ANTI-CD28 AND / OR ANTI-B7H3 COMPOSITIONS
Anti-CD28 x anti-TAA antibodies selectively target tumor cells to enhance antitumor activity and minimize peripheral toxicity, addressing the limitations of non-discriminatory antibody therapies and checkpoint blockade failures.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-03-10
AI Technical Summary
Existing cancer therapies using monospecific full-length antibodies for costimulatory receptors are non-discriminatory, leading to peripheral toxicity and autoimmune issues, while checkpoint blockade therapies do not effectively target tumor cells in many patients.
Development of anti-CD28 x anti-TAA antibodies, specifically targeting CD28 on T cells and tumor-associated antigens like B7H3, to enhance antitumor activity selectively at tumor sites with minimal peripheral toxicity, potentially combined with checkpoint inhibitors.
The antibodies enhance tumor-specific immune responses, reducing peripheral toxicity and improving treatment efficacy by selectively targeting tumor cells, while allowing combination with other cancer therapies.
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Abstract
Description
[Technical Field]
[0001] Priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 067,834, filed August 19, 2020, and U.S. Provisional Patent Application No. 63 / 092,272, filed October 15, 2020, which are incorporated by reference in their entireties.
[0002] Intrinsic paragraphs in sequence listings This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on August 19, 2021, is named 067461-5272-WO_SL.txt and is 1,130,417 bytes in size. [Background technology]
[0003] The innate immune response to tumors deploys immune effector cells, such as natural killer (NK) cells and T cells, to attack and destroy tumor cells. Tumor-infiltrating lymphocytes (TILs) often express multiple immune checkpoint receptors (e.g., PD-1 and CTLA-4) and costimulatory receptors (e.g., ICOS, 4-1BB, OX40, GITR, and CD28). TILs lose their cytotoxicity over time due to the upregulation of inhibitory immune checkpoints. Although checkpoint blockade has demonstrated increased clinical response rates compared to other treatment options, many patients still fail to achieve a response to checkpoint blockade. Engagement of costimulatory receptors on TILs can provide positive signals that can overcome the negative signals of immune checkpoints. Preclinical and clinical studies of agonistic costimulatory receptor antibodies have demonstrated that costimulatory receptor agonism can result in impressive antitumor responses, activating T cells to attack tumor cells.
[0004] Enhancing antitumor activity by specifically destroying tumor cells while minimizing peripheral toxicity is also important for cancer therapy. In this context, it is important that only T cells receive costimulatory signals in the presence of target tumor cells. However, agonism of costimulatory receptors by monospecific full-length antibodies is likely non-discriminatory with respect to TILs, peripheral T cells, or autoantigen-reactive T cells, which contribute to autoimmune toxicity. For example, urelumab, a monospecific, non-discriminatory pan-4-1BB agonist antibody, demonstrated significant liver toxicity in early-phase clinical trials (Segal et al., 2016). Therefore, novel immune response-enhancing compositions for cancer treatment remain needed. Summary of the Invention
[0005] Provided herein are novel anti-CD28 compositions comprising anti-CD28 x anti-TAA (e.g., αCD28 x αB7H3) antibodies, and methods of using such antibodies for the treatment of cancer. The subject anti-CD28 x anti-TAA antibodies can agonistically bind to the CD28 costimulatory molecule on T cells and tumor-associated antigens (e.g., B7H3) on tumor cells. Thus, such antibodies selectively enhance anti-tumor activity at the tumor site while minimizing peripheral toxicity. The subject antibodies provided herein are particularly useful in combination with other anti-cancer therapies, including, for example, checkpoint inhibitors. Also provided herein are novel αCD28 and αB7H3 binding domains.
[0006] In a first aspect, the present invention provides a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 are first Fc domains, respectively. and c) a common light chain that comprises, from N- to C-terminus, a VH1-CH1 domain in the first monomer and a VH2-CH1 domain in the second monomer, wherein the VH1-CH1 domain in the first monomer binds to human CD28 and the VH2-CH1 domain in the second monomer binds to human B7H3.
[0007] In some embodiments, the first ABD binds to human CD28 and the second ABD binds to human B7H3. In certain embodiments, the first ABD binds to human B7H3 and the second ABD binds to human CD28.
[0008] In some embodiments, the amino acid sequence of the VH1 domain is SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, and the amino acid sequence of the VL domain is SEQ ID NO:874.
[0009] In some embodiments, the amino acid sequence of the VH2 domain is SEQ ID NO:585, SEQ ID NO:870, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617 , SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651. It is selected.
[0010] In certain embodiments, the first Fc domain and the second Fc domain are each variant Fc domains.
[0011] In some embodiments of the heterodimeric antibody, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In exemplary embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0012] In some embodiments, the first and second Fc domains each comprise one or more deletion variants, hi some embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0013] In some embodiments, one of the first or second monomers further comprises a pI variant, in some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variant N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0014] In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0015] In an exemplary embodiment, the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
[0016] In some embodiments, the second monomer comprises the amino acid sequence of SEQ ID NO:1019, the first monomer comprises the amino acid sequence of SEQ ID NO:1020, and the light chain has the amino acid sequence of SEQ ID NO:1021.
[0017] In another embodiment, the antibody comprises: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain; and c) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain. and a common light chain comprising, from the C-terminus to the VH domain, a VH domain and a VL domain together forming a first ABD, and a VL domain and a VH domain together forming a second ABD, wherein one of the first and second ABDs binds human CD28 and the other of the first and second ABDs binds human B7H3.
[0018] In certain embodiments, the amino acid sequence of the VH1 domain is SEQ ID NO: 518, SEQ ID NO: No. 928, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 500, SEQ ID NO: 501, SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO: 504, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 507, SEQ ID NO: 508, SEQ ID NO: 509, SEQ ID NO: 510, SEQ ID NO: 511, SEQ ID NO: 512, SEQ ID NO: 513, SEQ ID NO: 514, SEQ ID NO: 515, SEQ ID NO: 516, SEQ ID NO: 517, SEQ ID NO: 519, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 5 43, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584; and the amino acid sequence of the VL domain is SEQ ID NO:874.
[0019] In some embodiments, the amino acid sequence of the VH2 domain is SEQ ID NO:585, SEQ ID NO:870, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, S SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651.
[0020] In certain embodiments, the first Fc domain and the second Fc domain are each variant Fc domains.
[0021] In some embodiments, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In certain embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0022] In some embodiments, the first and second Fc domains each comprise one or more deletion variants. In certain embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, numbering follows EU numbering.
[0023] In certain embodiments, one of the first or second monomers further comprises a pI variant. In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the first monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0024] In some embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0025] In some embodiments, the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
[0026] In another embodiment, a heterodimer comprising: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-first domain linker-scFv-second domain linker-CH2-CH3, where VH1 is a first variable heavy domain, scFv is an anti-CD28 scFv, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, where VL1 is a variable light chain domain and CL is a constant light chain domain. Provided herein are antibodies, wherein each of a VH1 domain and a first VL1 domain together form a first antigen-binding domain (ABD), and the scFv comprises a second VH domain (VH2), an scFv linker, and a second VL domain (VL2), wherein the VH2 and VL2 form a second ABD, and one of the first and second ABDs binds to human CD28, and the other of the first and second ABDs binds to a tumor target antigen (TTA).
[0027] In certain embodiments, the first ABD binds human CD28 and the second ABD binds TTA, hi some embodiments, the first ABD binds TTA and the second ABD binds human CD28.
[0028] In some embodiments, the scFv comprises, from N- to C-terminus, VL2-scFv linker-VH2. In some embodiments, the scFv comprises, from N- to C-terminus, VH2-scFv linker-VL2.
[0029] In some embodiments, the amino acid sequence of VH2 is SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:6 28, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651, and the amino acid sequence of VL2 is selected from the group consisting of SEQ ID NO:674, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO 55, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685 686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717 7, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748Selected from the group consisting of SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752, SEQ ID NO: 753, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 1200 and SEQ ID NO: 756.
[0030] In certain embodiments, the TTA is human B7H3.
[0031] In some embodiments, the first Fc domain and the second Fc domain are each variant Fc domains.
[0032] In exemplary embodiments, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In some embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0033] In some embodiments, the first and second Fc domains each comprise one or more deletion variants, hi certain embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0034] In some embodiments, one of the first or second monomers further comprises a pI variant. In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, Numbering follows EU numbering.
[0035] In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0036] In certain embodiments, the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
[0037] In one aspect, a) a first monomer comprising i) an 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 linked to the N-terminus of the first Fc domain using a domain linker; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, wherein VH is the first variable heavy domain and CH2-CH3 is the second Fc domain; and c) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3 and a light chain comprising VL1-CL, where VL1 is a variable light chain domain and CL is a constant light chain domain, wherein VH1 and VL1 together form a first ABD, and the scFv comprises a second VH domain (VH2), an scFv linker, and a second VL domain (VL2), wherein VH2 and VL2 form a second ABD, and one of the first ABD and the second ABD binds to CD28, and the other of the first ABD and the second ABD binds to TTA.
[0038] In some embodiments, the scFv comprises, from N- to C-terminus, VL2-scFv linker-VH2. In certain embodiments, the scFv comprises, from N- to C-terminus, VH2-scFv linker-VL2.
[0039] In certain embodiments, the second ABD binds to human CD28 and the amino acid sequence of VH2 is SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604 , SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628 , SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651, and the amino acid sequence of VL2 is selected from the group consisting of: SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676 , SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO: SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.
[0040] In some embodiments, the first Fc domain and the second Fc domain are each variant Fc domains.
[0041] In certain embodiments, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In exemplary embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0042] In certain embodiments, the first and second Fc domains each comprise one or more deletion variants, hi some embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0043] In certain embodiments, one of the first or second monomers further comprises a pI variant. In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0044] In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0045] In some embodiments, the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
[0046] In another embodiment, a) from N-terminus to C-terminus, VH1-CH1-hinge-CH2- 1. A heterodimeric antibody comprising: a) a first monomer comprising a CH3-domain linker-scFv, where VH1 is a first variable heavy domain, the scFv is an anti-CD28 scFv, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, where VL1 is a variable light domain and CL is a constant light domain. provided herein, wherein each of a VH1 domain and a first VL1 domain together form a first antigen-binding domain (ABD), and the scFv comprises a second VH domain (VH2), an scFv linker, and a second VL domain (VL2), wherein the VH2 and VL2 together form a second ABD, and one of the first and second ABDs binds to human CD28, and the other of the first and second ABDs binds to a tumor target antigen (TTA).
[0047] In certain embodiments, the first ABD binds human CD28 and the second ABD binds TTA, hi some embodiments, the first ABD binds TTA and the second ABD binds human CD28.
[0048] In certain embodiments, the scFv comprises, from N- to C-terminus, VL2-scFv linker-VH2. In some embodiments, the scFv comprises, from N- to C-terminus, VH2-scFv linker-VL2.
[0049] In certain embodiments, the amino acid sequence of VH2 is SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID Sequence number 612, sequence number 613, sequence number 614, sequence number 615, sequence number 616, sequence number 617, sequence number 618, sequence number 619, sequence number 620, sequence number 621, sequence number 622, sequence number 623, sequence number 624, sequence number 1198, sequence number 1199, sequence number 625, sequence number 626, sequence number 627, sequence number 628, sequence number 629, sequence number 630, sequence number 631, sequence number 632, sequence number 633, sequence number 634, sequence number 635, sequence number 636, sequence number 637, sequence number 638, sequence number 639, sequence number 640, sequence number and the amino acid sequence of VL2 is selected from the group consisting of SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651; and the amino acid sequence of VL2 is selected from the group consisting of SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, S SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698SEQ ID NO: 699, SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.
[0050] In some embodiments, the TTA is human B7H3.
