sequences, the variable heavy domain (vh or VH; containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containing the vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domain being attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain being attached to the N-terminus of the constant light domain (and thus forming the light chain). In a scFv format, the vh and vl domains are covalently attached, generally through the use of a linker (a “scFv linker”) as outlined herein, into a single polypeptide sequence, which can be either (starting from the N-terminus) vh-linker-vl or vl-linker-vh, with the former being generally preferred (including optional domain linkers on each side, depending on the format used (e.g., from
By “variable region” or “variable domain” as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively, and contains the CDRs that confer antigen specificity. Thus, a “variable heavy domain” pairs with a “variable light domain” to form an antigen binding domain (“ABD”). In addition, each variable domain comprises three hypervariable regions (“complementary determining regions,” “CDRs”) (VHCDR1, VHCDR2 and VHCDR3 for the variable heavy domain and VLCDR1, VLCDR2 and VLCDR3 for the variable light domain) and four framework (FR) regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDR numbering of the invention are described in Table 2.
By “single domain Fv”, “sdFv” or “sdABD” herein is meant an antigen binding domain that only has three CDRs, generally based on camelid antibody technology. See Protein Engineering 9(7):1129-35 (1994); Rev Mol Biotech 74:277-302 (2001); Ann Rev Biochem 82:775-97 (2013).
By “Fab” or “Fab region” as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on the other). Fab may refer to this region in isolation, or this region in the context of a bispecific antibody described herein. In the context of a Fab, the Fab comprises an Fv region in addition to the CH1 and CL domains.
By “Fv” or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of an ABD. Fv regions can be formatted as both Fabs (as discussed above, generally two different polypeptides that also include the constant regions as outlined above) and scFvs, where the VL and VH domains are combined (generally with a linker as discussed herein) to form an scFv. (In some cases, the Fv region is a sdABD, as appropriate herein).
By “single chain Fv” or “scFv” herein is meant a variable heavy domain covalently attached to a variable light domain, generally using a scFv linker as discussed herein, to form a scFv or scFv domain. A scFv domain can be in either orientation from N- to C-terminus (VH-linker-VL or VL-linker-VH). In the sequences depicted in the sequence listing and in the figures, the order of the VH and VL domain is indicated in the name, e.g. H.X_L.Y means N- to C-terminal is VH-linker-VL, and L.Y_H.X is VL-linker-VH.
Some embodiments of the subject antibodies provided herein comprise at least one scFv domain, which, while not naturally occurring, generally includes a variable heavy domain and a variable light domain, linked together by a scFv linker. As outlined herein, while the scFv domain is generally from N- to C-terminus oriented as VH-scFv linker-VL, this can be reversed for any of the scFv domains (or those constructed using vh and vl sequences from Fabs), to VL-scFv linker-VH, with optional linkers at one or both ends depending on the format.
By “modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. By “amino acid modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. For clarity, unless otherwise noted, the amino acid modification is always to an amino acid coded for by DNA, e.g. the 20 amino acids that have codons in DNA and RNA.
By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.
By “amino acid insertion” or “insertion” as used herein is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, −233E or 233E designates an insertion of glutamic acid after position 233 and before position 234. Additionally, −233ADE or A233ADE designates an insertion of AlaAspGlu after position 233 and before position 234.
By “amino acid deletion” or “deletion” as used herein is meant the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233− or E233 #, E233( ) or E233del designates a deletion of glutamic acid at position 233. Additionally, EDA233− or EDA233 # designates a deletion of the sequence GluAspAla that begins at position 233.
By “variant protein” or “protein variant”, or “variant” as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification. The protein variant has at least one amino acid modification compared to the parent protein, yet not so many that the variant protein will not align with the parental protein using an alignment program such as that described below. In general, variant proteins (such as variant Fc domains, etc., outlined herein, are generally at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the parent protein, using the alignment programs described below, such as BLAST. “Variant” as used herein also refers to particular amino acid modifications that confer particular function (e.g., a “heterodimerization variant,” “pI variant,” “ablation variant,” etc.).
As described below, in some embodiments the parent polypeptide, for example an Fc parent polypeptide, is a human wild type sequence, such as the heavy constant domain or Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as “parent polypeptides”, for example the IgG1/2 hybrid of US Publication 2006/0134105 can be included. The protein variant sequence herein will preferably possess at least about 80% identity with a parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. Accordingly, by “antibody variant” or “variant antibody” as used herein is meant an antibody that differs from a parent antibody by virtue of at least one amino acid modification, “IgG variant” or “variant IgG” as used herein is meant an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by virtue of at least one amino acid modification, and “immunoglobulin variant” or “variant immunoglobulin” as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification. “Fc variant” or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain as compared to an Fc domain of human IgG1, IgG2 or IgG4.
“Fc variant” or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain. The modification can be an addition, deletion, or substitution. The Fc variants are defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S is an Fc variant with the substitution for serine at position 434 relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. Likewise, M428L/N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, 428L/434S is the same Fc variant as 434S/428L, and so on. For all positions discussed herein that relate to antibodies or derivatives and fragments thereof (e.g., Fc domains), unless otherwise noted, amino acid position numbering is according to the EU index. The “EU index” or “EU index as in Kabat” or “EU numbering” scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference).
In general, variant Fc domains have at least about 80, 85, 90, 95, 97, 98 or 99 percent identity to the corresponding parental human IgG Fc domain (using the identity algorithms discussed below, with one embodiment utilizing the BLAST algorithm as is known in the art, using default parameters). Alternatively, the variant Fc domains can have from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications as compared to the parental Fc domain. Alternatively, the variant Fc domains can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications as compared to the parental Fc domain. Additionally, as discussed herein, the variant Fc domains described herein still retain the ability to form a dimer with another Fc domain as measured using known techniques as described herein, such as non-denaturing gel electrophoresis.
By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
By “residue” as used herein is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in the human antibody IgG1.
By “IgG subclass modification” or “isotype modification” as used herein is meant an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, because IgG1 comprises a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an IgG subclass modification.
By “non-naturally occurring modification” as used herein is meant an amino acid modification that is not isotypic. For example, because none of the human IgGs comprise a serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.
By “amino acid” and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids that are coded for by DNA and RNA.
By “effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC.
By “IgG Fc ligand” as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include but are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirely incorporated by reference). Fc ligands may include undiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. By “Fc ligand” as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex.
By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRT (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes.
By “FcRn” or “neonatal Fc Receptor” as used herein is meant a protein that binds the IgG antibody Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, the functional FcRn protein comprises two polypeptides, often referred to as the heavy chain and light chain. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn protein refers to the complex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRn variants used to increase binding to the FcRn receptor, and in some cases, to increase serum half-life. An “FcRn variant” is one that increases binding to the FcRn receptor, and suitable FcRn variants are shown below.
By “parent polypeptide” as used herein is meant a starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Accordingly, by “parent immunoglobulin” as used herein is meant an unmodified immunoglobulin polypeptide that is modified to generate a variant, and by “parent antibody” as used herein is meant an unmodified antibody that is modified to generate a variant antibody. It should be noted that “parent antibody” includes known commercial, recombinantly produced antibodies as outlined below. In this context, a “parent Fc domain” will be relative to the recited variant; thus, a “variant human IgG1 Fc domain” is compared to the parent Fc domain of human IgG1, a “variant human IgG4 Fc domain” is compared to the parent Fc domain human IgG4, etc.
By “position” as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for antibody numbering.
By “target antigen” as used herein is meant the molecule that is bound specifically by the antigen binding domain comprising the variable regions of a given antibody.
By “strandedness” in the context of the monomers of the heterodimeric antibodies described herein is meant that, similar to the two strands of DNA that “match”, heterodimerization variants are incorporated into each monomer so as to preserve the ability to “match” to form heterodimers. For example, if some pI variants are engineered into monomer A (e.g. making the pI higher) then steric variants that are “charge pairs” that can be utilized as well do not interfere with the pI variants, e.g. the charge variants that make a pI higher are put on the same “strand” or “monomer” to preserve both functionalities. Similarly, for “skew” variants that come in pairs of a set as more fully outlined below, the skilled artisan will consider pI in deciding into which strand or monomer one set of the pair will go, such that pI separation is maximized using the pI of the skews as well.
By “target cell” as used herein is meant a cell that expresses a target antigen.
By “host cell” in the context of producing a bispecific antibody according to the antibodies described herein is meant a cell that contains the exogeneous nucleic acids encoding the components of the bispecific antibody and is capable of expressing the bispecific antibody under suitable conditions. Suitable host cells are discussed below.
By “wild type or WT” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.
Provided herein are a number of antibody domains that have sequence identity to human antibody domains. Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as that of Smith, T. F. & Waterman, M. S. (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S. B. & Wunsch, CD. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol. 48:443 [homology alignment algorithm], Pearson, W. R. & Lipman, D. J. (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [search for similarity method]; or Altschul, S. F. et al, (1990) “Basic Local Alignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST” algorithm, see https://blast.ncbi.nlm.nih.gov/Blast.cgi. When using any of the aforementioned algorithms, the default parameters (for Window length, gap penalty, etc.) are used. In one embodiment, sequence identity is done using the BLAST algorithm, using default parameters
The antibodies described herein are generally isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. “Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells, and they can be isolated as well.
“Specific binding” or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10−4 M, at least about 10−5 M, at least about 10−6 M, at least about 10−7 M, at least about 10−8 M, at least about 10−9 M, alternatively at least about 10−10 M, at least about 10−11 M, at least about 10−12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope.
Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction. Binding affinity is generally measured using a Biacore, SPR or BLI assay.
The antibodies provided herein are listed in several different formats. In some instances, each monomer of a particular antibody is given a unique “XENP” number, although as will be appreciated in the art, a longer sequence might contain a shorter one. For example, a “scFv-Fc” monomer of a 1+1 Fab-scFv-Fc format antibody may have a first XENP number, while the scFv domain itself will have a different XENP number. Some molecules have three polypeptides, so the XENP number, with the components, is used as a name. Thus, the molecule XENP33041, which is an anti-TGFßRII×anti-PD1 bsAb in the “1+1 Fab-scFv-Fc” format, as depicted in
Therapeutic antibodies directed against immune checkpoint inhibitors such as PD-1 are showing great promise in limited circumstances in the clinic for the treatment of cancer. Cancer can be considered as an inability of the patient to recognize and eliminate cancerous cells. In many instances, these transformed (e.g. cancerous) cells counteract immunosurveillance. There are natural control mechanisms that limit T-cell activation in the body to prevent unrestrained T-cell activity, which can be exploited by cancerous cells to evade or suppress the immune response. Restoring the capacity of immune effector cells-especially T cells—to recognize and eliminate cancer is the goal of immunotherapy. The field of immuno-oncology, sometimes referred to as “immunotherapy” is rapidly evolving, with several recent approvals of T cell checkpoint inhibitory antibodies such as Yervoy, Keytruda and Opdivo. These antibodies are generally referred to as “checkpoint inhibitors” because they block normally negative regulators of T cell immunity. It is generally understood that a variety of immunomodulatory signals, both costimulatory and coinhibitory, can be used to orchestrate an optimal antigen-specific immune response.
Generally, these monoclonal antibodies bind to checkpoint inhibitor proteins such as CTLA-4 and PD-1, which under normal circumstances prevent or suppress activation of cytotoxic T cells (CTLs). By inhibiting the checkpoint protein, for example through the use of antibodies that bind these proteins, an increased T cell response against tumors can be achieved. That is, these cancer checkpoint proteins suppress the immune response; when the proteins are blocked, for example using antibodies to the checkpoint protein, the immune system is activated, leading to immune stimulation, resulting in treatment of conditions such as cancer and infectious disease.