[0051] In some embodiments, the first Fc domain and the second Fc domain are each variant Fc domains. In some embodiments, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In certain embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0052] In some embodiments, the first and second Fc domains each comprise one or more deletion variants, hi certain embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0053] In some embodiments, one of the first or second monomers further comprises a pI variant. In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0054] In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0055] In an exemplary embodiment, the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
[0056] In another aspect, provided herein is a nucleic acid composition comprising: a) a first nucleic acid encoding a first monomer of any of the heterodimeric antibodies described herein; b) a second nucleic acid encoding a second monomer of the heterodimeric antibody; and c) a third nucleic acid encoding a light chain of the heterodimeric antibody. Also provided herein are expression vector compositions comprising expression vectors comprising one or more of the first, second, and third nucleic acids, host cells comprising such expression vector compositions, and methods of making the heterodimeric antibodies described herein.
[0057] In another aspect, there is provided a method of treating cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of Provided herein are methods that include administering to a patient a heterodimeric antibody described herein.
[0058] In another aspect, provided herein is a method of treating cancer in a patient in need thereof, the method comprising administering to the patient: a) a heterodimeric antibody described herein, wherein the TTA is human B7H3; and b) a bispecific antibody that binds CD3 and B7H3.
[0059] In yet another aspect, provided herein is a method of treating cancer in a patient in need thereof, the method comprising administering to the patient: a) a heterodimeric antibody as described herein, wherein the TTA is human B7H3; and b) a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody and an anti-PD-L1 antibody.
[0060] In yet another aspect, a) the amino acid sequence of SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:6 13, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, 642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 and SEQ ID NO:672; and b) a variable heavy chain domain having an amino acid sequence selected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 and SEQ ID NO:672. No. 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO: No. 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, SEQ ID NO: 731, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736, SEQ ID NO: 737, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, and a variable light chain domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752, SEQ ID NO: 753, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 1200, and SEQ ID NO: 756. In some embodiments, the composition is an antibody comprising a) a heavy chain comprising VH-CH1-hinge-CH2-CH3, and b) a light chain comprising VL-CL.
[0061] In another aspect, a) the sequences of SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, Column number 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 524, SEQ ID NO: 525, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584; and a) a variable heavy chain domain having an amino acid sequence selected from the group consisting of SEQ ID NO:874 and SEQ ID NO:932. Provided herein are compositions comprising an ABD.
[0062] In one aspect, provided herein is a composition comprising an anti-B7H3 ABD comprising: a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 946; and b) a variable light chain domain having the amino acid sequence of SEQ ID NO: 950.
[0063] In another aspect, provided herein is a composition comprising an anti-B7H3 ABD comprising: a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 956; and b) a variable light chain domain having the amino acid sequence of SEQ ID NO: 960.
[0064] In one aspect, provided herein is a composition comprising an anti-B7H3 ABD comprising: a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 964; and b) a variable light chain domain having the amino acid sequence of SEQ ID NO: 968.
[0065] In another aspect, provided herein is a composition comprising an anti-B7H3 ABD comprising: a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO:972; and b) a variable light chain domain having the amino acid sequence of SEQ ID NO:976.
[0066] In some embodiments, the composition is an antibody comprising: a) a heavy chain comprising a VH linked to -CH1-hinge-CH2-CH3; and b) a light chain comprising a VL linked to -CL.
[0067] In another embodiment, a first nucleic acid encoding a) a VH of any anti-CD28 ABD or anti-B7H3 ABD described herein; and b) a VL of an anti-CD28 or anti-B7H3 ABD. and a second nucleic acid encoding the first or second nucleic acid, respectively. Also provided herein are expression vector compositions comprising an expression vector containing one or more of the first and second nucleic acids, host cells comprising such nucleic acid compositions or expression vector compositions, and methods of making the anti-CD28 ABD or anti-B7H3 ABD compositions described herein.
[0068] In one aspect, provided herein is a composition comprising a CD28 antigen binding domain (ABD), wherein the CD28 ABD comprises the variable heavy chain complementarity determining regions 1-3 (vhCDR1-3) and variable light chain complementarity determining regions (vlCDR1-3) of any of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD2 8]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28] _H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1.
[0069] In some embodiments, the CD28 ABD comprises the variable heavy and variable light domains of any of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3 [CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 28 1VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1. In exemplary embodiments, the CD28 antigen binding domain is selected from the following CD28 binding domains: CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, CD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1 , 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1.
[0070] In another aspect, provided herein are anti-CD28 antibodies comprising a CD28 antigen-binding domain (ABD) comprising the variable heavy chain complementarity-determining regions 1-3 (vhCDR1-3) and variable light chain complementarity-determining regions (vlCDR1-3) of any of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, and CD28.3. In some embodiments, the CD28 ABD comprises the variable heavy and light domains of any of the following CD28 binding domains: 1A7[CD28]_H1L1, ... 8]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1. In exemplary embodiments, the CD28 antigen binding domain is selected from the following CD28 binding domains: CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, CD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1 , 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1.
[0071] In some embodiments, the anti-CD28 antibody comprises a) a first monomer comprising a first antigen-binding domain and a first constant domain; and b) a second monomer comprising a second antigen-binding domain and a second constant domain, wherein either the first antigen-binding domain or the second antigen-binding domain is a CD28 antigen-binding domain.
[0072] In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to different antigens.
[0073] In certain embodiments, the CD28 antigen-binding domain is an anti-CD28 single-chain fragment (scFv). In exemplary embodiments, the scFv comprises a charged scFv linker.
[0074] In some embodiments, the first and second constant domains each comprise CH2-CH3. In exemplary embodiments, the first and second constant domains each are variant constant domains. In certain embodiments, the first and second constant domains comprise a set of heterodimerization variants selected from the group consisting of S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V. In certain embodiments, the first and second monomers each further comprise one or more deletion variants. In exemplary embodiments, the deletion variants are E233P / L234V / L235A / G236del / S267K. In some embodiments, at least one of the first or second monomers further comprises one or more pI variants, hi certain embodiments, the pI variants are N208D / Q295E / N384D / Q418E / N421D.
[0075] In one aspect, provided herein is a composition comprising a B7H3 antigen binding domain (ABD), wherein the B7H3 binding domain comprises the variable heavy chain complementarity determining regions 1-3 (vhCDR1-3) and variable light chain complementarity determining regions (vlCDR1-3) of any of the following B7H3 binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, or 2E4A3.189[B7H3]_H1.22L1. , 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m85 7, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chA b8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0076] In some embodiments, the B7H3 ABD comprises the variable heavy and variable light domains of any of the following B7H3 binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S 10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb1 0, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0077] In exemplary embodiments, the B7H3 ABD is selected from the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58 , 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chA b7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0078] In yet another aspect, provided herein is an anti-B7H3 antibody comprising a B7H3 antigen binding domain, wherein the B7H3 antigen binding domain comprises the variable heavy chain complementarity determining regions 1-3 (vhCDR1-3) and variable light chain complementarity determining regions (vlCDR1-3) of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D. , BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L5 8, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, ch Ab18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-38 03, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0079] In some embodiments, the anti-B7H3 antibody comprises a B7H3 antigen binding domain, which comprises the variable heavy and variable light domains of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, S P265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, ch Ab10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.In exemplary embodiments, the B7H3 antigen binding domain is selected from any one of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP 265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chA b10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0080] In some embodiments, the antibody comprises a) a first monomer comprising a first antigen-binding domain and a first constant domain, and b) a second monomer comprising a second antigen-binding domain and a second constant domain, wherein either the first antigen-binding domain or the second antigen-binding domain is a B7H3 antigen-binding domain. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain bind to different antigens.
[0081] In exemplary embodiments, the first antigen-binding domain is a B7H3 antigen-binding domain and the second antigen-binding domain is a CD28 binding domain. In some embodiments, the CD28 binding domain comprises the vhCDR1-3 and vlCDR1-3 of any of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L In some embodiments, the CD28 binding domain comprises the variable heavy and light domains of any of the following CD28 binding domains: hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1. Contains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD 28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1. In certain embodiments, the CD28 binding domain is an anti-CD28 scFv. In exemplary embodiments, the scFv comprises a charged scFv linker.
[0082] In some embodiments, the first and second constant domains each comprise CH2-CH3. In exemplary embodiments, the first and second constant domains each are variant constant domains.
[0083] In certain embodiments, the first and second constant domains comprise a set of heterodimerization variants selected from S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V. In certain embodiments, the first and second monomers each comprise one or more deletion variants. In certain embodiments, the deletion variant is E233P / L234V / L235A / G236del / S267K. In some embodiments, at least one of the first or second monomers further comprises one or more pI variants. In a particular embodiment, the pI variant is N208D / Q295E / N384D / Q418E / N421D.
[0084] In another aspect, provided herein is an anti-CD28 x anti-TAA 1+1 Fab-scFv-Fc heterodimeric antibody. In one embodiment, the heterodimeric antibody comprises: a) a first monomer comprising: i) an anti-CD28 scFv comprising a first variable light domain, an scFv linker, and the first variable light domain; and ii) a first Fc domain, wherein the scFv is covalently linked to the N-terminus of the first Fc domain using a domain linker; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain; and c) a third monomer comprising a second variable light domain, wherein the second variable heavy domain and the second variable light domain form a tumor-associated antigen (TAA) binding domain.
[0085] In some embodiments, the anti-CD28 scFv comprises vhCDR1-3 and vlCDR1-3 of any of the following CD28 antigen binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28. 3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.
[0086] In certain embodiments, the first variable heavy chain domain and the first variable light chain domain of the anti-CD28 scFv are the variable heavy chain domain and the variable light chain domain, respectively, of any of the following CD28 antigen binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1L1, 1A7[CD28]_H1L2, 1A7[CD28]_H1L3, 1A7[CD28]_H1L4, 1A7[CD28]_H1L5, 1A7[CD28]_H1L6, 1A7[CD28]_H1L7, 1A7[CD28]_H1L8, 1A7[CD28]_H1L9, 1A7[CD28]_H1L1 ...2, 1A7[ ]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_ L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN14 12_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, Hu TN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.
[0087] In certain embodiments, the TAA binding domain is a B7H3 binding domain. In some embodiments, the B7H3 binding domain comprises the vhCDR1-3 and vlCDR1-3 of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1 -L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0088] In exemplary embodiments, the second variable heavy chain domain and the second variable light chain domain are, respectively, the variable heavy chain domain and the variable light chain domain of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M3 0, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chA b16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0089] In exemplary embodiments, the anti-CD28 scFv is oriented from N- to C-terminus as follows: first variable light domain-scFv linker-first variable heavy domain. In other embodiments, the anti-CD28 scFv is oriented from N- to C-terminus as follows: first variable heavy domain-scFv linker-first variable light domain. In many embodiments, the scFv linker is a charged scFv linker.
[0090] In certain embodiments, the first and second Fc domains are variant Fc domains. In some embodiments, the first and second Fc domains are heterodimerization domains selected from the group consisting of S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V. In an exemplary embodiment, the first and second Fc domains comprise a heterodimerized variant S364K / E357Q:L368D / K370S, where the numbering is according to EU numbering.
[0091] In certain embodiments, the first and second Fc domains each comprise one or more deletion variants. In exemplary embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0092] In some embodiments, one of the first or second monomers comprises one or more pI variants. In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0093] In an exemplary embodiment, the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K and the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0094] In certain embodiments, the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS)4.
[0095] In a specific embodiment, the first and second Fc domains each further comprise the amino acid variant 428 / 434S.
[0096] In some embodiments, the anti-CD28 x anti-TAA 1+1 Fab-scFv-Fc heterodimeric antibody comprises: a) a first monomer comprising, from N-terminus to C-terminus, anti-CD28 scFv-linker-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising VL-CL, where the first variant Fc domain comprises the amino acid variant S364K / E357Q and the second the variant Fc domain of the first monomer comprises the amino acid variants L368D / K370S, the first and second variant Fc domains each comprise the amino acid variants E233P / L234V / L235A / G236del / S267K, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants N208D / Q295E / N384D / Q418E / N421D, the VH and VL form a tumor-associated antigen (TAA) binding domain, and the anti-CD28 The scFvs contain the following CD28 antigen-binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, and TGN14 12_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1, where numbering is according to EU numbering.
[0097] In certain embodiments, the TAA binding domain is a B7H3 binding domain. In some embodiments, VH and VL are the variable heavy and variable light domains, respectively, of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1- L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0098] In exemplary embodiments, the scFv comprises a charged scFv linker having the amino acid sequence (GKPGS)4. In some embodiments, the first and second variant Fc domains each further comprise the amino acid variant 428 / 434S, where numbering is according to EU numbering.
[0099] In another aspect, an anti-CD28 x anti-TAA 2+1 antibody comprises: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker1-anti-CD28 scFv-linker2-CH2-CH3, where VH1 is a first variable heavy domain, linker1 and linker2 are first and second domain linkers, respectively, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain; and c) a common light chain comprising a variable light domain. Provided herein is a Fab2-scFv-Fc, wherein a first variable heavy chain domain and a variable light chain domain form a first tumor-associated antigen (TAA)-binding domain, and a second variable heavy chain domain and a variable light chain domain form a second TAA-binding domain.
[0100] In an exemplary embodiment, the first TAA binding domain and the second TAA binding domain are each B7H3 binding domains. In exemplary embodiments, the first and second B7H3 binding domains each comprise the vhCDR1-3 and vlCDR1-3 of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M 30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb In some embodiments, the first and second variable heavy chain domains each comprise a variable heavy chain domain of a B7H3 binding domain, the variable light chain domain comprises a variable light chain domain of a B7H3 binding domain, and the B7H3 binding domain comprises a B7H3 binding domain selected from the group consisting of: The following antibodies were detected: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852 , m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, c hAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0101] In some embodiments of the anti-CD28 x anti-TAA 2+1 Fab2-scFv-Fc antibody, the anti-CD28 scFv comprises an scFv variable heavy domain, an scFv variable light domain, and an scFv linker connecting the scFv variable heavy domain and the scFv variable light domain. In certain embodiments, the anti-CD28 scFv comprises vhCDR1-3 and vlCDR1-3 of any of the following CD28 antigen-binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28. 3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1. The scFv variable heavy chain domain and the scFv variable light chain domain of the scFv comprise the variable heavy chain domain and the variable light chain domain, respectively, of any of the following CD28 antigen-binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD 28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.
[0102] In some embodiments, the scFv variable heavy domain is linked to the C-terminus of the CH1 of the first monomer using a first domain linker, and the scFv variable light domain is covalently linked to the N-terminus of the first Fc domain using a second Fc domain linker. In other embodiments, the scFv variable light domain is linked to the C-terminus of the CH1 of the first monomer using a first domain linker, and the scFv variable heavy domain is covalently linked to the N-terminus of the first Fc domain using a second Fc domain linker. In some embodiments, the scFv linker is a charged scFv linker.
[0103] In certain embodiments, the first and second Fc domains are variant constant domains, wherein the first and second Fc domains are heterodimers selected from the following heterodimerizing variants: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V. In some embodiments, the first and second Fc domains comprise a heterodimeric variant S364K / E357Q:L368D / K370S, where the numbering is according to EU numbering.
[0104] In some embodiments, the first and second Fc domains each comprise one or more deletion variants. In exemplary embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0105] In some embodiments, one of the first or second monomers comprises one or more pI variants, hi certain embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0106] In an exemplary embodiment, the first Fc domain of the first monomer comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K and the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants N208D / E233P / L234V / L235A / G236del / S267K / Q295E / L368D / K370S / N384D / Q418E / N421D, where numbering is according to EU numbering.
[0107] In some embodiments, the anti-CD28 scFv comprises a charged scFv linker having the amino acid sequence (GKPGS)4. In certain embodiments, the first and second variant Fc domains each further comprise the amino acid variant 428 / 434S, where numbering is according to EU numbering.
[0108] In some embodiments, the anti-CD28 x anti-TAA 2+1 Fab2-scFv-Fc antibody comprises: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker1-anti-CD28 scFv-linker2-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and c) a common light chain comprising VL-CL, where the first variant Fc domain comprises the amino acid variants S364K / E357Q and the second the variant Fc domain of the first monomer comprises the amino acid variant L368D / K370S, the first and second variant Fc domains each comprise the amino acid variants E233P / L234V / L235A / G236del / S267K, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants N208D / Q295E / N384D / Q418E / N421D, the VH1 and VL each form a tumor-associated antigen (TAA) binding domain, and the anti-CD28 The scFvs contain the following CD28 antigen-binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, and TGN1 The antibody comprises the variable heavy chain domain and variable light chain domain of any of 412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1, wherein the numbering is according to EU numbering.
[0109] In some embodiments, VH1 and VL form a B7H3 binding domain. In exemplary embodiments, VH1 and VL are the variable heavy and light domains of any of the following B7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, and M30-H1-L4. , SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, c hAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0110] In some embodiments, the scFv comprises a charged scFv linker having the amino acid sequence (GKPGS) 4. In certain embodiments, the first and second variant Fc domains each further comprise the amino acid variant 428 / 434S.
[0111] In another embodiment, an anti-CD28 x anti-TAA 1+1 antibody comprises: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 are a second Fc domain; and c) a common light chain comprising, from N-terminus to C-terminus, VL-CL, where VL is a variable light domain and CL is a constant light domain. Provided herein are CLC heterodimeric antibodies, wherein a first variable heavy chain domain and a variable light chain domain form a first antigen-binding domain, and a second variable heavy chain domain and a variable light chain domain form a second antigen-binding domain.
[0112] In some embodiments, the first Fc domain and the second Fc domain are each variant Fc domains. In certain embodiments, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In exemplary embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0113] In certain embodiments, the first and second Fc domains each comprise one or more deletion variants. In exemplary embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0114] In some embodiments, one of the first or second monomers further comprises a pI variant, hi certain embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0115] In certain embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0116] In some embodiments, the first and second variant Fc domains each comprise the amino acid variant 428 / 434S.
[0117] In certain embodiments, the first antigen-binding domain or the second binding domain binds to CD28, and the other antigen-binding domain binds to a tumor-associated antigen (TAA).
[0118] In certain embodiments, the second antigen-binding domain binds to CD28, and VH2 and VL comprise the variable heavy and variable light chain domains, respectively, of any one of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28 .3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.
[0119] In some embodiments, the first antigen-binding domain binds to a TTA. In an exemplary embodiment, the TAA is B7H3. In an exemplary embodiment, VH1 and VL comprise the variable heavy and variable light domains, respectively, of any one of the following B7H3 binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1 -L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0120] In an exemplary embodiment, the first antigen-binding domain binds to B7H3, the second antigen-binding domain binds to CD28, VH1 is the variable heavy chain domain 2E4A3.189[B7H3]_H1.22, VH2 is the variable heavy chain domain 1A7[CD28]_H1.14, and VL is the variable light chain domain 1A7[CD28]_L1.
[0121] In one aspect, the present invention provides a method for producing a fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 are a first Fc domain; and b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 are Provided herein is an anti-CD28 x anti-TAA 2+1 CLC heterodimeric antibody comprising: a second monomer that is a second Fc domain; and c) a common light chain that comprises, from N-terminus to C-terminus, VL-CL, where VL is a variable light chain domain and CL is a constant light chain domain, wherein the first variable heavy chain domain and variable light chain domain, respectively, form a first antigen-binding domain, and the second variable heavy chain domain and variable light chain domain form a second antigen-binding domain.
[0122] In some embodiments, the first Fc domain and the second Fc domain are each variant Fc domains. In some embodiments, the first and second Fc domains comprise a set of heterodimerization 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, where numbering is according to EU numbering. In certain embodiments, the first and second Fc domains comprise the heterodimerization variant S364K / E357Q:L368D / K370S.
[0123] In some embodiments, the first and second Fc domains each comprise one or more deletion variants. In exemplary embodiments, the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, where the numbering is according to EU numbering.
[0124] In certain embodiments, one of the first or second monomers further comprises a pI variant. In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the first monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0125] In some embodiments of the anti-CD28 x anti-TAA 2+1 CLC heterodimeric antibody, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0126] In some embodiments, the first and second variant Fc domains each comprise the amino acid variant 428 / 434S.
[0127] In certain embodiments, the first antigen-binding domain binds to CD28 and the second antigen-binding domain binds to a tumor-associated antigen (TAA). In exemplary embodiments, VH1 and VL comprise the variable heavy and light chain domains, respectively, of any one of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0 L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.
[0128] In some embodiments, the TAA is B7H3. and VL each comprise the variable heavy and variable light domains of any one of the following B7H3 binding domains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, and SP26. 5, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb 10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704.
[0129] In some embodiments, VH1 is the variable heavy chain domain 1A7[CD28]_H1.14, VH2 is the variable heavy chain domain 2E4A3.189[B7H3]_H1.22, and VL is the variable light chain domain 1A7[CD28]_L1.
[0130] In another aspect, provided herein is a heterodimeric antibody selected from the following heterodimeric antibodies: XENP34730, XENP34389, XENP34728, XENP34717, and XENP34339.
[0131] Also provided herein are nucleic acid compositions encoding the compositions and antibodies provided herein, expression vectors containing such nucleic acids, and host cells containing the expression vectors.