However, as discussed above, studies have shown that TILs commonly express multiple checkpoint receptors; this may suggest that single checkpoint blockade could be insufficient to promote a complete T cell response. Moreover, it is likely that TILs that express multiple checkpoints are in fact the most tumor-reactive, thus suggesting that therapies that engage more than one checkpoint antigen could be very useful.
Another mechanism by which tumors evade immune surveillance is by producing the immunosuppressive cytokine TGFß which directly inhibits the expression of cytolytic proteins such as IFNγ which are necessary for T cell-mediated tumor cytotoxicity. Additionally, TGFß is pro-fibrotic and promotes the expansion of fibroblasts. Cancer-associated fibroblasts (CAFs) have been reported to promote tumor survival and proliferation (Orimo et al., 2006; Xing et al., 2011), for example by providing growth factors for angiogenesis and by further encouraging an immunosuppressive environment, and have been associated with poor prognosis (Underwood et al, 2015).
Accordingly, the present invention provides bispecific heterodimeric antibodies, that bind to cells expressing the two antigens and methods of activating T cells and/or NK cells to treat diseases such as cancer and infectious diseases, and other conditions where increased immune activity results in treatment.
In some instances, it is beneficial to target TGFßRII blockade in broader cell populations. As described herein CD5 is a promiscuous cell-surface phosphatase that is expressed in many activated and unactivated T cells. Thus, bispecific heterodimeric antibodies that bind TGFßRII and CD5 can advantageously block TGFß activity in a broad population of cells, wherein such blockage of TGFß activity is desirable, for example, for the treatment of cancers.
Thus, the invention is directed, in some instances, to solving the issue of toxicity and expense of administering multiple antibodies by providing bispecific antibodies that bind to two different molecules on a single cell and advantageously requiring administration of only one therapeutic substance.
Bispecific antibodies, which can bind two different targets simultaneously, offer the potential to improve the selectivity of targeting particular cell types (e.g., high CD5+ cells or TILs), while also reducing cost of therapy. The bivalent interaction of an antibody with two targets on a cell surface should—in some cases—lead to a higher binding avidity relative to a monovalent interaction with one target at a time. Because of this, normal bivalent antibodies tend to have high avidity for their target on a cell surface. With bispecific antibodies, the potential exists to create higher selectivity for cells that simultaneously express two different targets, utilizing the higher avidity afforded by simultaneous binding to both targets.
Accordingly, the present invention is directed to novel constructs to provide heterodimeric antibodies that allow binding to more than one antigen or ligand, e.g. to allow for bispecific binding. The heterodimeric bispecific antibodies of the invention are useful to treat a variety of types of cancers. As will be appreciated by those in the art, in contrast to traditional monoclonal antibodies that bind to tumor antigens, or to the newer classes of bispecific antibodies that bind, for example, CD3 and tumor antigens (such as described in U.S. Ser. No. 15/141,350, for example), checkpoint antibodies are used to increase the immune response but are not generally tumor specific in their action. That is, the bispecific antibodies of the invention inhibit the suppression of the immune system, generally leading to T cell activation, which in turn leads to greater immune response to cancerous cells and thus treatment. Such antibodies can therefore be expected to find utility for treatment of a wide variety of tumor types. For example, the FDA recently approved Keytruda®, an anti-PD-1 monospecific antibody on the basis of a genetic feature, rather than a tumor type.
Additionally, the bispecific heterodimeric antibodies of the present invention that bind to TGFßRII and PD-1 can have two different functional components. In some embodiments, the anti-PD-1 antigen binding domain (ABD) competes for binding with approved anti-PD-1 antibodies such as nivolumab (OPDIVO®) and pembrolizumab (KEYTRUDA®); that is, the anti-PD-1 ABD of the bispecific antibody serves to prevent the binding of PD-1 to its cognate ligands such as PD-L1. That is, the anti-PD-1 ABD is used to both target T cells but also to block interaction with PD-1 ligands. In other embodiments, the anti-PD-1 ABD of the bispecific antibody serves only to target the bispecific antibody to the T cell and does not interfere with the association of PD-1 with its ligands; that is, it does not compete with nivolumab or permbrolizumab, and thus, in some embodiments, can be co-administered with a standard anti-PD-1 antibody such as nivolumab or permbrolizumab to give unexpectedly better results. Anti-PD-1 ABDs that do not compete are referred to herein as “non-competing PD-1 ABDs” or “NCPD-1 ABDs”.
Accordingly, in some embodiments the present invention provides heterodimeric immunomodulatory antibodies that rely on the use of two different heavy chain variant Fc sequences, that will self-assemble to form heterodimeric Fc domains and heterodimeric antibodies.
The present invention is directed to novel constructs to provide heterodimeric antibodies that allow binding to more than one immunomodulatory antigen or ligand, e.g. to allow for bispecific binding. The heterodimeric antibody constructs are based on the self-assembling nature of the two Fc domains of the heavy chains of antibodies, e.g. two “monomers” that assemble into a “dimer”. Heterodimeric antibodies are made by altering the amino acid sequence of each monomer as more fully discussed below. Thus, the present invention is generally directed to the creation of heterodimeric immunomodulatory antibodies which can co-engage antigens in several ways, relying on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and/or allow for ease of purification of heterodimers over the homodimers.
Thus, the present invention provides bispecific antibodies. An ongoing problem in antibody technologies is the desire for “bispecific” antibodies that bind to two different antigens simultaneously, in general thus allowing the different antigens to be brought into proximity and resulting in new functionalities and new therapies. In general, these antibodies are made by including genes for each heavy and light chain into the host cells. This generally results in the formation of the desired heterodimer (A-B), as well as the two homodimers (A-A and B-B (not including the light chain heterodimeric issues)). However, a major obstacle in the formation of bispecific antibodies is the difficulty in purifying the heterodimeric antibodies away from the homodimeric antibodies and/or biasing the formation of the heterodimer over the formation of the homodimers. In some embodiments, the bispecific antibodies include a TGFßRII binding domain. Any suitable TGFßRII binding domain can be included in the bispecific antibody provided herein, including those disclosed in the Figures (e.g.,
There are a number of mechanisms that can be used to generate the heterodimers of the present invention. In addition, as will be appreciated by those in the art, these mechanisms can be combined to ensure high heterodimerization. Thus, amino acid variants that lead to the production of heterodimers are referred to as “heterodimerization variants”. As discussed below, heterodimerization variants can include steric variants (e.g. the “knobs and holes” or “skew” variants described below and the “charge pairs” variants described below) as well as “pI variants”, which allows purification of homodimers away from heterodimers. As is generally described in WO2014/145806, hereby incorporated by reference in its entirety and specifically as below for the discussion of “heterodimerization variants”, useful mechanisms for heterodimerization include “knobs and holes” (“KIH”; sometimes herein as “skew” variants (see discussion in WO2014/145806), “electrostatic steering” or “charge pairs” as described in WO2014/145806, pI variants as described in WO2014/145806, and general additional Fc variants as outlined in WO2014/145806 and below.
In the present invention, there are several basic mechanisms that can lead to ease of purifying heterodimeric antibodies; one relies on the use of pI variants, such that each monomer has a different pI, thus allowing the isoelectric purification of A-A, A-B and B-B dimeric proteins. Alternatively, some scaffold formats, such as the “triple F” format, also allows separation on the basis of size. As is further outlined below, it is also possible to “skew” the formation of heterodimers over homodimers. Thus, a combination of steric heterodimerization variants and pI or charge pair variants find particular use in the invention.
In general, embodiments of particular use in the present invention rely on sets of variants that include skew variants, that encourage heterodimerization formation over homodimerization formation, coupled with pI variants, which increase the pI difference between the two monomers.
Additionally, as more fully outlined below, depending on the format of the heterodimer antibody, pI variants can be either contained within the constant and/or Fc domains of a monomer, or charged linkers, either domain linkers or scFv linkers, can be used. That is, scaffolds that utilize scFv(s) such as the Triple F format can include charged scFv linkers (either positive or negative), that give a further pI boost for purification purposes. As will be appreciated by those in the art, some Triple F formats are useful with just charged scFv linkers and no additional pI adjustments, although the invention does provide pI variants that are on one or both of the monomers, and/or charged domain linkers as well. In addition, additional amino acid engineering for alternative functionalities may also confer pI changes, such as Fc, FcRn and KO variants.
In the present invention that utilizes pI as a separation mechanism to allow the purification of heterodimeric proteins, amino acid variants can be introduced into one or both of the monomer polypeptides; that is, the pI of one of the monomers (referred to herein for simplicity as “monomer A”) can be engineered away from monomer B, or both monomer A and B change be changed, with the pI of monomer A increasing and the pI of monomer B decreasing. As discussed, the pI changes of either or both monomers can be done by removing or adding a charged residue (e.g. a neutral amino acid is replaced by a positively or negatively charged amino acid residue, e.g. glycine to glutamic acid), changing a charged residue from positive or negative to the opposite charge (e.g. aspartic acid to lysine) or changing a charged residue to a neutral residue (e.g. loss of a charge; lysine to serine.). A number of these variants are shown in the Figures.
Accordingly, this embodiment of the present invention provides for creating a sufficient change in pI in at least one of the monomers such that heterodimers can be separated from homodimers. As will be appreciated by those in the art, and as discussed further below, this can be done by using a “wild type” heavy chain constant region and a variant region that has been engineered to either increase or decrease its pI (wt A−+B or wt A−−B), or by increasing one region and decreasing the other region (A+−B− or A−B+).
Thus, in general, a component of some embodiments of the present invention are amino acid variants in the constant regions of antibodies that are directed to altering the isoelectric point (pI) of at least one, if not both, of the monomers of a dimeric protein to form “pI antibodies” by incorporating amino acid substitutions (“pI variants” or “pI substitutions”) into one or both of the monomers. As shown herein, the separation of the heterodimers from the two homodimers can be accomplished if the pIs of the two monomers differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in the present invention.
As will be appreciated by those in the art, the number of pI variants to be included on each or both monomer(s) to get good separation will depend in part on the starting pI of the components, for example in the triple F format, the starting pI of the scFv and Fab of interest. That is, to determine which monomer to engineer or in which “direction” (e.g. more positive or more negative), the Fv sequences of the two target antigens are calculated and a decision is made from there. As is known in the art, different Fvs will have different starting pIs which are exploited in the present invention. In general, as outlined herein, the pIs are engineered to result in a total pI difference of each monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred as outlined herein. Furthermore, as will be appreciated by those in the art and outlined herein, in some embodiments, heterodimers can be separated from homodimers on the basis of size. Several of the formats provided herein allow separation of heterodimers and homodimers on the basis of size.
1. Heterodimerization Variants
The present invention provides heterodimeric proteins, including heterodimeric antibodies in a variety of formats, which utilize heterodimeric variants to allow for heterodimeric formation and/or purification away from homodimers. A number of heterodimerization variants are shown in the Figures (e.g.,
There are a number of suitable pairs of sets of heterodimerization skew variants. These variants come in “pairs” of “sets”. That is, one set of the pair is incorporated into the first monomer and the other set of the pair is incorporated into the second monomer. It should be noted that these sets do not necessarily behave as “knobs in holes” variants, with a one-to-one correspondence between a residue on one monomer and a residue on the other; that is, these pairs of sets form an interface between the two monomers that encourages heterodimer formation and discourages homodimer formation, allowing the percentage of heterodimers that spontaneously form under biological conditions to be over 90%, rather than the expected 50% (25 homodimer A/A:50% heterodimer A/B:25% homodimer B/B).
2. Steric Variants
In some embodiments, the formation of heterodimers can be facilitated by the addition of steric variants. That is, by changing amino acids in each heavy chain, different heavy chains are more likely to associate to form the heterodimeric structure than to form homodimers with the same Fc amino acid sequences. Suitable steric variants are included in in the Figures.
One mechanism is generally referred to in the art as “knobs and holes”, referring to amino acid engineering that creates steric influences to favor heterodimeric formation and disfavor homodimeric formation can also optionally be used; this is sometimes referred to as “knobs and holes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated by reference in their entirety. The Figures identify a number of “monomer A-monomer B” pairs that rely on “knobs and holes”. In addition, as described in Merchant et al., Nature Biotech. 16:677 (1998), these “knobs and hole” mutations can be combined with disulfide bonds to skew formation to heterodimerization.
An additional mechanism that finds use in the generation of heterodimers is sometimes referred to as “electrostatic steering” as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), hereby incorporated by reference in its entirety. This is sometimes referred to herein as “charge pairs”. In this embodiment, electrostatics are used to skew the formation towards heterodimerization. As those in the art will appreciate, these may also have an effect on pI, and thus on purification, and thus could in some cases also be considered pI variants. However, as these were generated to force heterodimerization and were not used as purification tools, they are classified as “steric variants”. These include, but are not limited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g. these are “monomer corresponding sets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.
Additional monomer A and monomer B variants that can be combined with other variants, optionally and independently in any amount, such as pI variants outlined herein or other steric variants that are shown in FIG. 37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which are incorporated expressly by reference herein.
In some embodiments, the steric variants outlined herein can be optionally and independently incorporated with any pI variant (or other variants such as Fc variants, FcRn variants, etc.) into one or both monomers, and can be independently and optionally included or excluded from the proteins of the invention.
A list of suitable skew variants is found in the Figures showing some pairs of particular utility in many embodiments. Of particular use in many embodiments are the pairs of sets including, but not limited to, S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L, K370S:S364K/E357Q and T366S/L368A/Y407V:T366W (optionally including a bridging disulfide, T366S/L368A/Y407V/Y349C:T366W/S354C). In terms of nomenclature, the pair “S364K/E357Q: L368D/K370S” means that one of the monomers has the double variant set S364K/E357Q and the other has the double variant set L368D/K370S; as above, the “strandedness” of these pairs depends on the starting pI.
3. pI (Isoelectric Point) Variants for Heterodimers
In general, as will be appreciated by those in the art, there are two general categories of pI variants: those that increase the pI of the protein (basic changes) and those that decrease the pI of the protein (acidic changes). As described herein, all combinations of these variants can be done: one monomer may be wild type, or a variant that does not display a significantly different pI from wild-type, and the other can be either more basic or more acidic. Alternatively, each monomer is changed, one to more basic and one to more acidic.
Preferred combinations of pI variants are shown in the Figures. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be altered this way, as well as isotype hybrids. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used.
In one embodiment, for example in the bottle opener format, a preferred combination of pI variants has one monomer (the negative Fab side) comprising 208D/295E/384D/418E/421D variants (N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a second monomer (the positive scFv side) comprising a positively charged scFv linker, including (GKPGS)4. However, as will be appreciated by those in the art, the first monomer includes a CH1 domain, including position 208. Accordingly, in constructs that do not include a CH1 domain (for example for heterodimeric Fc fusion proteins that do not utilize a CH1 domain on one of the domains, for example in a dual scFv format), a preferred negative pI variant Fc set includes 295E/384D/418E/421D variants (Q295E/N384D/Q418E/N421D when relative to human IgG1).
Accordingly, in some embodiments, one monomer has a set of substitutions from the Figures and the other monomer has a charged linker (either in the form of a charged scFv linker because that monomer comprises an scFv or a charged domain linker, as the format dictates).
4. Isotypic Variants
In addition, many embodiments of the invention rely on the “importation” of pI amino acids at particular positions from one IgG isotype into another, thus reducing or eliminating the possibility of unwanted immunogenicity being introduced into the variants. A number of these are shown in FIG. 21 of US Publ. 2014/0370013, hereby incorporated by reference. That is, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the heavy constant region of IgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). By introducing IgG2 residues at particular positions into the IgG1 backbone, the pI of the resulting monomer is lowered (or increased) and additionally exhibits longer serum half-life. For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI 3.22); importing the glutamic acid will affect the pI of the resulting protein. As is described below, a number of amino acid substitutions are generally required to significant affect the pI of the variant antibody. However, it should be noted as discussed below that even changes in IgG2 molecules allow for increased serum half-life.
In other embodiments, non-isotypic amino acid changes are made, either to reduce the overall charge state of the resulting protein (e.g. by changing a higher pI amino acid to a lower pI amino acid), or to allow accommodations in structure for stability, etc. as is more further described below.
In addition, by pI engineering both the heavy and light constant domains, significant changes in each monomer of the heterodimer can be seen. As discussed herein, having the pIs of the two monomers differ by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point.
5. Calculating pI
The pI of each monomer can depend on the pI of the variant heavy chain constant domain and the pI of the total monomer, including the variant heavy chain constant domain and the fusion partner. Thus, in some embodiments, the change in pI is calculated on the basis of the variant heavy chain constant domain, using the chart in the FIG. 19 of US Pub. 2014/0370013. As discussed herein, which monomer to engineer is generally decided by the inherent pI of the Fv and scaffold regions. Alternatively, the pI of each monomer can be compared.
6. pI Variants that Also Confer Better FcRn In Vivo Binding
In the case where the pI variant decreases the pI of the monomer, they can have the added benefit of improving serum retention in vivo.
Although still under examination, Fc regions are believed to have longer half-lives in vivo, because binding to FcRn at pH 6 in an endosome sequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598, entirely incorporated by reference). The endosomal compartment then recycles the Fc to the cell surface. Once the compartment opens to the extracellular space, the higher pH, ˜7.4, induces the release of Fc back into the blood. In mice, Dall'Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180, entirely incorporated by reference). The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid the release of the Fc back into the blood. Therefore, the Fc mutations that will increase Fc's half-life in vivo will ideally increase FcRn binding at the lower pH while still allowing release of Fc at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is not surprising to find His residues at important positions in the Fc/FcRn complex.
Recently it has been suggested that antibodies with variable regions that have lower isoelectric points may also have longer serum half-lives (Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated by reference). However, the mechanism of this is still poorly understood. Moreover, variable regions differ from antibody to antibody. Constant region variants with reduced pI and extended half-life would provide a more modular approach to improving the pharmacokinetic properties of antibodies, as described herein.
7. Additional Fc Variants for Additional Functionality
In addition to pI amino acid variants, there are a number of useful Fc amino acid modification that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcγR receptors, altered binding to FcRn receptors, etc.
Accordingly, the proteins of the invention can include amino acid modifications, including the heterodimerization variants outlined herein, which includes the pI variants and steric variants. Each set of variants can be independently and optionally included or excluded from any particular heterodimeric protein.
8. FcγR Variants
Accordingly, there are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcγRIIIa results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Ser. No. 11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495, 11/538,406, all of which are expressly incorporated herein by reference in their entirety and specifically for the variants disclosed therein. Particular variants that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.
In addition, there are additional Fc substitutions that find use in increased binding to the FcRn receptor and increased serum half-life, as specifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporated by reference in its entirety, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.
9. Ablation Variants
Similarly, another category of functional variants are “FcγR ablation variants” or “Fc knock out (FcKO or KO)” variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or remove the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. That is, for example, in many embodiments, particularly in the use of bispecific immunomodulatory antibodies desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity such that one of the Fc domains comprises one or more Fcγ receptor ablation variants. These ablation variants are depicted in the figures, and each can be independently and optionally included or excluded, with preferred aspects utilizing ablation variants selected from the group consisting of G236R/L328R, E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del. It should be noted that the ablation variants referenced herein ablate FcγR binding but generally not FcRn binding.
10. Combination of Heterodimeric and Fc Variants
As will be appreciated by those in the art, all of the recited heterodimerization variants (including skew and/or pI variants) can be optionally and independently combined in any way, as long as they retain their “strandedness” or “monomer partition”. In addition, all of these variants can be combined into any of the heterodimerization formats.
In the case of pI variants, while embodiments finding particular use are shown in the Figures, other combinations can be generated, following the basic rule of altering the pI difference between two monomers to facilitate purification.
In addition, any of the heterodimerization variants, skew and pI, are also independently and optionally combined with Fc ablation variants, Fc variants, FcRn variants, as generally outlined herein.
In one aspect, provided herein are antigen binding domains (ABDs) that bind CD5 (also referred to herein as “anti-CD5 antigen binding domains” or CD5 antigen binding domains” or “CD5 binding domains”) and related antibodies that include such anti-CD5 binding domains (e.g., anti-CD5×anti-TGFßRII bispecific antibodies). The subject anti-CD5 binding domains described herein are capable of binding to CD5 expressing cells.
In some embodiments, antibodies that include the anti-CD5 antigen binding domains provided herein are capable of selectively binding to cells expressing high levels of CD5 over cells expressing low levels of CD5. In some embodiments, the antibodies that include such anti-CD5 antigen binding domains are useful in the treatment of cancers. For example, anti-CD5×anti-TGFßRII bispecific antibodies that include the subject anti-CD5 antigen binding domains find use as cancer therapeutics by blocking the TGFß/TGFßR axis in cells that express CD5. In particular embodiments, the subject anti-CD5×anti-TGFßRII bispecific antibodies are capable of enhancing blocking activity on both on activated and unactivated T cells.
As will be appreciated by those in the art, suitable anti-CD5 binding domains can include a set of 6 CDRs as depicted in the figures (
In one embodiment, the anti-CD5 antigen binding domain includes the 6 CDRs (i.e., vhCDR1-3 and vlCDR1-3) of an anti-CD5 antigen binding domain formed by any combination of an anti-CD5 VH and VL described herein, including the figures (
In addition to the parental CDR sets disclosed in the figures and sequence listing that form an ABD to CD5, provided herein are variant anti-CD5 ABDS having CDRs that include at least one modification of the anti-CD5 ABD CDRs disclosed herein. In one embodiment, the anti-CD5 ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of an anti-CD5 antigen binding domain formed by any combination of an anti-CD5 variable heavy (VH) domain and variable light (VL) domain described herein, including the figures (
In one embodiment, the anti-CD5 ABD includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of an anti-CD5 antigen binding domain formed by any combination of an anti-CD5 variable heavy (VH) domain and variable light (VL) domain described herein, including the figures (
In another exemplary embodiment, the anti-CD5 ABD include the variable heavy (VH) domain and variable light (VL) domain of any one of the anti-CD5 VH domains and VL domains described herein, including the figures (
In addition to the parental anti-CD5 binding domain variable heavy and variable light domains disclosed herein, provided herein are anti-CD5 ABDs that include a variable heavy domain and/or a variable light domain that are variants of an anti-CD5 ABD variable heavy (VH) domain and variable light (VL) domain disclosed herein. In one embodiment, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of an anti-CD5 variable heavy (VH) domain and variable light (VL) domain described herein, including the figures (
In one embodiment, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of an anti-CD5 ABD as described herein, including the figures (
Such CD5 binding domains can be included in any of the heterodimeric antibodies provided herein including, for example, the “1+1 Fab-scFv-Fc,” “1+1 Fab-VHH-Fc,” “1+1 VHH-scFv-Fc,” “2+1 Fab2-scFv-Fc,” and “2+1 Fab2-VHH-Fc” format antibodies disclosed herein.
Provided herein are antigen binding domains that bind TGFßRII (also referred to herein as “anti-TGFßRII antigen binding domains” or “TGFßRII antigen binding domains”) and variants thereof, as well as related antibodies that include such anti-TGFßRII binding domains (e.g., anti-CD5×anti-TGFßRII bispecific antibodies and anti-PD-1×anti-TGFßRII bispecific antibodies). As outlined herein, there are two different types of anti-TGFßRII ABDs, those that contain a VH and VL and those that are VHH domains and only contain a single heavy variable domain.