[0132] In another aspect, provided herein are methods of treating cancer, comprising administering an antibody provided herein (e.g., an anti-CD28 x anti-TAA antibody) to a patient in need thereof. In some embodiments, the patient is also administered a cancer therapeutic agent. In certain embodiments, the therapeutic agent is a checkpoint inhibitor (e.g., an anti-PD1 antibody) or an anti-CD3 x anti-TAA bispecific antibody. [Brief explanation of the drawings]
[0133] [Figure 1] The sequences of human, mouse, and cynomolgus CD28 are shown. Such CD28s are useful for developing cross-reactive CD28 antigen-binding domains to facilitate clinical development. [Figure 2A-2B] The sequences of human, mouse, and cynomolgus monkey B7H3 are shown. Such B7H3s are useful for developing cross-reactive B7H3 antigen-binding domains that facilitate clinical development. [Figures 3A-3F] Useful pairs of heterodimerization variant sets (including skew and pI variants) are shown. Figure 3F shows variants for which there is no corresponding "monomer 2" variant. Such variants are pI variants that can be used alone in either monomer of the αB7H3xαCD28 bsAb, or can be included, for example, on the non-scFv side of a format utilizing an scFv as a building block, with an appropriate charged scFv linker used in the second monomer utilizing an scFv as the CD28-binding domain. Suitable charged linkers are shown in Figure 6. [Figure 4] A list of isosteric variant antibody constant regions and their respective substitutions is provided below. pI_(-) indicates a lower pI variant and pI_(+) indicates a higher pI variant. These variants can be optionally and independently combined with other variants, including heterodimerization variants, as outlined herein. [Figure 5]Useful deletion variants that eliminate FcγR binding are shown (also referred to as "knockout" or "KO" variants). In some embodiments, such deletion variants are contained in both monomeric Fc domains of the subject antibodies described herein. In other embodiments, deletion variants are contained in only one variant Fc domain. [Figure 6] As described herein, several charged scFv linkers are shown for use in increasing or decreasing the pI of the subject heterodimeric αB7H3×αCD28 bsAb, which utilize one or more scFvs as building blocks. (+H) Positive linkers are particularly used herein, particularly with the anti-CD28 VL and VH sequences shown herein. A single prior art scFv linker bearing a single charge is referred to as "Whitlow" from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used to reduce aggregation and increase proteolytic stability in scFvs. Such charged scFv linkers can be used in any of the subject antibody formats disclosed herein that include scFvs (e.g., 1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fc formats). [Figure 7] Some exemplary domain linkers are shown. In some embodiments, these linkers find use in linking a single-chain Fv to an Fc chain. In some embodiments, these linkers can be combined in any orientation. For example, a GGGGS linker can be combined with a "lower half-hinge" linker at the N- or C-terminus. In some embodiments, two or more of the domain linkers shown in Figure 7 can be combined to form longer domain linkers for use in the heterodimeric antibodies described herein. [Figure 8] A particularly useful embodiment of the heterodimeric Fc domain (i.e., CH2-CH3 in this embodiment) of the αB7H3×αCD28 bsAb of the present invention is shown. [Figure 9]1A-1D show various heterodimeric variant amino acid substitutions that may be used in the heterodimeric antibodies described herein. [Figures 10A-10C]
[0039] Figure 1 shows the sequences of several useful heterodimeric αB7-H3xαCD28 bsAb scaffolds based on human IgGl, without cytokine sequences. Heterodimeric Fc scaffold 1 is based on human IgGl (356E / 358M allotype) and contains the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K / E357Q scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Heterodimeric Fc scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Heterodimeric Fc scaffold 3 is based on human IgG1 (356E / 358M allotype) and contains the L368E / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Heterodimeric Fc scaffold 4 is based on human IgG1 (356E / 358M allotype) and contains the K360E / Q362E / T411E scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the D401K scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variants in both chains.Heterodimeric Fc scaffold 5 is based on human IgG1 (356D / 358L allotype) and contains the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K / E357Q scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Heterodimeric Fc scaffold 6 is based on human IgG1 (356E / 358M allotype) and contains the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K / E357Q scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variant and the N297A variant that abolishes glycosylation in both chains. Heterodimeric Fc scaffold 7 is based on human IgG1 (356E / 358M allotype) and contains the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K / E357Q scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variant and the N297S variant to ablate glycosylation in both chains. Heterodimeric Fc scaffold 8 is based on human IgG4 and comprises the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K / E357Q scuba variant in the second heterodimeric Fc chain, and the S228P (according to EU numbering, S241P in Kabat) variant (known in the art) that eliminates Fab arm exchange in both chains. Heterodimeric Fc scaffold 9 is based on human IgG2 and comprises the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D scuba variant in the first heterodimeric Fc chain, and the S364K / E357Q scuba variant in the second heterodimeric Fc chain.The heterodimeric Fc scaffold 10 is based on human IgG2 and comprises an L368D / K370S scuba variant and a Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, and an S364K / E357Q scuba variant and an S267K deletion variant in both chains in the second heterodimeric Fc chain. Heterodimeric Fc scaffold 11 is based on human IgG1 (356E / 358M allotype) and contains the L368D / K370S scuba variant and the Q295E / N384D / Q418E / N421D pI variant in the first heterodimeric Fc chain, the S364K / E357Q scuba variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variant and the M428L / N434S Xtend variant in both chains. Heterodimeric Fc scaffold 12 is based on human IgG1 (356E / 358M allotype) and contains the L368D / K370S variant in the first heterodimeric Fc chain, the S364K / E357Q variant and the P217R / P229R / N276K pI variant in the second heterodimeric Fc chain, and the E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Included in each of these scaffolds are sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequence and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the figure, which already contains some amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4, depending on the scaffold), as will be understood by those skilled in the art). That is, the listed scaffolds can contain additional amino acid modifications (generally amino acid substitutions) in addition to, or as an alternative to, the scaffold variants, pI variants, and deletion variants contained within the scaffolds in this figure. Additionally, the scaffolds shown herein can contain a C-terminal glycine (K446_) and / or lysine (K447_) deletion. C-terminal glycine and / or lysine deletion can be purposefully engineered to reduce heterogeneity or in the context of certain bispecific formats, such as the mAb-scFv format.Additionally, deletion of C-terminal glycine and / or lysine may occur naturally, for example, during production and storage. [Figure 11]
[0033] Figure 1 shows an exemplary sequence of a heterodimeric B7H3xCD28 bsAb scaffold for use in a 2+1 mAb-scFv format. The format shown here is based on heterodimeric Fc scaffold 1 shown in Figure X, but further includes G446 in monomer 1 (-) and G446 / K447 in monomer 2 (+). Note that any of the additional scaffolds shown in Figure X can be adapted for use in a 2+1 mAb-scFv format, with or without K447 in one or both chains. Note that these sequences may further include the M428L / N434S variant. [Figure 12]
[0039] Figure 39 shows the sequence of the "CH1 + hinge" used in embodiments of the αB7H3xαCD28 bsAb utilizing the Fab a-binding domain. The "CH1 + hinge" sequence is used to link the variable heavy domain (VH) to the Fc backbone (as shown in Figure 39). In certain embodiments where the Fab is on the (+) side, a "CH1(+) + hinge" sequence may be used. In certain embodiments where the Fab is on the (-) side, a "CH1(-) + hinge" sequence may be used. [Figure 13] Figure 1 shows the sequence of the "CH1 + half-hinge" domain linker used in embodiments of the αB7H3 x αCD28 bsAb in a 2 + 1 Fab2-scFv-Fc format or a 2 + 1 CLC format. In the 2 + 1 Fab2-scFv-Fc format, the "CH1 + half-hinge" sequence is used to link the variable heavy domain (VH) to the scFv domain on the Fab-scFv-Fc side of the bispecific antibody. In the 2 + 1 CLC format, the "CH1 + half-hinge" sequence is used to link the first variable heavy domain (VH) to the second VH domain on the Fab-Fab-Fc side of the bispecific antibody. Note that other linkers may be used instead of the "CH1 + half-hinge." Note also that while the sequences here are based on IgG1 sequences, equivalents may be constructed based on IgG2 or IgG4 sequences. [Figure 14]1 shows the sequence of "CH1" used in an embodiment of the αB7H3xαCD28 bsAb. [Figure 15] 1 shows the sequence of the "hinge" used in an embodiment of the αB7H3xαCD28 bsAb. [Figure 16] 1 shows the constant domains of the cognate light chains used in the subject αB7H3×αCD28 bsAb, which utilizes a Fab binding domain. [Figure 17] 1 shows the sequence of XENP16432, an anti-PD-1 mAb based on nivolumab and an IgG1 backbone with the E233P / L234V / L235A / G236del / S267K deletion variant. CDRs are underlined and slashes indicate the boundary between the variable and constant domains. [Figure 18] The variable heavy and variable light chain sequences of an exemplary phage-derived CD28 binding domain, 1A7, and the sequences of 1A7 with the E233P / L234V / L235A / G236del / S267K deletion variant and XENP28428, an anti-CD28 mAb based on an IgG1 backbone, are shown. CDRs are underlined, and a slash indicates the boundary between the variable region and the constant domain. As is true for all sequences described herein and containing CDRs herein, the exact identification of CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab formats. [Figure 19] The sequences of exemplary affinity-optimized 1A7 VH variants are shown. Note that the VHs shown herein can be paired with any of the other variable light chain domains shown herein. [Figure 20] The sequence of an exemplary affinity-optimized 1A7-derived variable light domain is shown. Note that this VL can be paired with any of the other variable heavy domains shown herein. [Figures 21A-21B]The sequences of exemplary affinity-optimized 1A7 VH / VH pairs are shown. Note that these pairs can be formatted as Fab or scFv. [Figure 22] FIG. 1 shows an exemplary affinity engineered 1A7 VH / VL pair and its binding affinity in the context of A) scFv (associated with a 1+1 Fab-scFv-Fc bsAb format) and B) Fab (associated with a 2+1 CLC bsAb format). [Figure 23]
[0033] Figure 2 shows the sequences of XENP27181, a bivalent anti-CD28 mAb based on an IgG1 backbone with a HuTN228-binding domain and an E233P / L234V / L235A / G236del / S267K deletion variant, and XENP27656, a monovalent anti-CD28 mAb based on an IgG1 backbone with a HuTN228-binding domain (formatted as an scFv) and an E233P / L234V / L235A / G236del / S267K deletion variant. CDRs are underlined, and a slash indicates the boundary between the variable and constant domains. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 24] Figure 1 shows the KDapp (apparent KD due to bivalent binding) of various CD28-binding phage clones (formatted as bivalent mAbs) against human CD28 as determined by Octet. The first 60 seconds of dissociation were used to fit the data. [Figure 25]
[0033] Figure 1 shows binding of exemplary bivalent anti-CD28 mAbs based on phage-derived clones on human PBMCs. The data indicate that the phage campaign generated a CD28-binding domain with weaker maximal binding than prior art HuTN228 (which is related to the humanized CD28-binding domain described in Example 1A). [Figure 26] The variable heavy and variable light chain sequences of an exemplary phage-derived B7H3 binding domain, 2E4A3.189, are shown, as well as the sequences of 2E4A3.189 with the E233P / L234V / L235A / G236del / S267K deletion variant and XENP32637, an anti-B7H3 mAb based on an IgG1 backbone. CDRs are underlined, and a slash indicates the boundary between the variable and constant domains. As is true for all sequences described herein and containing CDRs herein, the exact identification of CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab formats. [Figure 27] The sequence of the affinity-optimized variable heavy chain 2E4A3.189_H1.22 is shown. Note that this VH can be paired with any of the other variable light domains (VL) shown herein. [Figure 28] The variable heavy and variable light chain sequences of an exemplary rat hybridoma-derived B7H3 binding domain, humanized 6A1, and the sequence of XENP33383, an anti-B7H3 mAb based on 6A1 and an IgG1 backbone with the E233P / L234V / L235A / G236del / S267K deletion variant, are shown. CDRs are underlined, and a slash indicates the boundary between the variable region and the constant domain. As is true for all sequences described herein and containing CDRs herein, the exact identification of CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab formats. [Figure 29]The variable heavy and variable light chain sequences of an exemplary rat hybridoma-derived B7H3 binding domain, humanized 3C4, are shown. The CDRs are underlined, and a slash indicates the boundary between the variable and constant domains. As is true for all sequences described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab format. [Figure 30] The variable heavy and variable light chain sequences of an exemplary rabbit hybridoma-derived B7H3 binding domain, humanized 4F12, are shown. The CDRs are underlined, and a slash indicates the boundary between the variable and constant domains. As is true for all sequences described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab format. [Figure 31] The variable heavy and variable light chain sequences of an exemplary rabbit hybridoma-derived B7H3 binding domain, humanized 38E2, are shown. The CDRs are underlined, and a slash indicates the boundary between the variable and constant domains. As is true for all sequences described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab format. [Figure 32]Figure 1 shows the monovalent binding affinities (KD) of various B7H3 binding domains in the context of a 1+1 bispecific format. Note that 2E4A3_H1.22_1A7_L1 and 2E4A3_H1.3_1A7_L1 utilize the VL of anti-CD28 clone 1A7. [Figures 33A-33E]
[0013] Exemplary formats of the invention are shown. Figure 33A shows a "1+1 Fab-scFv-Fc" format, in which a first Fab arm binds a first antigen and a second scFv arm binds a second antigen. The 1+1 Fab-scFv-Fc format includes a first monomer comprising a first heavy chain variable region (VH1) covalently linked (optionally via a linker) to the N-terminus of a first heterodimeric Fc scaffold, a second monomer comprising a single-chain Fv covalently linked (optionally via a linker) to the N-terminus of a second corresponding heterodimeric Fc scaffold, and a third monomer comprising a light chain variable region covalently linked to a light chain constant domain, where the light chain variable region is complementary to VH1. Figure 33B shows a "2+1 Fab2-scFv-Fc" format having a first Fab arm and a second Fab-scFv arm, where the Fab binds a first antigen and the scFv binds a second antigen. The 2+1 Fab2-scFv-Fc format includes a first monomer comprising a first heavy chain variable region (VH1) covalently linked (optionally via a linker) to the N-terminus of a first heterodimeric Fc scaffold, a second monomer comprising VH1 covalently linked (optionally via a linker) to a single-chain Fv that is covalently linked (optionally via a linker) to the N-terminus of a second, corresponding heterodimeric Fc scaffold, and a third monomer comprising a light chain variable region covalently linked to a light chain constant domain, where the light chain variable region is complementary to VH1. Figure 33C shows a "1 + 1 common light chain" or "1 + 1 CLC" format, in which a first Fc contains a first Fab arm that binds a first antigen, and a second Fc contains a second Fab arm that binds a second antigen. The 1 + 1 CLC format includes a first monomer containing VH1-CH1-hinge-CH2-CH3, a second monomer containing VH2-CH1-hinge-CH2-CH3, and a third monomer containing VL-CL. The VL pairs with VH1 to form a binding domain with a first antigen-binding specificity, and the VL pairs with VH2 to form a binding domain with a second antigen-binding specificity. Figure 33D shows a "2 + 1 common light chain" or "2 + 1 CLC" format, in which a first Fc contains two Fab arms, each binding a first antigen, and a second Fc contains one Fab arm that binds a second antigen.The 2+1 CLC format comprises a first monomer comprising VH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VL pairs with the first and second VH1 to form a binding domain with a first antigen-binding specificity, and the VL pairs with the VH2 to form a binding domain with a second antigen-binding specificity. Figure 33E shows a "2+1 mAb-scFv" format, in which the first Fc comprises an N-terminal Fab arm that binds the first antigen, and the second Fc comprises an N-terminal Fab arm that binds the first antigen and a C-terminal scFv that binds the second antigen. The 2+1 mAb-scFv format comprises a first monomer comprising VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH1-CH1-hinge-CH2-CH3-scFv, and a third monomer comprising VL-CL, where the VL pairs with the first and second VH1 to form a binding domain with binding specificity for a first antigen. [Figures 34A-34E]
[0013] Figure 34A shows an exemplary format of the invention utilized for CD28 bispecific antibodies. Figure 34A shows a "1+1 Fab-scFv-Fc" format, in which a first Fab arm binds to a tumor-associated antigen and a second scFv arm binds to CD28. The 1+1 Fab-scFv-Fc format includes a first monomer comprising a first heavy chain variable region (VH1) covalently linked (optionally via a linker) to the N-terminus of a first heterodimeric Fc scaffold, a second monomer comprising a single-chain Fv covalently linked (optionally via a linker) to the N-terminus of a second corresponding heterodimeric Fc scaffold, and a third monomer comprising a light chain variable region covalently linked to a light chain constant domain, where the light chain variable region is complementary to VH1. Figure 34B shows a "2+1 Fab2-scFv-Fc" format having a first Fab arm and a second Fab-scFv arm, where the Fab binds a tumor-associated antigen and the scFv binds CD28. The 2+1 Fab2-scFv-Fc format includes a first monomer comprising a first heavy chain variable region (VH1) covalently linked (optionally via a linker) to the N-terminus of a first heterodimeric Fc scaffold, a second monomer comprising VH1 covalently linked (optionally via a linker) to a single-chain Fv that is covalently linked (optionally via a linker) to the N-terminus of a second, corresponding heterodimeric Fc scaffold, and a third monomer comprising a light chain variable region covalently linked to a light chain constant domain, where the light chain variable region is complementary to VH1. Figure 34C shows a "1+1 common light chain" or "1+1 CLC" format, in which a first Fc comprises a first Fab arm that binds to a tumor-associated antigen, and a second Fc comprises a second Fab arm that binds to CD28. The 1+1 CLC format comprises a first monomer comprising VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VL pairs with VH1 to form a binding domain with a first antigen-binding specificity, and the VL pairs with VH2 to form a binding domain with a second antigen-binding specificity.Figure 34D shows a "2+1 common light chain" or "2+1 CLC" format, in which a first Fc contains two Fab arms, each binding to a tumor-associated antigen, and a second Fc contains one Fab arm that binds to CD28. The 2+1 CLC format includes a first monomer comprising VH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VL pairs with the first and second VH1 to form a binding domain with a first antigen-binding specificity, and the VL pairs with the VH2 to form a binding domain with a second antigen-binding specificity. Figure 34E shows a "2+1 mAb-scFv" format, in which the first Fc comprises an N-terminal Fab arm that binds to a tumor-associated antigen, and the second Fc comprises an N-terminal Fab arm that binds to a tumor-associated antigen and a C-terminal scFv that binds to CD28. The 2+1 mAb-scFv format includes a first monomer comprising VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH1-CH1-hinge-CH2-CH3-scFv, and a third monomer comprising VL-CL. The VL pairs with the first and second VH1 to form a binding domain with binding specificity for the tumor-associated antigen. [Figure 35A-35B] The sequence of an exemplary αB7H3×αCD28 bsAb in a 1+1 Fab-scFv-Fc format is shown. CDRs are underlined, and slashes indicate the boundaries between the variable region, Fc region, and constant domain. Note that αB7H3×αCD28 bsAb can utilize variable regions, Fc regions, and constant domains that are 90, 95, 98, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Additionally, each sequence outlined herein can include or exclude the M428L / N434S variant in one or, preferably, both Fc domains, which confers a longer half-life in serum. [Figures 36A-36C]Figure 1 shows the sequence of an exemplary αB7H3 x αCD28 bsAb in a 2+1 Fab2-scFv-Fc format. CDRs are underlined, and slashes indicate the boundaries between the variable, Fc, and constant domains. The scFv domain has a VH-scFv linker-VL orientation (N- to C-terminus), although this can be reversed. Note that the scFv domain sequence contains the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 796) as the scFv linker between the variable heavy and variable light regions; however, this linker can be replaced with any of the scFv linkers in Figure 6. Note also that the chain 2 sequence contains the sequence GGGGSGGGGSKTHTCPPCP (SEQ ID NO: 818), a "flex half-hinge" domain linker, as the domain linker between the C-terminus of the scFv and the N-terminus of the CH2 domain; however, this linker can be replaced with any of the "useful domain linkers" in Figure 7. Note that αB7H3xαCD28 bsAbs can utilize variable regions, Fc regions, and constant domains that are 90, 95, 98, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Additionally, each sequence outlined herein can include or exclude the M428L / N434S variant in one or preferably both Fc domains, which confers a longer half-life in serum. [Figure 37] The sequence of an exemplary αB7H3×αCD28 bsAb in a 1+1 CLC format is shown. CDRs are underlined, and slashes indicate the boundaries between the variable region, linker, Fc region, and constant domain. Note that αB7H3×αCD28 bsAbs can utilize variable regions, Fc regions, and constant domains that are 90, 95, 98, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Additionally, each sequence outlined herein can include or exclude the M428L / N434S variant in one or, preferably, both Fc domains, which confers a longer half-life in serum. [Figures 38A-38E] The sequence of an exemplary αB7H3×αCD28 bsAb in a 2+1 CLC format is shown. CDRs are underlined, and slashes indicate the boundaries between the variable region, Fc region, and constant domain. The scFv domains have a VH-scFv linker-VL orientation (N- to C-terminus), although this can be reversed. Note that the chain 2 sequence contains the sequence EPKSCGKPGSGKPGS (SEQ ID NO: 1182) as the domain linker (double underlined); however, this linker can be replaced with any domain linker, including any of the "useful domain linkers" in Figure 6. Note that αB7H3×αCD28 bsAbs can utilize variable regions, Fc regions, and constant domains that are 90, 95, 98, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein can include or exclude the M428L / N434S variant in one or preferably both Fc domains, which confers a longer half-life in serum. [Figures 39A-39C]The sequence of an exemplary αB7H3×αCD28 bsAb in a 2+1 mAb-scFv format is shown. CDRs are underlined, and slashes indicate the boundaries between the variable region, Fc region, and constant domain. The scFv domains have a VH-scFv linker-VL orientation (N- to C-terminus), although this can be reversed. Note that the chain 2 sequence contains the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 796) as a domain linker; however, this linker can be replaced with any domain linker, including any of the "useful domain linkers" in Figure 6. Note that αB7H3×αCD28 bsAbs can utilize variable regions, Fc regions, and constant domains that are 90, 95, 98, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein can include or exclude the M428L / N434S variant in one or preferably both Fc domains, which confers a longer half-life in serum. [Figure 40A-40B] Combining a CD3 bispecific antibody with a CD28 bispecific antibody replicates A) and B) classical T cell / APC interactions. In classical T cell / APC interactions, there is a first signal (signal 1) provided by TCR reactivity with peptide-MHC, and a second signal (signal 2) provided by CD28 cross-linking by CD80 / CD86 expressed on the APC, which together fully activate the T cell. In contrast, treatment with a CD3 bispecific provides only the first signal. A CD28 signal can be provided by a CD28 bispecific, with the idea being to promote activation and proliferation through CD28 costimulation. [Figure 41] 1 shows induction of CD28 signaling by a CD28 bispecific antibody and relief of any checkpoint-mediated suppression of the added CD28 signal by checkpoint blockade (e.g., PD-1 blockade). [Figure 42]Figure 1 shows induction of IL-2 release by effector cells in the presence of MCF7 cancer cells transfected with anti-CD3 scFv (effector:target ratio 1:1) and B7H3xCD3 bsAbs XENP34339, XENP35612, XENP35611, and XENP34336 (with CD28 binding affinities of 77 nM, 270 nM, 610 nM, and 440 nM, respectively). The data demonstrate that decreasing CD28 binding affinity reduces the potency of B7H3xCD28 bispecific antibodies. [Figures 43A-44C] Shown is the induction of IL-2 secretion from T cells by B7H3xCD28 bsAb in the presence of A) MDA-MB-231, B) LnCAP, and C) DU145 target cells (E:T ratio 1:1) and a fixed dose of an exemplary B7H3xCD3 bsAb. [Figure 44] Consensus framework regions (FR) and complementarity determining regions (CDR) (as in Kabat) of anti-CD28 clone 1A7 variable heavy and variable light domain variants are shown. [Figures 45A-45D] Figure 1 shows the pharmacokinetics of B7H3xCD28 bsAb in various antibody formats