Anti-TGFßRII ABD variable heavy and variable light domains are depicted in
As will be appreciated by those in the art, suitable anti-TGFßRII binding domains can include a set of 6 CDRs as depicted in the figures (
In one embodiment, the anti-TGFßRII antigen binding domain includes the 6 CDRs (i.e., vhCDR1-3 and vlCDR1-3) of an anti-TGFßRII antigen binding domain formed by any combination of an anti-TGFßRII VH and VL described herein, including the figures (
In addition to the parental CDR sets disclosed in the figures and sequence listing that form an ABD to TGFßRII, provided herein are variant anti-TGFßRII ABDS having CDRs that include at least one modification of the anti-TGFßRII ABD CDRs disclosed herein. In one embodiment, the anti-TGFßRII ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of an anti-TGFßRII antigen binding domain formed by any combination of an anti-TGFßRII variable heavy (VH) domain and variable light (VL) domain described herein, including the figures (
In one embodiment, the anti-TGFßRII ABD includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of an anti-TGFßRII antigen binding domain formed by any combination of an anti-TGFßRII variable heavy (VH) domain and variable light (VL) domain described herein, including the figures (
In another exemplary embodiment, the anti-TGFßRII ABD include the variable heavy (VH) domain and variable light (VL) domain of any one of the anti-TGFßRII VH domains and VL domains described herein, including the figures (
In addition to the parental anti-TGFßRII binding domain variable heavy and variable light domains disclosed herein, provided herein are anti-TGFßRII ABDs that include a variable heavy domain and/or a variable light domain that are variants of an anti-TGFßRII ABD variable heavy (VH) domain and variable light (VL) domain disclosed herein. In one embodiment, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of an anti-TGFßRII variable heavy (VH) domain and variable light (VL) domain described herein, including the figures (
In one embodiment, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of an anti-TGFßRII ABD as described herein, including the figures (
Such TGFßRII binding domains can be included in any of the heterodimeric antibodies provided herein including, for example, the “1+1 Fab-scFv-Fc,” “1+1 Fab-VHH-Fc,” “1+1 VHH-scFv-Fc,” “2+1 Fab2-scFv-Fc,” and “2+1 Fab2-VHH-Fc” format antibodies disclosed herein.
The invention provides bispecific heterodimeric antibodies that bind to human PD-1, the sequence of which are depicted in the sequence listing. As outlined herein, there are two different types of anti-PD-1 ABDs, those that compete for binding with nivolumab and pembrolizumab (e.g. those that interfere with the binding of the PD-1 protein with its cognate functional ligands but target the antibody to the T cells), and those that do not compete (and thus can be co-administered with anti-PD-1 antibodies as well).
As will be appreciated by those in the art, there are a large number of suitable anti-PD-1 ABDs that bind human PD-1, including those depicted in the sequence listing. Additionally, these anti-PD-1 variable heavy (VH) domain and variable light (VL) domain domains can be utilized either as Fab constructs, or as scFv constructs. The VH/VL of competing PD-1 ABDs that are useful in the subject in the heterodimeric antibodies provided herein include, but are not limited to: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479 and variants thereof.
Alternatively, anti-PD-1 ABDs that bind human PD-1 but do not compete for binding with nivolumab and pembrolizumab, “NCPD-1 ABDs”, can also be used in the present invention. Non-competing PD-1 ABDs that are useful in the subject in the heterodimeric antibodies provided herein include, but are not limited to, those that include a variable heavy domain selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123 or a variant thereof, and a variable light domain selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127 or a variant thereof.
In one embodiment, the anti-PD-1 antigen binding domain includes the 6 CDRs (i.e., vhCDR1-3 and vlCDR1-3) of an anti-PD-1 antigen binding domain formed by any combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, including the sequence listing. In exemplary embodiments, the variable heavy domain is selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123, and the variable light domain is selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127. In exemplary embodiments, the variable heavy domain and variable light domain are selected from the following VH/VL combinations: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479.
In addition to the parental CDR sets disclosed in the figures and sequence listing that form an ABD to PD-1, provided herein are variant anti-PD-1 ABDS having CDRs that include at least one modification of the anti-PD-1 ABD CDRs disclosed herein. In one embodiment, the anti-PD-1 ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of an anti-PD-1 antigen binding domain formed by any combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, including the sequence listing. In exemplary embodiments, the anti-PD-1 ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of an anti-PD-1 antigen binding domain formed by any combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, wherein the variable heavy domain is selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123, and the variable light domain is selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127. In exemplary embodiments, the anti-PD-1 ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of an anti-PD-1 antigen binding domain formed by any combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, including any of following VH/VL combinations: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479.
In one embodiment, the anti-PD-1 ABD includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of an anti-PD-1 antigen binding domain formed by any combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, including the sequence listing. In exemplary embodiments, the anti-PD-1 ABD includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of an anti-PD-1 antigen binding domain formed by a combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, wherein the variable heavy domain is selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123, and the variable light domain is selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127. In exemplary embodiments, the anti-PD-1 ABD includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of an anti-PD-1 antigen binding domain formed by a combination of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, including any of following VH/VL combinations: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479. In certain embodiments, the anti-PD-1 ABD is capable of binding to PD-1 antigen, as measured by at least one of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the anti-PD-1 ABD is capable of binding human PD-1.
In another exemplary embodiment, the anti-PD-1 ABD include the variable heavy (VH) domain and variable light (VL) domain of any one of the anti-PD-1 VH domains and VL domains described herein, including the sequence listing. In exemplary embodiments, the variable heavy domain is selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123, and the variable light domain is selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127. In exemplary embodiments, the variable heavy domain and variable light domain is any of following VH/VL combinations: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479.
In addition to the parental anti-PD-1 binding domain variable heavy and variable light domains disclosed herein, provided herein are anti-PD-1 ABDs that include a variable heavy domain and/or a variable light domain that are variants of an anti-PD-1 ABD variable heavy (VH) domain and variable light (VL) domain disclosed herein. In one embodiment, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of an anti-PD-1 variable heavy (VH) domain and variable light (VL) domain described herein, including the sequence listing. In exemplary embodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain, wherein the variable heavy domain is selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123, and the variable light domain selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127. In exemplary embodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain selected from any of following VH/VL combinations: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479. In certain embodiments, the anti-PD-1 ABD is capable of binding to PD-1, as measured by at least one of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the anti-PD-1 ABD is capable of binding human PD-1.
In one embodiment, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of an anti-PD-1 ABD as described herein, including the sequence listing. In exemplary embodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to a VH and/or VL, wherein the variable heavy domain is selected from the group consisting of: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, and SEQ ID NO:123, and the variable light domain selected from the group consisting of: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, and SEQ ID NO: 127. In exemplary embodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to a VH and/or VL selected from any of following VH/VL combinations: SEQ ID NOs:131 and 135, SEQ ID NOs:139 and 143, SEQ ID NOs:147 and 151, SEQ ID NOs:155 and 159, SEQ ID NOs:163 and 167, SEQ ID NOs:171 and 175, SEQ ID NOs:179 and 183, SEQ ID NOs:187 and 191, SEQ ID NOs:195 and 199, SEQ ID NOs:203 and 207, SEQ ID NOs:211 and 215, SEQ ID NOs:219 and 223, SEQ ID NOs:227 and 231, SEQ ID NOs:235 and 239, SEQ ID NOs:243 and 247, SEQ ID NOs:251 and 255, SEQ ID NOs:259 and 264, SEQ ID NOs:267 and 271, SEQ ID NOs:275 and 279, SEQ ID NOs:283 and 287, SEQ ID NOs:291 and 295, SEQ ID NOs:299 and 303, SEQ ID NOs:307 and 211, SEQ ID NOs:315 and 319, SEQ ID NOs:323 and 327, SEQ ID NOs:331 and 335, SEQ ID NOs:339 and 343, SEQ ID NOs:347 and 351, SEQ ID NOs:355 and 359, SEQ ID NOs:363 and 367, SEQ ID NOs:371 and 375, SEQ ID NOs:379 and 384, SEQ ID NOs:387 and 391, SEQ ID NOs:395 and 399, SEQ ID NOs:403 and 407, SEQ ID NOs:411 and 415, SEQ ID NOs:419 and 423, SEQ ID NOs:427 and 431, SEQ ID NOs:435 and 439, SEQ ID NOs:443 and 447, SEQ ID NOs:451 and 455, SEQ ID NOs:459 and 463, SEQ ID NOs:467 and 471, and SEQ ID NOs:475 and 479. In certain embodiments, the anti-PD-1 ABD is capable of binding to PD-1, as measured by at least one of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the anti-PD-1 ABD is capable of binding human PD-1.
Such PD-1 binding domains can be included in any of the heterodimeric antibodies provided herein including, for example, the “1+1 Fab-scFv-Fc,” “1+1 Fab-VHH-Fc,” “1+1 VHH-scFv-Fc,” “2+1 Fab2-scFv-Fc,” and “2+1 Fab2-VHH-Fc” format antibodies disclosed herein.
As will be appreciated by those in the art and discussed more fully below, the bispecific heterodimeric antibodies of the present invention can take on a wide variety of configurations, as are generally depicted in
As will be appreciated by those in the art, the heterodimeric formats (see
1. 1+1 Fab-scFv-Fc Format
One heterodimeric scaffold that finds particular use in the antibodies described herein is the “1+1 Fab-scFv-Fc” or “bottle-opener” format as shown in
There are several distinct advantages to the present “1+1 Fab-scFv-Fc” format. As is known in the art, antibody analogs relying on two scFv constructs often have stability and aggregation problems, which can be alleviated in the antibodies described herein by the addition of a “regular” heavy and light chain pairing. In addition, as opposed to formats that rely on two heavy chains and two light chains, there is no issue with the incorrect pairing of heavy and light chains (e.g. heavy 1 pairing with light 2, etc.).
Many of the embodiments outlined herein rely in general on the 1+1 Fab-scFv-Fc or “bottle opener” format antibody that comprises a first monomer comprising an scFv, comprising a variable heavy and a variable light domain, covalently attached using an scFv linker (charged, in many but not all instances), where the scFv is covalently attached to the N-terminus of a first Fc domain usually through a domain linker The domain linker can be either charged or uncharged and exogenous or endogenous (e.g., all or part of the native hinge domain). Any suitable linker can be used to attach the scFv to the N-terminus of the first Fc domain. In some embodiments, the domain linker is chosen from the domain linkers in
In
In addition, the Fc domains of the antibodies described herein generally include skew variants (e.g. a set of amino acid substitutions as shown in
In certain embodiments, the 1+1 Fab-scFv-Fc format includes a first monomer that includes, from N- to C-terminus, a scFv-domain linker-CH2-CH3 monomer, a second monomer that includes a first variable heavy domain-CH1-hinge-CH2-CH3 monomer and a third monomer that includes a first variable light domain and a constant light domain. In some embodiments, the CH2-CH3 of the first monomer is a first variant Fc domain and the CH2-CH3 of the second monomer is a second variant Fc domain. In some embodiments, the scFv includes a scFv variable heavy domain and a scFv variable light domain that form a binding moiety. In certain embodiments, the scFv variable heavy domain and scFv variable light domain are covalently attached using an scFv linker (charged, in many but not all instances. See, e.g.,
In some embodiments, the 1+1 Fab-scFv-Fc format antibody includes any one of the anti-CD5 antigen binding domains provided herein. In exemplary embodiments, the 1+1 Fab-scFv-Fc format antibody includes a Fab side that includes any of the anti-CD5 antigen binding domains provided herein. In exemplary embodiments, the 1+1 Fab-scFv-Fc format antibody includes an scFv side that includes any of the anti-CD5 antigen binding domains provided herein. In some embodiments, the anti-CD5 antigen binding domain includes the 6 CDRs or the VH and VL of any of the anti-CD5 antigen binding domains described herein, including those depicted in
In some embodiments, the 1+1 Fab-scFv-Fc format antibody includes any one of the anti-TGFßRII antigen binding domains provided herein. In some embodiments of the 1+1 Fab-scFv-Fc format antibody, the anti-TGFßRII includes the 6 CDRs or the VH and VL of any of the anti-TGFßRII antigen binding domains described herein, including those depicted in
In some embodiments of the 1+1 Fab-scFv-Fc format antibody, the antibody is an anti-CD5×anti-TGFßRII bispecific antibody, wherein the Fab side is the anti-CD5 ABD and the scFv side is the anti-TGFßRII ABD. In some embodiments of the 1+1 Fab-scFv-Fc format antibody, the Fab side is the anti-TGFßRII ABD and the scFv side is the CD5 ABD.
In some embodiments of the anti-CD5×anti-TGFßRII 1+1 Fab-scFv-Fc format antibody, the anti-TGFßRII ABD has a variable heavy and a variable light domain selected from those described herein, including those depicted in
In some embodiments of the anti-CD5×anti-TGFßRII 1+1 Fab-scFv-Fc format antibody, the anti-CD5 ABD has a variable heavy and a variable light domain selected from those described herein, including those depicted in
In some embodiments of the 1+1 Fab-scFv-Fc format antibody, the antibody is an anti-PD-1×anti-TGFßRII bispecific antibody, wherein the Fab side is the anti-PD-1 ABD and the scFv side is the anti-TGFßRII ABD. In some embodiments of the 1+1 Fab-scFv-Fc format antibody, the Fab side is the anti-TGFßRII ABD and the scFv side is the anti-PD-1 ABD.
In some embodiments of the anti-PD-1×anti-TGFßRII 1+1 Fab-scFv-Fc format antibody, the TGFßRII binding domain comprises a variable heavy domain selected from: SEQ ID NOs: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, 1859, 1863, 1871, 1875, and 1323-1605, or a variant thereof and a variable light domain selected from: SEQ ID NO:1867, SEQ ID NO:1879, and SEQ ID NOs:1606-1703 or a variant thereof. In exemplary embodiments, the TGFßRII binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:2389 and 1867, and SEQ ID NOs:2393 and 1867, respectively.
In some embodiments of the anti-PD-1×anti-TGFßRII 1+1 Fab-scFv-Fc format antibody, the anti-PD-1 ABD has a variable heavy and a variable light domain selected from those described herein, including those depicted in the sequence listing or a variant thereof. In some embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, and SEQ ID NO:521 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In some embodiments, the 1+1 Fab-scFv-Fc format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 1+1 Fab-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence of
In some embodiments, the 1+1 Fab-scFv-Fc format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 1+1 Fab-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence of
Exemplary anti-TGFßRII×anti-PD-1 bsAbs in the 1+1 Fab-scFv-Fc format are depicted in
Exemplary anti-TGFßRII×anti-CD5 bsAbs in the 1+1 Fab-scFv-Fc format are depicted in
2. 1+1 Fab-VHH-Fc Format
In addition to the
In some embodiments, the 1+1 Fab-VHH-Fc format antibody is an anti-CD5×anti-TGFßRII antibody. In
In addition, the Fc domains of the antibodies described herein generally include skew variants (e.g. a set of amino acid substitutions as shown in
In some embodiments, the 1+1 Fab-VHH-Fc format antibody includes any one of the anti-CD5 antigen binding domains provided herein. In exemplary embodiments, the 1+1 Fab-VHH-Fc format antibody includes a Fab side that includes any of the anti-CD5 antigen binding domains provided herein. In some embodiments, the anti-CD5 antigen binding domain includes the 6 CDRs or the VH and VL of any of the anti-CD5 antigen binding domains depicted in
In some embodiments, the 1+1 Fab-VHH-Fc format antibody includes any one of the anti-PD-1 antigen binding domains provided herein. In exemplary embodiments, the 1+1 Fab-VHH-Fc format antibody includes a Fab side that includes any of the anti-PD-1 antigen binding domains provided herein. In some embodiments, the anti-PD-1 antigen binding domain includes the 6 CDRs or the VH and VL of any of the anti-PD-1 antigen binding domains depicted in the sequence listing or a variant thereof. In some embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, ora variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, and SEQ ID NO:521 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In some embodiments of the 1+1 Fab-VHH-Fc format antibody is an anti-CD5×anti-TGFßRII bispecific antibody, wherein the Fab side is the anti-CD5 ABD and the VHH side is the TGFßRII ABD. In some embodiments of the 1+1 Fab-VHH-Fc format antibody is an anti-PD-1×anti-TGFßRII bispecific antibody, wherein the Fab side is the anti-PD-1 ABD and the VHH side is the TGFßRII ABD.
In some embodiments of the 1+1 Fab-VHH-Fc format antibody, the anti-TGFßRII ABD is the VHH and has any of the following sequences: SEQ ID NO: 580, 584, 588, 592, 596, 600, 604, 608, 612, 616, 620, 624, 628, 632, 636, 640, 644, 648, 652, 656, 660, 664, 668, 672, 676, 680, 684, 688, 692, 696, 700, 704, 708, 712, 716, 720, 724, 728, 732, 736, 740, 744, 748, 752, 756, 760, 764, 768, 772, 776, 780, 784, 788, 792, 796, 800, 804, 808, 812, 816, 820, 824, 828, 832, 836, 840, 844, 848, 852, 856, 860, 864, 868, 872, 876, 880, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, and 936 or a variant thereof. In certain embodiments, the TGFßRII ABD is the VHH and has any of the following sequences: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NO:1863, SEQ ID NO:1871, SEQ ID NO:1875, SEQ ID NOs:1323-1605 or a variant thereof.
In some embodiments, the 1+1 Fab-VHH-Fc format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 1+1 Fab-VHH-Fc formats that comprise: a) a first monomer that comprises a charged scFv linker (with the +H sequence of
In some embodiments, the 1+1 Fab-VHH-Fc format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 1+1 Fab-VHH-Fc formats that comprise: a) a first monomer (the “VHH monomer”) that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a VHH as outlined herein; b) a second monomer (the “Fab monomer”) that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S, and a variable heavy domain; and c) a light chain that includes a variable light domain (VL) and a constant light domain (CL), wherein numbering is according to EU numbering.
Exemplary anti-TGFßRII×anti-PD-1 bsAbs in the 1+1 Fab-VHH-Fc format are depicted in
Exemplary anti-TGFßRII×anti-CD5 bsAbs in the 1+1 Fab-VHH-Fc format are depicted in
3. 1+1 VHH-scFv Fc Format
In addition to the
In some embodiments, the 1+1 VHH-scFv-Fc format antibody is an anti-CD5×anti-TGFßRII antibody. In
In addition, the Fc domains of the antibodies described herein generally include skew variants (e.g. a set of amino acid substitutions as shown in
In some embodiments, the 1+1 VHH-scFv-Fc format antibody includes any one of the anti-CD5 antigen binding domains provided herein. In exemplary embodiments, the 1+1 VHH-scFv-Fc antibody includes an anti-CD5 scFv that includes the 6 CDRs or the VH and VL of any of the anti-CD5 antigen binding domains depicted in
In some embodiments, the 1+1 VHH-scFv-Fc format antibody includes any one of the anti-PD-1 antigen binding domains provided herein. In exemplary embodiments, the 1+1 VHH-scFv-Fc format antibody includes a scFv side that includes any of the anti-PD-1 antigen binding domains provided herein. In some embodiments, the anti-PD-1 antigen binding domain includes the 6 CDRs or the VH and VL of any of the anti-PD-1 antigen binding domains depicted in the sequence listing or a variant thereof. In some embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, and SEQ ID NO:521 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In some embodiments of the 1+1 scFv-VHH-Fc format antibody is an anti-CD5×anti-TGFßRII bispecific antibody, wherein the scFv side is the anti-CD5 ABD and the VHH side is the TGFßRII ABD. In some embodiments of the 1+1 Fab-VHH-Fc format antibody is an anti-PD-1×anti-TGFßRII bispecific antibody, wherein the scFv side is the anti-PD-1 ABD and the VHH side is the TGFßRII ABD.
In some embodiments of the 1+1 scFv-VHH-Fc format antibody, the anti-TGFßRII ABD is the VHH and has any of the following sequences: SEQ ID NO: 580, 584, 588, 592, 596, 600, 604, 608, 612, 616, 620, 624, 628, 632, 636, 640, 644, 648, 652, 656, 660, 664, 668, 672, 676, 680, 684, 688, 692, 696, 700, 704, 708, 712, 716, 720, 724, 728, 732, 736, 740, 744, 748, 752, 756, 760, 764, 768, 772, 776, 780, 784, 788, 792, 796, 800, 804, 808, 812, 816, 820, 824, 828, 832, 836, 840, 844, 848, 852, 856, 860, 864, 868, 872, 876, 880, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, and 936 or a variant thereof. In certain embodiments, the TGFßRII ABD is the VHH and has any of the following sequences: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NO:1863, SEQ ID NO:1871, SEQ ID NO:1875, SEQ ID NOs:1323-1605 or a variant thereof.
In some embodiments, the 1+1 VHH-scFv-Fc format antibody includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 1+1 VHH-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence of
In some embodiments, the 1+1 VHH-scFv-Fc format antibody includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 1+1 VHH-scFv-Fc formats that comprise: a) a first monomer that comprises a charged scFv linker (with the +H sequence of
Exemplary anti-TGFßRII×anti-PD-1 bsAbs in the 1+1 Fab-VHH-Fc format are depicted in
Exemplary anti-TGFßRII×anti-CD5 bsAbs in the 1+1 scFv-VHH-Fc format are depicted in
In addition to antibodies that are bivalent and bispecific, the present invention also provides formats that include trivalent bispecific constructs, wherein the antibodies bind one antigen (either TGFßRII or CD5) bivalently (e.g. contain two ABDs) and the other antigen monovalently (with one ABD), such as are generally depicted in
4. 2+1 Fab2-scFv-Fc Format
One heterodimeric scaffold that finds particular use in the present invention is the 2+1 Fab2-scFv-Fc format shown in
In this embodiment, one monomer comprises a first monomer comprising a first variable heavy domain, a CH1 domain (and optional hinge) and Fc domain, with a scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain. The scFv is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using optional domain linkers (VH1-CH1-[optional linker]-VH2-scFv linker-VL2-[optional linker including the hinge]-CH2-CH3, or the opposite orientation for the scFv, VH1-CH1-[optional linker]-VL2-scFv linker-VH2-[optional linker including the hinge]-CH2-CH3). The other monomer (the second monomer) is a standard Fab side (i.e., a VH1-CH1-hinge-CH2-CH3 monomer). This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain that associates with the VH1 variable heavy domains of the two monomers to form two identical Fabs that bind a checkpoint inhibitor. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein.
In some embodiments, the 2+1 Fab2-scFv-Fc format antibody includes any one of the anti-CD5 antigen binding domains provided herein. In exemplary embodiments, the 2+1 Fab2-scFv-Fc format antibody includes two Fabs that includes the 6 CDRs or the VH and VL of any of the anti-CD5 antigen binding domains depicted in
In some embodiments of the 2+1 Fab2-scFv-Fc format antibody, each of the Fabs are anti-CD5 ABD and the scFv is the anti-TGFßRII ABD. In some embodiments of the 2+1 Fab2-scFv-Fc format antibody, the two Fab sides are anti-TGFßRII ABDs and the scFv is the anti-CD5 ABD.
In some embodiments of the 2+1 Fab2-scFv-Fc format antibody, the anti-TGFßRII ABD has variable heavy and variable light domains selected from those depicted in in
In some embodiments of the 2+1 Fab2-scFv-Fc format antibody, each of the Fabs are anti-PD-1 and the scFv is the anti-TGFßRII ABD. In some embodiments of the 2+1 Fab2-scFv-Fc format antibody, the two Fab sides are anti-TGFßRII ABDs and the scFv is the anti-PD-1 ABD.