in a cynomolgus monkey study. The data indicate that the 2+1 common light chain format had the best half-life and pharmacokinetics at each dose level investigated. [Figures 46A-46H] Figure 1 shows the change in serum concentration levels over time in cynomolgus monkeys administered A) XENP34398, B) XENP37808, C) XENP37810, D) XENP34732, E) XENP35151, F) XENP351535, G) XENP37807, and H) XENP37982. Relative doses are represented as follows: circle, 0.5x dose; inverted triangle, 1.3x dose; hexagon, 1.8x dose; square, 2x dose; diamond, 3.25x dose; star, 4.5x dose; triangle, 5x dose. [Figure 47]Summary of the properties of B7H3xCD28 bsAbs XENP34398, XENP37808, XENP34732, and XENP35153. Please note that some of the data presented in this summary table represents experimental data from early stages of development, and therefore some of such data may not be the same as experimental data presented elsewhere in the Examples. [Figure 48A-48B] Figure 1 shows IFNγ release after incubation of A) A549 cancer cells and B) SKOV-3 cancer cells and CD3+ T cells (effector:target ratio of 10:1) with the indicated concentrations of the B7H3xCD28 bispecific antibody XENP34339 or XENP34717. The data show that both XENP34339 and XENP34717 induced cytokine release by T cells. XENP34339, which has bivalent B7H3 binding, induced cytokine release more potently than XENP34717, which has monovalent B7H3 binding. [Figure 49] Restoration of CD28 signaling in a mixed lymphocyte reaction (following incubation with 1 μg / mL of CTLA-4-Fc) by XENP34339. Error bars represent the average expression in culture supernatants from one MLR reaction tested in technical quadruplicate. [Figure 50A-50B] Figure 1 shows IFNγ release after incubation of NLV-loaded MDA-MB-231 cancer cells and CD3+ T cells purified from A) the first donor and B) the second donor at a 10:1 effector:target ratio with the indicated combinations of XENP16432, XENP34339, and XENP34389. The data show that incubation with XENP34339 alone induces cytokine release from T cells and synergistically combines with PD-1 blockade to enhance cytokine release. [Figure 51]Figure 1 shows the proliferation of NLV tetramer-positive cells after incubation of NLV-loaded MCF7 cancer cells and purified CD3+ T cells at an effector:target ratio of 10:1 with the indicated combinations of XENP16432 and XENP34339. The data show that the combination of XENP34339 and PD-1 blockade promoted the proliferation of NLV tetramer-positive CD8+ T cells. [Figure 52] Dissociation constants (KD, and corresponding sensorgrams) of anti-B7H3 clone 2E4A3.189 and clone 6A1 for the complete B7H3 extracellular V1C1-V2V2 domain or individual V1C1 or V2C2 domains are shown. [Figure 53] The dissociation constant (KD, and corresponding sensorgram) of anti-CD28 clone 1A7 affinity variant H1.14_L1 is shown as Fab in 2+1 CLC format to CD28 antigen or as scFv format in 2+1 Fab2-scFv. [Figure 54A-54B] Figure 1 shows the sequence of an exemplary αPSMAxαCD3 bsAb, shown in a 2+1 Fab2-scFv-Fc format and including the H1.30_L1.47 anti-CD3 scFv (also known as CD3 High [VHVL]). CDRs are underlined, and slashes indicate boundaries between the variable region and other chain components (e.g., constant regions and domain linkers). Note that αPSMAxαCD3 bsAbs can utilize variable regions, Fc regions, and constant domains that are 90, 95, 98, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Additionally, each sequence outlined herein can include or exclude the M428L / N434S variant in one or, preferably, both Fc domains, which confers a longer half-life in serum. [Figure 55]Figure 1 shows cell killing over time following incubation of LNCaP cancer cells (PSMA+B7H3+) and CD3+ T cells at a 1:1 effector:target ratio with the indicated concentrations of an exemplary CD3 bispecific (αPSMA×αCD3 XENP31602) alone or in combination with XENP34339. The data show that XENP31602 αPSMA×αCD3 alone minimally enhances cell killing compared to incubation of cancer and T cells alone. The addition of XENP34339 αB7H3×αCD28 overcomes cancer cell resistance to the CD3 bispecific. [Figures 56A-56I]
[0039] Figure 1 shows the sequence of an exemplary anti-CD3 binding domain suitable for use in a CD3 bispecific antibody that can be combined with the CD28 bispecific antibody of the present invention. The CDRs are underlined, the scFv linker is double underlined (in the sequence, the scFv linker is a positively charged scFv(GKPGS)4 linker (SEQ ID NO: 796), although one of skill in the art will understand that this linker can be replaced with other linkers, including uncharged or negatively charged linkers, some of which are shown in Figure 6), and the slash indicates the boundary of the variable domain. Additionally, the nomenclature indicates the orientation of the scFv from N-terminus to C-terminus. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 2; therefore, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figures 57A-57C] Shown are A) IFNγ release, B) IL-2 release, and C) CD3+ T cell proliferation following incubation of LNCaP cancer cells (PSMA+B7H3+) and CD3+ T cells with 1 μg / ml XENP34339 at a 1:1 effector:target ratio in combination with dose titration of an exemplary CD3 bispecific (αPSMA×αCD3 XENP31602). [Figures 58A-58C]Shown are A) IFNγ release, B) IL-2 release, and C) CD3+ T cell proliferation following incubation of 22Rv1 cancer cells (PSMA+B7H3+) and CD3+ T cells with 1 μg / ml XENP34339 at a 1:1 effector:target ratio in combination with a dose titration of an exemplary CD3 bispecific (αPSMA×αCD3 XENP31602). [Figures 59A-59C] Shown are A) IFNγ release, B) IL-2 release, and C) CD3+ T cell proliferation following incubation of SKOV-3 cancer cells (PSMA-B7H3+) and CD3+ T cells with 1 μg / ml XENP34339 at a 1:1 effector:target ratio in combination with dose titration of an exemplary CD3 bispecific (αPSMA×αCD3 XENP31602). [Figures 60A-60C] Shown are A) IFNγ release, B) IL-2 release, and C) CD3+ T cell proliferation following incubation of OVCAR-8 cancer cells (PSMA-B7H3+) and CD3+ T cells with 1 μg / ml XENP34339 at a 1:1 effector:target ratio in combination with dose titration of an exemplary CD3 bispecific (αPSMA×αCD3 XENP31602). [Figures 61A-61E] Figure 1 shows the change in tumor volume over time (in days) for individual mice in pp65-MDA-MB-231 and huPBMC-engrafted NSG mice treated with A) the first exemplary B7H3xCD3 bispecific antibody (CD3bsAb1) (0.5 mg / kg) alone, B) the second exemplary B7H3xCD3 bispecific antibody (CD3bsAb2) (0.5 mg / kg) alone, C) XENP34339 (5.0 mg / kg) in combination with CD3bsAb1 (0.5 mg / kg), D) XENP34339 (5.0 mg / kg) in combination with CD3bsAb2 (0.5 mg / kg), or E) PBS (determined by caliper measurement, baseline corrected). [Figure 62]1 shows the group median change in tumor volume over time (in days) in pp65-MDA-MB-231 and huPBMC-engrafted NSG mice treated with a first exemplary B7H3xCD3 bispecific antibody (CD3bsAb1) (0.5 mg / kg) alone, a second exemplary B7H3xCD3 bispecific antibody (CD3bsAb2) (0.5 mg / kg) alone, the combination of XENP34339 (5.0 mg / kg) and CD3bsAb1 (0.5 mg / kg), the combination of XENP34339 (5.0 mg / kg) and CD3bsAb2 (0.5 mg / kg), or a PBS control (determined by caliper measurement, baseline corrected). [Figure 63] 1 shows CD45+ cell counts in the blood of pp65-MDA-MB-231 and huPBMC-engrafted NSG mice administered a first exemplary B7H3xCD3 bispecific antibody (CD3bsAb1) (0.5 mg / kg) alone, a second exemplary B7H3xCD3 bispecific antibody (CD3bsAb2) (0.5 mg / kg) alone, the combination of XENP34339 (5.0 mg / kg) and CD3bsAb1 (0.5 mg / kg), the combination of XENP34339 (5.0 mg / kg) and CD3bsAb2 (0.5 mg / kg), or a PBS control 14 days after the first dose. [Figures 64A-64E] Figure 1 shows proliferation of A) CD45+, B) CD4+ (total), C) CD8+ (total), D) CD4+ (Ki67+), and E) CD8+ (Ki67+) cells (shown as cell counts) in the blood of 22RV1 and huPBMC-engrafted NSG-DKO mice treated with low or high doses of the exemplary PSMAxCD3 bsAb XENP32220 alone or in combination with XENP34339. Treatment with both the CD3 bsAb and CD28 bsAb enhanced T cell proliferation compared to treatment with the CD3 bsAb alone. [Figures 65A-65E]Figure 1 shows A) CD4+ cell activation (indicated by CD25 expression), B) CD4+ cell activation (indicated by PD1 expression), C) CD8+ cell activation (indicated by CD25 expression), and D) CD8+ cell activation (indicated by PD1 expression) in the blood of 22RV1 and huPBMC-engrafted NSG-DKO mice administered low or high doses of the exemplary PSMAxCD3 bsAb XENP32220 alone or in combination with XENP34339. Treatment with both the CD3 and CD28 bsAb enhanced T cell activation compared to treatment with the CD3 bsAb alone. [Figure 66] Shown is the group median change in tumor volume over time (in days) in pp65-MDA-MB-231-implanted CD34+ Hu-NSG mice treated with an exemplary B7H3xCD3 bispecific antibody (0.5 mg / kg) alone, XENP35612 alone (1 mg / kg) alone, XENP34339 (0.3 mg / kg) in combination with B7H3xCD3 bsAb (0.5 mg / kg), XENP35612 (1 mg / kg) in combination with B7H3xCD3 bsAb (0.5 mg / kg), or PBS control (determined by caliper measurement, baseline corrected). [Figure 67A-67B] Figure 1 shows baseline-corrected tumor volumes on A) day 6 and B) day 9 (post-treatment) in pp65-MDA-MB-231-implanted CD34+ Hu-NSG mice treated with an exemplary B7H3xCD3 bispecific antibody (0.5 mg / kg) alone, XENP35612 (1 mg / kg) alone, XENP34339 (0.3 mg / kg) in combination with B7H3xCD3 bsAb (0.5 mg / kg), XENP35612 (1 mg / kg) in combination with B7H3xCD3 bsAb (0.5 mg / kg), or PBS control. Statistics performed on baseline-corrected data using a Mann-Whitney test. [Figure 68A-68B]Figure 1 shows proliferation of A) CD45+ and B) CD8+ cells in tumors of pp65-MDA-MB-231-implanted CD34+ Hu-NSG mice treated with an exemplary B7H3xCD3 bispecific antibody (0.5 mg / kg) alone, XENP35612 alone (1 mg / kg) alone, the combination of XENP35612 (1 mg / kg) and B7H3xCD3 bsAb (0.5 mg / kg), or a PBS control. Statistics performed on log-transformed data using an unpaired t-test. [Figure 69] The sequence of XENP29154, an in-house produced TGN1412, is shown. [Figures 70A-70C] Shown is the release of A) IFNγ, B) IL-6, and C) TNFα from air-dried human PBMCs treated with XENP34339, TGN1412 (XENP29154), or negative control PBS. [Figures 71A-71C] Shown is the release of A) IFNγ, B) IL-2, and C) TNFα from air-dried human PBMCs treated with XENP37808, TGN1412 (XENP29154), or negative control PBS. [Figure 72A-72B] Figure 1 shows the induction of IL-2 release by A) PBMCs from human donors or B) PBMCs from cynomolgus donors by XENP37808 in the presence of HEK cells transfected with αCD3 scFv (with or without B7H3 knockout). [Figure 73A-73B] Figure 1 shows the induction of RTCC on A) 22RV-NLR (with approximately 170K B7H3 antigen density) and B) DU145-NLR (with approximately 270K B7H3 antigen density) target cells with B7H33 x CD3 mAb alone or in combination with either XENP34398 or XENP37808. The data show that XENP34398 and XENP37808 (in combination with B7H3 x CD3) induce very similar levels of RTCC. [Figures 74A-74C]Shown is the induction of IL-2 release by T cells in the presence of XENP34398 or XENP37808 in combination with A) OVCAR8 (with approximately 20K B7H3 antigen density), B) 22RV1-NLR (with approximately 170K B7H3 antigen density), and C) DU145-NLR (with approximately 270K B7H3 antigen density), and B7H3xCD3 bsAb. [Figure 75] Consensus framework regions (FR) and complementarity determining regions (CDR) (as in Kabat) of anti-B7H3 clone 2E4A3.189 variable heavy and variable light domain variants are shown. DETAILED DESCRIPTION OF THE INVENTION
[0134] I. Overview T cell activation in cancer treatment has been extensively studied. T cells require multiple signals for full activation and differentiation. As shown in Figure 40, signal 1, promoted by recognition of peptide-MHC (pMHC) complexes by the T cell receptor (TCR), is absolutely required for T cell activation. Signal 2, which synergizes with and amplifies signal 1, is typically provided by the interaction of CD28 itself with the CD28 ligands CD80 and CD86. While CD28 engagement alone is typically inactive, its combination with signal 1 activation promotes further activation, survival, and proliferation signals, including IL-2 secretion. Because CD80 and CD86 are naturally expressed only by professional antigen-presenting cells (APCs), the degree of CD28 costimulation in the tumor environment can be highly variable. Thus, the present invention is directed to a novel class of tumor-targeting CD28 bispecific antibodies (including B7H3xCD28, described more fully herein) that can mimic CD80 / CD86 engagement of CD28 to provide an artificial source of signal 2. In particular, the signal can be provided by natural TCR:pMHC recognition of tumor cells or by a combination of CD28 bispecific and CD3 bispecific (which can mimic signal 1).
[0135] Accordingly, provided herein are novel anti-CD28 x anti-B7H3 (also referred to as "αCD28 x αB7H3" and sometimes "CD28 x B7H3") bispecific antibodies and methods of using such antibodies for the treatment of cancer. Often, these bispecific antibodies are heterodimers. The subject αCD28 x αB7H3 antibodies can agonistically bind to the CD28 costimulatory molecule on T cells and target B7H3 on tumor cells. Thus, such antibodies selectively enhance anti-tumor activity at the tumor site while minimizing peripheral toxicity. The subject antibodies provided herein are particularly useful for enhancing anti-tumor activity when used alone, as monotherapy, or in combination with other anti-cancer therapies, as more fully described herein.