In some embodiments, the 2+1 Fab2-scFv-Fc format antibody includes any one of the anti-PD-1 antigen binding domains provided herein. In exemplary embodiments, the 2+1 Fab2-scFv-Fc format antibody includes a Fab side that includes any of the anti-PD-1 antigen binding domains provided herein. In some embodiments, the anti-PD-1 antigen binding domain includes the 6 CDRs or the VH and VL of any of the anti-PD-1 antigen binding domains depicted in the sequence listing or a variant thereof. In some embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, and SEQ ID NO:521 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In some embodiments, the 2+1 Fab2-scFv-Fc format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 2+1 Fab2-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence of
In some embodiments, the 2+1 Fab2-scFv-Fc format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 2+1 Fabz-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence of
5. 2+1 Fab2-VHH-Fc Format
An additional heterodimeric scaffold that finds particular use in the present invention is the 2+1 Fab2-VHH-Fc format shown in
In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain (and optional hinge) and Fc domain, with a VHH domain. The VHH is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using optional domain linkers (vh1-CH1-[optional linker]-VHH-[optional linker including the hinge]-CH2-CH3. The other monomer is a standard Fab side. This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, which associates with the heavy chains to form two identical Fabs that bind a target. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein.
In some embodiments, the 2+1 Fab2-VHH-Fc format antibody includes any one of the anti-CD5 antigen binding domains provided herein. In exemplary embodiments, the 2+1 Fab2-VHH-Fc format antibody includes two Fabs that includes the 6 CDRs or the VH and VL of any of the anti-CD5 antigen binding domains depicted in
In some embodiments of the 2+1 Fab2-VHH-Fc format antibody, the two Fabs are the anti-CD5 ABDs and the VHH side is the anti-TGFßRII ABD.
In some embodiments of the 2+1 Fab2-VHH-Fc format antibody, the anti-TGFßRII VHH ABD has the VHH and has any of the following sequences: SEQ ID NO: 580, 584, 588, 592, 596, 600, 604, 608, 612, 616, 620, 624, 628, 632, 636, 640, 644, 648, 652, 656, 660, 664, 668, 672, 676, 680, 684, 688, 692, 696, 700, 704, 708, 712, 716, 720, 724, 728, 732, 736, 740, 744, 748, 752, 756, 760, 764, 768, 772, 776, 780, 784, 788, 792, 796, 800, 804, 808, 812, 816, 820, 824, 828, 832, 836, 840, 844, 848, 852, 856, 860, 864, 868, 872, 876, 880, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, and 936 or a variant thereof. In certain embodiments, the TGFßRII ABD is the VHH and has any of the following sequences: SEQ ID NO:2389, SEQ ID NO:2393, SEQ ID NO:2369, SEQ ID NO:2373, SEQ ID NO:2377, SEQ ID NO:2381, SEQ ID NO:2385, SEQ ID NO:2397, SEQ ID NO:1859, SEQ ID NO:1863, SEQ ID NO:1871, SEQ ID NO:1875, SEQ ID NOs:1323-1605 or a variant thereof.
In some embodiments of the 2+1 Fab2-VHH-Fc format antibody, the two Fabs are the anti-PD-1 ABDs and the VHH side is the anti-TGFßRII ABD.
In some embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, SEQ ID NO:195, SEQ ID NO:203, SEQ ID NO:211, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:235, SEQ ID NO:243, SEQ ID NO:251, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:275, SEQ ID NO:283, SEQ ID NO:291, SEQ ID NO:299, SEQ ID NO:307, SEQ ID NO:315, SEQ ID NO:323, SEQ ID NO:331, SEQ ID NO:339, SEQ ID NO:347, SEQ ID NO:355, SEQ ID NO:363, SEQ ID NO:371, SEQ ID NO:379, SEQ ID NO:387, SEQ ID NO:395, SEQ ID NO:403, SEQ ID NO:411, SEQ ID NO:419, SEQ ID NO:427, SEQ ID NO:435, SEQ ID NO:443, SEQ ID NO:451, SEQ ID NO:459, SEQ ID NO:467, and SEQ ID NO:475, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, SEQ ID NO:521, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:351, SEQ ID NO:359, SEQ ID NO:367, SEQ ID NO:375, SEQ ID NO:383, SEQ ID NO:391, SEQ ID NO:399, SEQ ID NO:407, SEQ ID NO:415, SEQ ID NO:423, SEQ ID NO:431, SEQ ID NO:439, SEQ ID NO:447, SEQ ID NO:455, SEQ ID NO:463, SEQ ID NO:471, and SEQ ID NO:479 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain selected from: SEQ ID NO:483, SEQ ID NO:959, SEQ ID NO:487, SEQ ID NO:491, SEQ ID NO:495, SEQ ID NO:499, SEQ ID NO:503, SEQ ID NO:943, SEQ ID NO:947, SEQ ID NO:951, SEQ ID NO:955, SEQ ID NO:963, SEQ ID NO:967, and SEQ ID NO:971, or a variant thereof, and a variable light domain selected from: SEQ ID NO:979, SEQ ID NO:517, SEQ ID NO:975, SEQ ID NO:983, SEQ ID NO:987, SEQ ID NO:501, SEQ ID NO:505, SEQ ID NO:509, SEQ ID NO:513, and SEQ ID NO:521 or a variant thereof. In exemplary embodiments, the PD-1 binding domain comprises a variable heavy domain and a variable light domain selected from the group consisting of: SEQ ID NOs:483 and 979, and SEQ ID NOs:959 and 517, respectively.
In some embodiments, the 2+1 Fab2-VHH-Fc format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 1+1 Fab-VHH-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence of
In some embodiments, the 2+1 Fab2-VHH-Fc format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 2+1 Fabz-VHH-Fc formats that comprise: a) a first monomer (the “VHH monomer”) that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and an scFv as outlined herein; b) a second monomer (the “Fab monomer”) that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S, and a variable heavy domain; and c) a light chain that includes a variable light domain (VL) and a constant light domain (CL), wherein numbering is according to EU numbering.
6. Monospecific, Monoclonal Antibodies
As will be appreciated by those in the art, the novel Fv sequences outlined herein for anti-CD5, anti-TGFßRII and anti-PD-1 ABDs can also be used in both monospecific antibodies (e.g. “traditional monoclonal antibodies”) or non-heterodimeric bispecific formats. Accordingly, the present invention provides monoclonal (monospecific) antibodies comprising the 6 CDRs and/or the vh and vl sequences from the figures, generally with IgG1, IgG2, IgG3 or IgG4 constant regions, with IgG1, IgG2 and IgG4 (including IgG4 constant regions comprising a S228P amino acid substitution) finding particular use in some embodiments. That is, any sequence herein with a “H_L” designation can be linked to the constant region of a human IgG1 antibody.
The invention further provides nucleic acid compositions encoding the bispecific antibodies of the invention (or, in the case of “monospecific” antibodies, nucleic acids encoding those as well).
As will be appreciated by those in the art, the nucleic acid compositions will depend on the format and scaffold of the heterodimeric protein. Thus, for example, when the format requires three amino acid sequences, such as for
As is known in the art, the nucleic acids encoding the components of the invention can be incorporated into expression vectors as is known in the art, and depending on the host cells used to produce the heterodimeric antibodies of the invention. Generally the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). The expression vectors can be extra-chromosomal or integrating vectors.
The nucleic acids and/or expression vectors of the invention are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with mammalian cells (e.g. CHO cells), finding use in many embodiments.
In some embodiments, nucleic acids encoding each monomer and the optional nucleic acid encoding a light chain, as applicable depending on the format, are each contained within a single expression vector, generally under different or the same promoter controls. In embodiments of particular use in the present invention, each of these two or three nucleic acids are contained on a different expression vector. As shown herein and in 62/025,931, hereby incorporated by reference, different vector ratios can be used to drive heterodimer formation. That is, surprisingly, while the proteins comprise first monomer:second monomer:light chains (in the case of many of the embodiments herein that have three polypeptides comprising the heterodimeric antibody) in a 1:1:2 ratio, these are not the ratios that give the best results.
The heterodimeric antibodies of the invention are made by culturing host cells comprising the expression vector(s) as is well known in the art. Once produced, traditional antibody purification steps are done, including an ion exchange chromatography step. As discussed herein, having the pIs of the two monomers differ by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point. That is, the inclusion of pI substitutions that alter the isoelectric point (pI) of each monomer so that such that each monomer has a different pI and the heterodimer also has a distinct pI, thus facilitating isoelectric purification of the “triple F” heterodimer (e.g., anionic exchange columns, cationic exchange columns). These substitutions also aid in the determination and monitoring of any contaminating dual scFv-Fc and mAb homodimers post-purification (e.g., IEF gels, cIEF, and analytical IEX columns).
Formulations of the antibodies and compositions provided herein are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (as generally outlined in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions.
Once made, the compositions of the invention find use in a number of oncology applications, by treating cancer, generally by inhibiting the suppression of T cell activation (e.g., T cells are no longer suppressed) with the binding of the subject antibodies described herein.
Accordingly, the heterodimeric compositions of the invention find use in the treatment of these cancers.
In some embodiments, the bispecific antibody can be co-administered with a separate anti-PD-1 antibody such as pembrolizumab (Keytruda®) or nivolumab (Opdivo®). Co-administration can be done simultaneously or sequentially, as will be appreciated by those in the art.
As described above, tumors can evade immune surveillance by producing the immunosuppressive cytokine TGFß. Accordingly, in vitro assays were established to probe these immunosuppressive effects and eventually to test the molecules of the invention.
1. TGFß Induces Phosphorylation of SMAD2/3 in T Cells In Vitro
TGFß binds to the TGFßRII receptor which subsequently recruits, phosphorylates and activates TGFßRI. TGFßRI in turn phosphorylates SMAD2/3 which then associates with SMAD4. The SMAD2/3:SMAD4 heterocomplex translocate into the nucleus to regulate further downstream processes. Phosphorylation of SMAD2/3 as an indicator of TGFß1 biological activity was demonstrated in vitro as follows. Human PBMC was seeded on 0.5 μg/ml anti-CD3 for 48 hours for activation, then serum deprived for 16 hours in 0.1% FBS (to remove confounding effect of TGFß in serum). The PBMC was then incubated with indicated dose of TGFß1 for 30 minutes at 37° C. Following incubation, intracellular phospho-flow cytometry was performed to measure phosphorylated SMAD2/3 (pSMAD2/3). The data as depicted in
2. TGFß Suppresses T Cell Proliferation, IFNγ Secretion, and CD95 Expression Induced by Nivolumab
The suppressive effects of TGFß was modeled in vitro in a mixed lymphocyte reaction (MLR). In an MLR, allogeneic (different MHC haplotype) lymphocytes are cultured together resulting in an immune response and corresponding T cell proliferation, activation, and exhaustion (from upregulation of checkpoint receptors). T cells from 11 unique donors were mixed with DC cells from 2 unique donors to make 21 MLR reactions. 10 μg/ml XENP16432 (bivalent anti-PD-1 mAb based on nivolumab with PVA/S267K) was also added (to reverse T cell exhaustion) in the presence or absence of 1 ng/ml soluble TGFß1. 5 days post T cell seeding, release of IFNγ, proliferation of CD3+ T cells (as indicated by percentage Ki67+), and CD95 expression on CD3+ T cells were measured as depicted in
As described above, an anti-TGFßRII antibody which was in clinic resulted in systemic toxicity and uncontrolled cytokine release syndrome.
Immune checkpoint proteins such as PD-1 are up-regulated in tumor-infiltrating lymphocytes. According, anti-TGFßRII×anti-PD1 bispecific antibodies (bsAbs) were conceived to target TGFßRII blockade antibodies to the tumor environment and subsequently circumvent toxicity and increase therapeutic index.
1. TGFßRII Binding Domains
Illustrative TGFßRII binding domains are depicted in
To confirm binding to cell-surface TGFßRII, PBMCs were activated with 500 ng/mL plate-bound anti-CD3 (OKT3) for 48 hours then incubated with illustrative anti-TGFßRII mAbs XENP28297, XENP33038, and XENP33040 as well as commercial antibodies C-4 (sc-17991) and D-2 (sc-17799) (both from Santa Cruz Biotechnology, Dallax, Tex.) (all labeled with Alexa647). Data showing binding of the various mAbs to activated T cells are depicted in
2. PD1 Binding Domains
As it would be useful to combine the anti-TGFßRII×anti-PD1 bsAbs of the invention with PD-1 blockade antibodies, or to administer anti-TGFßRII×anti-PD1 bsAbs of the invention subsequent to treatment with PD-1 blockade antibodies, it is important that the PD-1 targeting arm of the anti-TGFßRII×anti-PD1 bsAbs does not bind the same or similar epitope as the PD-1 blockade antibody. PD-1 blockade antibodies contemplated herein include, but are not limited to, nivolumab and pembrolizumab.
Illustrative non-competing anti-PD-1 binding domains contemplated for use in the anti-TGFßRII×anti-PD1 bsAbs of the invention are referred to as mAb A, mAb B, and mAb C (sequences for their humanized variable regions are depicted as SEQ ID NOs: 115-130, 483-524 and 940-989, humanized using string content optimization (see, e.g., U.S. Pat. No. 7,657,380, issued Feb. 2, 2010)).
Tandem epitope binning was performed to demonstrate that the mAbs did not compete with nivolumab and pembrolizumab. Epitope binning was performed using the Octet HTX instrument. AMC (anti-mouse Fc) biosensors were first used to capture murine-Fc fusions of human PD-1, dipping into 100 nM of a first antibody (indicated on the left side of
However, PD1 binding domains which do compete for binding with PD-1 blockade antibodies such as nivolumab and pembrolizumab may still be suitable for use in the anti-TGFßRII×anti-PD1 bsAbs. Accordingly, additional PD1 binding domains contemplated for use are depicted as SEQ ID NOs: 131-482.
3. Engineering Prototype Anti-TGFßRII×Anti-PD1 bsAbs
Various formats for the anti-TGFßRII×anti-PD1 bsAbs of the invention were conceived and generated including bsAbs in the 1+1 Fab-scFv-Fc format (as depicted schematically in
Additionally, αRSV×αPD1 bsAbs were constructed to act as a surrogate for investigating the behavior of αTGFßRII×αPD1 bsAbs outside of the tumor environment, illustrative sequences for which are depicted in
1. Anti-TGFßRII×Anti-PD1 bsAbs Block SMAD2/3 Phosphorylation
Using the pSMAD2/3 assay described above, the biological activity of the prototype anti-TGFßRII×anti-PD1 bsAbs were investigated and compared with anti-TGFßRII mAbs. Human PBMC was seeded on 0.5 μg/ml anti-CD3 for 48 hours for activation, then serum deprived for 16 hours in 0.1% FBS (to remove confounding effect of TGFß in serum). The PBMC was then incubated with 20 μg/ml test articles (including E-6 (sc-17792) and D-2 (sc-17799) from Santa Cruz Biotechnology) for 30 minutes at room temperature followed by incubation with 10 μg/ml test articles and indicated dose of TGFß1 for 30 minutes at 37° C. Following incubation, intracellular phospho-flow cytometry was performed to measure phosphorylated SMAD2/3 (pSMAD2/3). The data as depicted in
In another pSMAD2/3 assay, human PBMCs were activated by seeding on 0.5 μg/ml anti-CD3 for 48 hours, then serum deprived for 16 hours (0.1% FBS). The PBMCs were then incubated with test articles for 30 minutes at room temperature followed by incubation with test articles+1 ng/ml TGFß1 for 30 minutes at 37° C. Following incubation, intracellular phospho-flow cytometry was performed to measure phosphorylated SMAD2/3 (pSMAD2/3). The data as depicted in
2. Anti-TGFßRII×Anti-PD1 bsAbs Block Suppressive Effects of TGFß
Next, the ability of the anti-TGFßRII×anti-PD1 bsAbs to reverse/block the suppressive effects of TGFß1 was investigated using the MLR assay as described above. T cells from 11 unique donors were mixed with DC cells from 2 unique donors to make 21 MLR reactions. 10 μg/ml of indicated test articles was also added in the presence or absence of 1 ng/ml soluble TGFß1. 5 days post T cell seeding, release IFNγ, proliferation of CD3+ T cells (as indicated by percentage Ki67+), and CD95 expression on CD3+ T cells were measured as depicted in
3. Anti-TGFßRII×Anti-PD1 bsAbs Enables Superior Blockade in Comparison to Corresponding Monospecific Anti-TGFßRII mAb at Higher TGFß1 Concentrations
In an experiment comparing the blockade efficacy of anti-TGFßRII×anti-PD1 bsAbs in comparison to monospecific anti-TGFßRII mAbs at higher TGFß1 concentrations, PBMCs were first activated by seeding on 0.5 μg/ml anti-CD3 for 48 hours, then serum deprived for 16 hours (0.1% FBS). The PBMCs were then incubated with test articles for 30 minutes at room temperature followed by incubation with test articles+10 ng/ml or 100 ng/ml TGFß1 for 30 minutes at 37° C. Following incubation, intracellular phospho-flow cytometry was performed to measure phosphorylated SMAD2/3 (pSMAD2/3). The data as depicted in
4. Anti-TGFßRII×Anti-PD1 bsAbs are Highly Selective for Activated (PD1-High) T Cells
To further investigate the selectivity of the anti-TGFßRII×anti-PD1 bsAbs of the invention, blocking experiments were performed utilizing unactivated (PD1 low) and activated PBMCs (PD1 high). For experiments utilizing unactivated PBMCs, PBMCs were thawed and recovered overnight for 24 hours, then serum deprived for 16 hours (in 0.1% FBS). In experiments utilizing activated PBMCs, PBMCs were activated by seeding on 0.5 μg/ml anti-CD3 for 48 hours, then serum deprived for 16 hours (in 0.1% FBS). In both types of experiments, following serum deprivation, PBMCs were incubated with the test articles at indicated concentrations for 30 minutes at room temperature followed by incubation with the test articles+1 ng/ml TGFß1 for 30 minutes at 37° C. The data as depicted in
1. Anti-TGFßRII×Anti-PD1 Bispecific Antibodies Enhance GVHD
The anti-TGFßRII×anti-PD1 bsAbs were evaluated in a Graft-versus-Host Disease (GVHD) model conducted in NSG (NOD-SCID-gamma) immunodeficient mice. When the NSG mice are engrafted with human PBMCs, the human PBMCs develop an autoimmune response against mouse cells and subsequently GVHD. As such, GVHD is a model for potential anti-tumor response. Treatment of huPBMC-engrafted NSG mice with anti-TGFßRII×anti-PD1 bsAbs should enhance proliferation and response of the engrafted T cells and enhance GVHD.
Accordingly in a pilot study, NSG mice were engrafted with 10×106 human PBMCs via IV-OSP on Day −1 and dosed intraperitoneally with XENP16432 (a bivalent anti-PD1 mAb based on nivolumab with PVA_S267K; a checkpoint inhibitor which enhances GVHD by de-repressing the engrafted human T cells), anti-TGFßRII mAb XENP28296 (clone TBRII-A_H1L1), XENP28296 in combination with XENP16432, and prototype anti-TGFßRII×anti-PD1 mAb XENP33045 in combination with XENP16432 on Days 0, 7, 14, and 20. Body weights were assessed twice per week as an indicator of GVHD (change in body weight as a percentage of initial body weight depicted in
The data show that both XENP28296 in combination with PD-1 blockade and XENP33045 in combination with PD-1 blockade significantly enhanced body weight loss by Day 13 in comparison to no treatment (i.e. PBS). Notably, by Day 17, XENP33045 in combination with PD-1 blockade significantly enhanced body weight loss in comparison to both PD-1 blockade alone as well as XENP28296 in combination with PD-1 blockade; and by Day 20, all mice treated with XENP33045 in combination with PD-1 blockade were dead. Finally, the data show that the test articles enhanced secretion of IFNγ and IL-10; and notably, XENP33045 in combination with PD-1 blockade induced significantly enhanced secretion of IFNγ by Day 7 in comparison to XENP28296 in combination with PD-1 blockade (statistics on log-transformed data). In a similar GVHD study, XENP34228 was found to promote significantly more GVHD weight loss, T cell expansion, and IFNγ release than PBS control or PD-1 blockade alone.
2. Anti-TGFßRII×Anti-PD1 Bispecific Antibodies Enhance Anti-Tumor Activity
To investigate anti-tumor activity of the anti-TGFßRII×anti-PD1 bsAbs, NSG mice that were MHC (NSG-DKO) and thus resistant to GVHD were used. NSG-DKO mice (10 per group) were intradermally inoculated with 2×106 pp65-transduced MDA-MB231 cells on Day −22. Mice were then intraperitoneally injected with 5×106 human PBMCs and treated with the indicated test articles/test article combinations on Day 0, and further treated on Days 8, 14, 21, and 28. Tumor volume was measured by caliper one to three times per week, body weights were measured once per week, and blood was drawn once per week.
Tumor volume on Days 20, 22, 25, 27, 29, 32, 34, and 36 as well as over time are depicted in
Data depicting the expansion of various lymphocyte population on Day 8 are depicted in
Collectively, this experiment show that anti-TGFßRII×anti-PD1 bispecific antibodies effectively enhance anti-tumor activity. Notably, the experiment also indicates that anti-TGFßRII×anti-PD1 having a non-competing PD-1 binding arm combines synergistically with PD-1 blockade.
TGFßRII binding domains described in Example 2A were engineered for enhanced stability in the context of an scFv for use in the bispecific antibody formats of the invention; and for modulated binding affinity to mitigate target-mediated drug disposition (TMDD) and to tune the efficacy, potency, and/or selectivity of the bispecific antibodies.
1. Round 1 Engineering
In a first round of engineering, variants of TBRII-A were engineered by introducing single or double mutations into the variable heavy region (VH) and the variable light region (VL) to generate 106 VH variants and 61 VL variants (sequences for which are depicted in SEQ ID NOs:1325-1430 TBRII-A_H1.2−TBRII-A_H1.107 and in SEQ ID NOs:1607-1667 as TBRII-A_L1.1−TBRII-A_L1.61).
His-tagged Fab domains, His-tagged scFvs, and TGFßRII bispecific antibodies (sequences depicted in
a. Stability of Variants
Stability of scFvs, Fabs, and bispecific antibodies comprising the variant VHs or VLs were evaluated using Differential Scanning Fluorimetry (DSF). DSF experiments were performed using a Bio-Rad CFX Connect Real-Time PCR Detection System. Proteins were mixed with SYPRO Orange fluorescent dye and diluted to 0.2 mg/mL in PBS. The final concentration of SYPRO Orange was 10×. After an initial 10 minute incubation period at 25° C., proteins were heated from 25 to 95° C. using a heating rate of 1° C./min. A fluorescence measurement was taken every 30 sec. Melting temperatures (Tm) were calculated using the instrument software. The stability results are depicted in
b. Binding Affinity of Variants
In a first experiment, binding affinity of the TBRII-A variants formatted as His-tagged Fabs for TGFßRII was screened using Octet, a BioLayer Interferometry (BLI)-based method. Experimental steps for Octet generally include the following: Immobilization (capture of ligand to a biosensor); Association (dipping of ligand-coated biosensors into wells containing the analyte); and Dissociation (returning of biosensors to well containing buffer). The resulting apparent dissociation constant (KD), association rate (ka), dissociation rate (kd), as well as sensorgram response are depicted in
In further experiments, binding affinity of select TBRII-A variants formatted as αTGFßRII×αPD1 or αTGFßRII×αRSV bsAbs for human TGFßRII (as well as for cynomolgus TGFßRII, as similar binding to cynomolgus antigen is useful for ease of clinical development) was screened using Octet as generally described above. The resulting apparent dissociation constant (KD), association rate (ka), dissociation rate (kd), as well as sensorgram response are depicted in
Collectively, the data show that a number of point mutations were identified which resulted in modulated binding affinity for TGFßRII, both in the context of Fabs but also more relevantly in the context of bispecific antibodies.