[0136] Thus, in one aspect, provided herein are heterodimeric antibodies that bind to two different antigens; e.g., the antibodies are "bispecific" in that they bind to two different target antigens, generally CD28 and B7H3, as described herein. These heterodimeric antibodies can bind to each of the target antigens either monovalently (e.g., with a single antigen-binding domain, such as a pair of variable heavy and variable light domains) or bivalently (with two antigen-binding domains, each of which independently binds to an antigen). In some embodiments, the heterodimeric antibodies provided herein comprise one CD28-binding domain and one B7H3-binding domain (e.g., a heterodimeric antibody in the "1+1 Fab-scFv-Fc" format described herein, which is therefore bispecific and bivalent). In other embodiments, the heterodimeric antibodies provided herein comprise one CD28 binding domain and two B7H3 binding domains (e.g., a heterodimeric antibody in the "2+1 Fab2-scFv-Fc" format described herein, which is therefore bispecific, but trivalent because it contains three antigen binding domains (ABDs). The provided heterodimeric antibodies are based on the use of distinct monomers that contain amino acid substitutions (i.e., "scubariant") that "skew" heterodimer formation on the homodimer, as outlined more fully below. In some embodiments, the heterodimeric antibodies are also linked to "pI variants" that allow for simple purification of the heterodimer away from the homodimer, as also outlined below. The provided heterodimeric bispecific antibodies generally rely on the use of engineered or variant Fc domains that can self-assemble in the production cell to produce the heterodimeric protein, and methods for generating and purifying such heterodimeric proteins.
[0137] II. Nomenclature The antibodies provided herein are listed in several different formats. In some cases, each monomer of a particular antibody is given a unique "XENP" number, although longer sequences may contain shorter ones, as understood in the art. For example, the "scFv-Fc" monomer of a 1+1 Fab-scFv-Fc format antibody may have a first XENP number, but the scFv domain itself has a different XENP number. Because some molecules have three polypeptides, the XENP number is used in conjunction with the components to designate them. Thus, the 2+1 Fab2-scFv-Fc format molecule XENP34389 contains three sequences (see Figure 28A): 1) a "Fab-Fc heavy chain" monomer; 2) a "Fab-scFv-Fc heavy chain" monomer; and 3) a "light chain" monomer, or equivalents, which one of skill in the art would readily identify through sequence alignment. These XENP numbers are found in the sequence listing as well as in the identifiers and figures. Additionally, a single molecule containing three components generates multiple sequence identifiers. For example, a Fab listing includes the complete heavy chain sequence, the variable heavy chain domain sequence, and three CDRs of the variable heavy chain domain sequence, as well as the complete light chain sequence, the variable light chain domain sequence, and three CDRs of the variable light chain domain sequence. A Fab-scFv-Fc monomer includes the full-length sequence, the variable heavy chain domain sequence, three heavy chain CDR sequences, and an scFv sequence (including the scFv variable heavy chain domain sequence, the scFv variable light chain domain sequence, and the scFv linker). Note that while a single charged scFv linker (+H) is used in some molecules containing scFv domains herein, others can be used. Additionally, the nomenclature for specific antigen-binding domains (e.g., B7H3 and CD28 binding domains) uses a format of "Hx.xx_Ly.yy," where the numbers are unique identifiers for the specific variable chain sequences. Thus, the variable domain on the Fab side of B7H3 binding domain 6A [B7H3] (e.g., Figure 28A) is "H1_L1," indicating that the variable heavy chain domain H1 is combined with the light chain domain L1.When these sequences are used as scFvs, the designation "H1_L1" indicates that the variable heavy chain domain H1 is combined with the light chain domain L1, in a VH-linker-VL orientation going N to C-terminus. A molecule with the same sequences of heavy and light variable domains but in the reverse order (VL-linker-VH orientation, going N to C-terminus) would be designated "L1_H1". Similarly, different constructs can be "mix and match" heavy and light chains, as will be apparent from the sequence listing and figures.
[0138] Additionally, with respect to the sequence listing, SEQ ID NOS: 1-88 correspond to the antigen-binding domain previously shown in Figure 17 of USSN 63 / 092,272, and SEQ ID NOS: 89-496 correspond to the antigen-binding domain previously shown in Figure 24 of USSN 63 / 092,272. Additionally, SEQ ID NOS: 497-584 are all variant variable heavy chain domains of the 2E4A3.189[B7H3] parent antibody, all of which find use in the present invention, as outlined more fully below. SEQ ID NOS: 585-651 are all variant variable heavy chain domains of the 1A7[CD28] parent antibody, all of which find use in the present invention. SEQ ID NOS: 652-756 are all variant variable light chain domains of the 1A7[CD28] parent antibody, all of which find use in the present invention.
[0139] III. Definition In order to facilitate a more complete understanding of this application, certain definitions are provided below. Such definitions are intended to encompass grammatical equivalents.
[0140] As used herein, "CD28," "cluster of differentiation 28," and "Tp44" (e.g., Genebank accession numbers NP_001230006 (human), NP_001230007 (human), NP_006130 (human), and NP_031668 (mouse)) refer to the B7 receptor expressed on T cells, which provides costimulatory signals necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) provides a potent signal for the production of various interleukins. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins. CD28 contains an intracellular domain with a YMNM motif that is important for the recruitment of SH2 domain-containing proteins, particularly PI3K. CD28 also contains two proline-rich motifs that can bind to SH3-containing proteins. An exemplary CD28 sequence is shown in Figure 1. Unless otherwise stated, references to CD28 are to the human CD28 sequence.
[0141] As used herein, "B7H3," "B7-H3," "B7RP-2," "CD276," and "cluster of differentiation 276" (e.g., Genebank accession numbers NP_001019907 (human), NP_001316557 (human), NP_001316558 (human), NP_079516 (human), and NP_598744 (mouse)) refer to a type 1 transmembrane protein that is a member of the B7 family and possesses an ectodomain composed of a single IgV-IgC domain pair. B7H3 is an immune checkpoint molecule that is aberrantly overexpressed in many types of cancer. An exemplary B7H3 sequence is shown in Figures 2A and B. Unless otherwise specified, references to B7H3 are to the human B7H3 sequence.
[0142] As used herein, "elimination" refers to reducing or eliminating activity. Thus, for example, "ablation of FcγR binding" means that an Fc region amino acid variant has less than 50% of the starting binding activity compared to an Fc region not containing the particular variant, preferably with 70-80-90-95-98% or more loss of activity, and generally with activity below detectable binding levels in Biacore, SPR, or BLI assays. Particularly useful in ablation of FcγR binding are those shown in Figure 5, which are generally added to both monomers.
[0143] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa results in increased ADCC activity.
[0144] As used herein, "ADCP" or "antibody-dependent cell-mediated phagocytosis" refers to a cell-mediated reaction in which non-specific phagocytes expressing FcγR recognize bound antibody on target cells and subsequently cause phagocytosis of the target cells.
[0145] As used herein, the term "antibody" is used generically. The antibodies provided herein can take several formats, as described herein, including traditional antibodies as well as antibody derivatives, fragments, and mimetics.
[0146] Conventional immunoglobulin (Ig) antibodies are "Y" shaped tetramers. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair consisting of one "light" chain monomer (typically having a molecular weight of about 25 kDa) and one "heavy" chain monomer (typically about 5 kDa). and having a molecular weight of 0 to 70 kDa.
[0147] Other useful antibody formats include, but are not limited to, the "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" formats provided herein (see, e.g., Figure 33). Additional useful antibody formats include, but are not limited to, antibodies in the "mAb-Fv," "mAb-scFv," "central-Fv," "one-arm scFv-mAb," "scFv-mAb," "dual-scFv," and "trident" formats, as disclosed in US20180127501A1, which is incorporated herein by reference, particularly the relevant portions relating to antibody formats (e.g., Figure 2 of US20180127501A1).
[0148] An antibody heavy chain typically comprises a variable heavy (VH) domain comprising vhCDR1-3 and an Fc domain comprising CH2-CH3 monomers. In some embodiments, an antibody heavy chain comprises a hinge and a CH1 domain. A conventional antibody heavy chain is organized from N- to C-terminus as monomers: VH-CH1-hinge-CH2-CH3. The CH1-hinge-CH2-CH3 are collectively referred to as the heavy chain "constant domain" or "constant region," although there are five different categories or "isotypes" of antibodies: IgA, IgD, IgG, IgE, and IgM.
[0149] In some embodiments, the antibodies provided herein comprise an IgG isotype constant domain, with several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The IgG subclass of immunoglobulins has several immunoglobulin domains in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin with a distinct tertiary structure. Heavy chain domains comprising the constant heavy (CH) domain and the hinge domain are of interest in the present invention. In the context of IgG antibodies, IgG isotypes each have three CH regions. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat; "hinge" refers to positions 216-230 according to the EU index as in Kabat; "CH2" refers to positions 231-340 according to the EU index as in Kabat; and "CH3" refers to positions 341-447 according to the EU index as in Kabat. The exact numbering and arrangement of heavy chain domains may vary in different numbering systems, as shown in Table 1. As shown herein and described below, pI variants may be in one or more CH regions, as well as in the hinge region, as discussed below.
[0150] It should be noted that IgG1 has different allotypes, with polymorphisms at 356 (D or E) and 358 (L or M). While the sequences presented herein use the 356E / 358M allotype, other allotypes are also encompassed herein. That is, any sequence comprising an IgG1 Fc domain included herein can have the 356D / 358L allotype instead of the 356E / 358M allotype. It should be understood that therapeutic antibodies can also comprise isotype and / or subclass hybrids. For example, as shown in U.S. Patent Application Publication No. 2009 / 0163699, incorporated by reference, the present antibodies in some embodiments comprise an IgG1 / G2 hybrid.
[0151] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody, in some cases excluding all or a portion of the first constant region immunoglobulin domain (e.g., CH1), and in some cases optionally including all or a portion of the hinge. In the case of an IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally CH1 (Cγ1) and The Fc domain may comprise all or part of the hinge region between CH2 (Cγ2). Thus, in some cases, an Fc domain comprises, from N- to C-terminus, CH2-CH3 and hinge-CH2-CH3. In some embodiments, the Fc domain is 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 a human IgG1 Fc domain, the hinge may comprise a C220S amino acid substitution. Further, in the case of a human IgG4 Fc domain, the hinge may comprise 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 at its carboxy-terminus, where numbering is according to the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region to, for example, alter binding to one or more FcγRs or FcRn, as described in more detail below.
[0152] As used herein, "heavy chain constant region" refers to the CH1-hinge-CH2-CH3 portion of an antibody (or a fragment thereof) excluding the variable heavy domain, which in the EU numbering system for human IgG1 corresponds to amino acids 118 to 447. As used herein, "heavy chain constant region fragment" refers to a heavy chain constant region having fewer amino acids from either or both of the N-terminus and C-terminus, but which still retains the ability to form a dimer with another heavy chain constant region.
[0153] Another type of domain in a heavy chain is the hinge region. As used herein, "hinge" or "hinge region" or "antibody hinge region" or "hinge domain" refers to the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, IgG The 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 defined herein to include positions 216 (E216 in IgG1) to 230 (P230 in IgG1), numbered according to the EU index as in Kabat. In some cases, "hinge fragments" are used, which contain fewer amino acids at either or both the N- and C-termini of the hinge domain. As described herein, pI variants can also be made in the hinge region. Many of the antibodies herein have at least one cysteine at position 220 according to EU numbering (hinge region) replaced with serine. Generally, this modification is on the "scFv monomer" side for most of the sequences shown herein (when a 1+1 or 2+1 format is used), but can also be on the "Fab monomer" side, or both, to reduce disulfide formation. Specifically included within the sequences herein are those in which one or both of these cysteines are replaced (C220S).
[0154] As will be appreciated by those skilled in the art, the exact numbering and arrangement of heavy chain constant region domains (i.e., CH1, hinge, CH2, and CH3 domains) may vary among different numbering systems. A useful comparison of EU and Kabat heavy constant region numbering is as follows: 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, which are incorporated by reference in their entireties. [Table 1]
[0155] Antibody light chains generally contain two domains: a variable light domain (VL) containing the light chain CDRs v1CDR1-3, and a constant light domain (often referred to as CL or Cκ). Antibody light chains are usually organized from N- to C-terminus: VL-CL.