2. Reduced Affinity TGFßRII Binding Reduces the Potency of αTGFßRII×αPD1 Bispecific Antibodies
The effect of reduced affinity TGFßRII binding was investigated using a pSMAD2/3 assay as generally described above. PBMCs were first activated by seeding on 0.5 μg/ml anti-CD3 for 48 hours, then serum deprived for 16 hours (0.1% FBS). The PBMCs were then incubated with test articles for 30 minutes at room temperature followed by incubation with test articles+1 ng/ml TGFß1 for 30 minutes at 37° C. Following incubation, intracellular phospho-flow cytometry was performed to measure phosphorylated SMAD2/3 (pSMAD2/3). The data depicted in
3. Further Engineering
In further rounds of engineering, additional VH variants and VL variants were engineered by combining substitutions identified in Example 5A as favorably enhancing stability (i.e. those that enable Tm>60° C.) or modulating binding affinity with an aim to identify variants having a Tm>70° C. (vs. initial Tm of 60° C.) while matching the affinity of the ladder identified in Round 1 Engineering (i.e. tighter affinity variants for more potent activity as well as weaker affinity variants aimed to reduce TMDD). Additionally, substitutions were explored to remove a possible VH oxidation site at Met109 (109L, T, V; Met98 in Kabat numbering), a possible VH deamidation motif NS Asn31Ser32 (31E, S, T, Q; 32A; *note: Asn31Ser32 in Kabat numbering), and a possible VL oxidation site Trp100 (100Y; Trp94 in Kabat numbering). The variant VHs were combined with WT VL and the variant VLs were combined with WT VH and formatted as His-tagged Fab domains and His-tagged scFvsto screen for stability and affinity. Notably, a core of E60G/S81N/P101A (as in H1.201 and H1.212; *note: E55G/S76N/P93A in Kabat numbering) was determined to provide minimal loss of affinity while providing high stability (Tm>70° C.); and are notably reversions to VH4-39 germline (which is expected to provide an additional benefit of reducing immunogenic potential). As described above, H1.201 further includes M109L (M98L in Kabat) and H1.212 further includes M109T (M98T in Kabat) to remove a potential oxidation site; and H1.201 further includes S32A (same in Kabat) while H1.212 further includes N31S (same in Kabat) to remove a deamidation motif. H1.212 further includes G37S (G35bS in Kabat) which was identified in Round 1 Engineering to enhance stability while decreasing TGFßRII binding affinity.
Affinity of illustrative such variants for TGFßRII were investigated in various contexts (e.g. in the context of αTGFßRII×αPD1 or αTGFßRII×αCD5) are depicted in
Additional engineering via the introduction of histidine substitutions was performed to generate pH-dependent variants. Weaker binding at low pH may allow for dissociation within the endosome after internalization and subsequent recycling to the cell surface by FcRn, thus lessening TMDD and improving pharmacokinetics. Alternatively, stronger binding at low pH may allow for selective binding and function in tumor microenvironments, which are often acidic.
Collectively, the further engineering generated an additional 175 VH variants and 36 VL variants (sequences for which are depicted in SEQ ID NOs: 1431-1605 as H1.108-H1.282 and in SEQ ID NOs: 1668-1703 as L1.62-L1.97).
4. VL-VH Swap
Another avenue explored was swapping the VH-VL orientation for the αTGFßR2 arm as in XENP35186. In the data shown in
In addition to modulating affinity of the TGFßRII binding domain, modulating affinity of the PD-1 binding domain was also explored. In particular, the aim was to restore potency through the PD1 binding domain to compensate for reduced binding affinity of the TGFßRII binding domain. In the data shown in
As described above, PD-1 is upregulated on TILs and so the anti-TGFßRII×anti-PD-1 bsAbs of the invention are selective for PD1-high TILs, and in particular, selective for activated T cells over unactivated T cells. However, it may also be useful in certain contexts to target broader T cell populations.
1. CD5 is Highly Expressed on Activated and Unactivated T Cells
CD5 has been reported as a highly expressed pan-T cell marker that is low or absent on most other immune cells. To confirm, CD5 expression level on numerous subsets of lymphocytes were analyzed (data depicted in
Additionally, in data not shown, it was found that TGFßRII bispecific antibodies internalize via TGFßRII and concomitantly induces internalization of the targeting receptor. Internalization of lower expressed targeting receptors may abrogate the ability of the targeted TGFßRII bispecific antibodies to bind their target cells. Targeting a highly expressed receptor such as CD5 may overcome this effect.
2. Anti-TGFßRII×Anti-CD5 Bispecific Antibodies
a. CD5 Binding Domains
Sequences for illustrative CD5 binding domains which may find use in the anti-TGFßRII×anti-CD5 bispecific antibodies of the invention are depicted in
b. Engineering Prototype Anti-TGFßRII×Anti-CD5 bsAbs
As described above, various formats for the anti-TGFßRII×anti-CD5 bsAbs of the invention were conceived and generated including bsAbs in the 1+1 Fab-scFv-Fc format (as depicted schematically in
c. Cynomolgus CD5 Cross-Reactive Binding Domains
For ease of clinical development, it is useful to investigate various parameters of the targeted TGFßRII bsAbs such as pharmacodynamics, pharmacodynamics, and toxicity in cynomolgus monkeys.
A first CD5 mAb previously described in U.S. App. No. 2008/0254027 as 5D7 (sequences depicted in
First, the binding affinity of the bispecific antibodies for human and cynomolgus CD5 antigen were determined using Octet as generally described above. In particular, His-tagged human and cynomolgus CD5 antigen were captured using HIS1K sensors and dipped into multiple concentrations of the bispecific antibodies. The resulting dissociation constant (KD) are depicted in
Next, cell binding of the bispecific antibodies were investigated. Unstimulated PBMCs were incubated with the indicated dose of bispecific antibodies for 1 hour on ice and washed. Next, the PBMCs were stained with goat anti-human Fc secondary antibody conjugated to Alexa647 (to assess binding of the bispecific antibodies) and antibodies against cell surface antigens (to define T cell populations) for 40 minutes on ice. Finally, the cells were analyzed by flow cytometry. As depicted in
3. Anti-TGFßRII×Anti-CD5 bsAbs Selectively Inhibit pSMAD Induction in a Broader T Cell Population
Using the pSMAD2/3 assay described above, the biological activity of the prototype anti-TGFßRII×anti-CD5 antibodies were investigated and compared with anti-TGFßRII×anti-RSV controls and anti-TGFßRII×anti-PD1 bsAbs. In experiments utilizing unactivated PBMCs, PBMCs were thawed and recovered overnight for 24 hours, then serum deprived for 16 hours (in 0.1% FBS). In experiments utilizing activated PBMCs, PBMCs were activated by seeding on 0.5 μg/ml anti-CD3 for 48 hours, then serum deprived for 16 hours (in 0.1% FBS). In both types of experiments, following serum deprivation, PBMCs were incubated with the test articles at indicated concentrations for 30 minutes at room temperature followed by incubation with the test articles+1 ng/ml TGFß1 for 30 minutes at 37° C. Following incubation, intracellular phospho-flow cytometry was performed to measure phosphorylated SMAD2/3 phosphorylation following incubation with the bsAbs. Data are depicted in
Consistent with the above, the anti-TGFßRII×anti-PD1 bsAbs having anti-TGFßRII arms based on TBRII-A show superior blocking of TGFß1-induced SMAD2/3 phosphorylation on activated CD4+ and CD8+ T cells compared to corresponding control anti-TGFßRII×anti-RSV bsAbs indicating that the PD-1-targeting enhances the blocking activity. Notably, the anti-TGFßRII×anti-CD5 bsAbs demonstrated further enhanced blocking activity in comparison to the anti-TGFßRII×anti-PD1 bsAbs. Additionally, the anti-TGFßRII×anti-CD5 bsAbs demonstrated enhanced blocking activity on both activated PBMCs and unactivated PBMCs. Finally, it was noted that both the anti-TGFßRII×anti-PD-1 bsAb and the anti-TGFßRII×anti-CD5 bsAb demonstrated little to no blocking activity on B cells and NK cells (CD5 and PD1 low/negative) except at very high concentrations.
4. Reducing Affinity TGFßRII and CD5 Binding Reduces the Potency of αTGFßRII×αCD5 Bispecific Antibodies
In another experiment, the biological activity of the additional anti-TGFßRII×anti-CD5 antibodies having alternative cynomolgus cross-reactive CD5-targeting arms as well as antibodies having reduced affinity TGFßRII binding were investigated. The experiment was performed as described above using activated PBMCs except using incubation with 10 ng/ml TGFß1.
The data as depicted in
Additional αTGFßRII×αCD5 bsAbs were generated using the stability/affinity optimized variants identified in Example 5C. In vitro activity of αTGFßRII×αCD5 bsAbs comprising illustrative such variants were investigated in SMAD2 phosphorylation assays using 10 ng/mL TGFß, data for which are depicted in
5. Humanization CD5 Binding Domains
Murine Cd5-A and Cd5-B binding domains were humanized using string content optimization (see, e.g., U.S. Pat. No. 7,657,380, issued Feb. 2, 2010), respectively depicted as Cd5-A_H1L1 and Cd5-B_H1L1. αTGFßRII×αCD5 bsAbs based on these humanized binders were generated and investigated in SMAD2 phosphorylation assay as described above. Data are depicted in
Binding of the murine, H1L1 humanized, and half-humanized (i.e. humanized VH+murine VL−H1L0 and murine VH+humanized VL−H0L1) Cd5-B Fvs were investigated in Octet, sensorgram depicted in
6. Anti-TGFßII×Anti-CD5 bsAbs are Active In Vivo and Combine with PD-1 Blockade
As above, the anti-TGFßRII×anti-CD5 bsAbs were evaluated in a Graft-versus-Host Disease (GVHD) model conducted in NSG (NOD-SCID-gamma) immunodeficient mice. NSG mice were engrafted with 10×106 human PBMCs via IV-OSP on Day −1 and dosed intraperitoneally with XENP16432 (a bivalent anti-PD1 mAb based on nivolumab with PVA_S267K; a checkpoint inhibitor which enhances GVHD by de-repressing the engrafted human T cells), anti-TGFßRII mAb XENP28297 (clone TBRII-A_H1.1_L1), prototype anti-TGFßRII×anti-CD5 mAb XENP35399 alone or in combination with XENP16432, and TGFßRII affinity-reduced anti-TGFßRII×anti-CD5 mAb XENP36132 alone or in combination with XENP16432 on Days 0, 8, and 16. Body weights were assessed twice per week as an indicator of GVHD (change in body weight as a percentage of initial body weight depicted in
In a mouse tumor model, activity of anti-TGFßRII×anti-CD5 bsAbs XENP40323 and XENP39131 having humanized (affinity-fixed) Cd5-B binding domain and stability/affinity-optimized TGFßRII binding domain (respectively high potency H1.201_L1 or mid-potency H1.212_L1) binding domains were investigated. On Day −16, NSG-DKO mice (n=10) were inoculated intradermally with 5×106 pp65-MDA-MB231 cancer cells. On Day 0, mice were intraperitoneally engrafted with 5×106 huPBMCs. Mice were dosed with indicated test articles at indicated concentrations on Days 0, 7, and 14. Blood was drawn over time to investigate lymphocyte expansion and tumor size was measured by caliper. The data as depicted in
This application claims the benefit of U.S. Provisional Patent Application No. 63/110,908, filed Nov. 6, 2020, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63110908 | Nov 2020 | US |