[0156] As used herein, the term "antigen-binding domain" or "ABD," when present as part of a polypeptide sequence, refers to a set of six complementarity-determining regions (CDRs) that specifically bind to a target antigen (e.g., B7H3 or CD28) as discussed herein. As known in the art, these CDRs generally exist as a first set of variable heavy chain CDRs (vhCDRs or VH CDRs) and a second set of variable light chain CDRs (vlCDRs or VL CDRs), each of which contains three CDRs: vhCDR1, vhCDR2, and vhCDR3, the variable heavy chain CDRs, and vlCDR1, vlCDR2, and vlCDR3, the variable light chain CDRs. The CDRs are present in the variable heavy chain domain (vhCDR1-3) and the variable light chain domain (vlCDR1-3). The variable heavy chain domain and the variable light chain domain from the Fv region.
[0157] The present invention provides a number of different CDR sets. In this case, a "complete CDR set" comprises three variable light chain CDRs and three variable heavy chain CDRs, e.g., vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These may be part of a larger variable light chain or variable heavy chain domain, respectively. In addition, as more fully outlined herein, the variable heavy and variable light chain domains may be on separate polypeptide chains when heavy and light chains are used (e.g., when Fabs are used), or on a single polypeptide chain in the case of scFv sequences.
[0158] As will be understood by those skilled in the art, the exact numbering and arrangement of CDRs may vary in different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light chain sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable heavy region is the disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is the disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). A useful comparison of CDR numbering follows; see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003). [Table 2]
[0159] Throughout this specification, the Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 for light chain variable regions and residues 1-113 for heavy chain variable regions), and the EU numbering system is for the Fc region (see, e.g., Kabat et al., supra (1991)).
[0160] CDRs contribute to antigen binding, or more specifically, to the formation of the antigen-binding domain and epitope-binding site of an antibody. "Epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. An epitope is a grouping of molecules, such as amino acids or sugar side chains, that usually has specific structural characteristics as well as specific charge characteristics. A single antigen may have two or more epitopes.
[0161] An epitope may include amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked by the specific antigen-binding peptide, in other words, amino acid residues that lie within the footprint of the specific antigen-binding peptide.
[0162] Epitopes may be either conformational or linear. Conformational epitopes are generated by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues within a polypeptide chain. Conformational and nonconformational epitopes can be distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.
[0163] An epitope typically comprises at least 3, 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 demonstrating the ability of one antibody to block the binding of another antibody to a target antigen, e.g., "binning." As outlined below, the present invention includes not only the recited antigen-binding domains and antibodies herein, but also those that compete for binding to the epitope bound by the recited antigen-binding domains.
[0164] In some embodiments, the six CDRs of an antigen-binding domain are contributed by a variable heavy chain and a variable light chain domain. In the "Fab" format, the set of six CDRs is provided by two different polypeptide sequences, a variable heavy chain domain (vh or VH; containing vhCDR1, vhCDR2, and vhCDR3) and a variable light chain domain (vl or VL; containing vlCDR1, vlCDR2, and vlCDR3), with the C-terminus of the vh domain attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain attached to the N-terminus of the constant light chain domain. (thereby forming a light chain). In the scFv format, the vh and vl domains are generally covalently linked into a single polypeptide sequence by the use of a linker ("scFv linker"), as outlined herein, which (starting from the N-terminus) can be either vh-linker-vl or vl-linker-vh, with the former generally preferred (including optional domain linkers on either side, depending on the format used). Generally, the C-terminus of the scFv domain is linked to the N-terminus of all or part of the hinge of the second monomer.
[0165] As used herein, a "variable region" or "variable domain" refers to a region of an immunoglobulin that includes one or more Ig domains substantially encoded by any of the Vκ, Vλ, and / or VH genes that constitute the kappa, lambda, and heavy chain immunoglobulin 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 contains three hypervariable regions ("complementarity-determining regions," "CDRs") (vhCDR1, vhCDR2, and vhCDR3 in the variable heavy chain domain and vlCDR1, vlCDR2, and vlCDR3 in the variable light chain domain) and four framework (FR) regions, arranged from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0166] As used herein, "Fab" or "Fab region" refers to an antibody region that generally comprises the VH, CH1, VL, and CL immunoglobulin domains of two different polypeptide chains (e.g., VH-CH1 of one chain, VL-CL of the other chain). Fab can refer to this region alone or in the context of a bispecific antibody of the invention. In the context of Fab, Fab includes the Fv region in addition to the CH1 and CL domains.
[0167] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to the antibody region comprising the VL and VH domains. The Fv region can be formatted as both a Fab (which, as noted above, are two distinct polypeptides that also generally comprise a constant region as outlined above) and a single-chain Fv (scFv), in which the vl and vh domains are generally comprised in a single peptide chain joined by a linker as discussed herein.
[0168] As used herein, "single-chain Fv" or "scFv" generally refers to a variable heavy domain covalently linked to a variable light domain, forming an scFv or scFv domain using an scFv linker as discussed herein. The scFv domain can be in either orientation (vh-linker-vl or vl-linker-vh) from N- to C-terminus. In the sequences shown in the sequence listing and figures, the order of the vh and vl domains is indicated in the name. For example, H.X_L.Y means, from N- to C-terminus, vh-linker-vl, and L.Y_H.X means vl-linker-vh.
[0169] Some embodiments of the subject antibodies provided herein comprise at least one scFv domain, which, while not naturally occurring, generally comprises a variable heavy domain and a variable light domain linked together by an scFv linker. As outlined herein, the scFv domains are generally oriented N- to C-terminus as VH-scFv linker-VL, although this can be reversed for any scFv domain (or domain constructed using vh and vl sequences from a Fab) to VL-scFv linker-VH, with optional linkers at one or both ends depending on the format.
[0170] As used herein, "modification" or "variant" refers to amino acid substitutions, insertions, and / or deletions in a polypeptide sequence, or to moieties chemically linked to a protein. For example, the modification may be a modified carbohydrate or PEG structure attached to the protein. As used herein, an "amino acid modification" refers to a substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence. For clarity, unless otherwise specified, an amino acid modification is always to an amino acid encoded by DNA, e.g., the 20 amino acids for which codons exist in DNA and RNA.
[0171] As used herein, an "amino acid substitution" or "substitution" refers to 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 for an amino acid that does not naturally occur (either does not occur naturally in an organism or does not occur in any organism) at the particular position. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which glutamic acid at position 272 has been replaced with tyrosine. For clarity, a protein that has been engineered to change the nucleic acid coding sequence but not the starting amino acid (e.g., replacing CGG (which encodes arginine) with CGA (which still encodes arginine) to increase host organism expression levels) is not an "amino acid substitution." That is, if a protein has the same amino acid at the particular position where it starts, despite the creation of a new gene encoding the same protein, it is not an amino acid substitution.
[0172] As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Additionally, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.
[0173] As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233#, E233() or E233del indicates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla beginning at position 233.
[0174] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that differs from that of a parent protein based on at least one amino acid modification. A protein variant has at least one amino acid modification compared to the parent protein, but not so many that the variant protein does not align with the parent protein using an alignment program such as those described below. Generally, the variant proteins outlined herein (such as variant Fc domains) are generally at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the parent protein using an alignment program such as BLAST, as described below.
[0175] "Variant" as used herein also refers to particular amino acid modifications that confer particular functions (e.g., "heterodimerization variants," "pI variants," "deletion variants," etc.).
[0176] As described below, in some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, is a human wild-type sequence, such as a heavy chain constant domain or Fc region from IgG1, IgG2, IgG3, or IgG4, although human sequences with variants can also serve as "parent polypeptides," including, for example, the IgG1 / 2 hybrids of U.S. Publication No. 2006 / 0134105. The sequences of the protein variants herein preferably share at least about 80% identity with the parent protein sequence, most preferably about 100% identity with the parent protein sequence. having at least about 90% identity, more preferably at least about 95-98-99% identity. Thus, as used herein, "antibody variant" or "variant antibody" refers to an antibody that differs from a parent antibody by at least one amino acid modification; as used herein, "IgG variant" or "variant IgG" refers to an antibody that differs from a parent IgG (again, often derived from a human IgG) by at least one amino acid modification; and as used herein, "immunoglobulin variant" or "variant immunoglobulin" refers to an immunoglobulin sequence that differs from that of the parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" refers to a protein that comprises an amino acid modification compared to the Fc domain of human IgG1, IgG2, or IgG4.
[0177] As used herein, "Fc variant" or "variant Fc" refers to a protein containing an amino acid modification in the Fc domain. The modification can be an addition, deletion, or substitution. Fc variants are defined according to the amino acid modification that constitutes them. Thus, for example, N434S or 434S is an Fc variant with a substituted serine at position 434 relative to the parent Fc polypeptide, where the numbering is according to the EU index. Similarly, M428L / N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acids may not be specified, in which case the variant is referred to as 428L / 434S. It is noted that the substitutions may be provided in any order; i.e., for example, 428L / 434S is the same Fc variant as 434S / 428L. For all positions discussed herein relating to antibodies or derivatives and fragments thereof (e.g., Fc domains), unless otherwise specified, the numbering of amino acid positions is according to the EU index. The "EU index" or "Kabat-like EU index" 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 incorporated by reference in its entirety). Modifications can be additions, deletions, or substitutions.
[0178] Generally, the variant Fc domain will have at least about 80, 85, 90, 95, 97, 98, or 99 percent identity to the corresponding parent human IgG Fc domain (using an identity algorithm discussed below (in one embodiment the BLAST algorithm known in the art) with default parameters). Alternatively, the variant Fc domain may have 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 relative to the parent Fc domain. Alternatively, the variant Fc domain may have 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 up to 20 amino acid modifications relative to the parent Fc domain. Additionally, as discussed herein, the variant Fc domains described herein still retain the ability to form dimers with another Fc domain as measured using known techniques described herein, such as non-denaturing gel electrophoresis.
[0179] As used herein, "protein" means at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. In addition, the polypeptides from which the antibodies of the invention are made can include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.
[0180] As used herein, "residue" refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297 or N297) is the residue at position 297 in the human antibody IgG1.
[0181] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different, matching IgG isotype. For example, because IgG1 contains tyrosine and IgG2 contains phenylalanine at EU position 296, an F296Y substitution in IgG2 is considered to be an IgG subclass modification.
[0182] As used herein, a "non-naturally occurring modification" refers to an amino acid modification that is not isotypic. For example, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered to be a non-naturally occurring modification because none of the human IgGs contain serine at position 434.
[0183] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.
[0184] As used herein, "effector function" refers to 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.
[0185] As used herein, "IgG Fc ligand" refers to any biologically derived molecule, preferably a polypeptide, 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, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors homologous to FcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, incorporated by reference in its entirety). Fc ligands may also include undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, "Fc ligand" refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.
[0186] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the 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), and any undiscovered human FcγR or FcγR isoform or allotype. FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.
[0187] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and β-2-microglobulin. Various FcRn variants can be used to increase binding to the FcRn receptor, and in some cases, to increase serum half-life. An "FcRn variant" refers to an amino acid modification that contributes to increased binding to the FcRn receptor. Suitable FcRn variants are listed below.
[0188] As used herein, a "parent polypeptide" refers to a starting polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a naturally occurring polypeptide or a variant or engineered version of a naturally occurring polypeptide. Thus, as used herein, a "parent immunoglobulin" refers to an unmodified immunoglobulin polypeptide that is modified to generate a variant, and a "parent antibody" refers to an unmodified antibody that is modified to generate a variant antibody. It should be noted that "parent antibody" includes known, commercially available, recombinantly produced antibodies, as outlined below. In this context, the "parent Fc domain" is 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 of human IgG4, etc.
[0189] As used herein, "position" means a location in the sequence of a protein. Positions may be numbered sequentially or according to established formats, such as the EU index for numbering antibody domains (e.g., CH1, CH2, CH3, or hinge domains).
[0190] "Target antigen," as used herein, means a molecule that is specifically bound by an antibody binding domain comprising the variable region of a given antibody.
[0191] "Strandedness" herein, in reference to the monomers of the heterodimeric antibodies of the invention, refers to the incorporation of heterodimerization variants into each monomer such that they retain the ability to "match" and form heterodimers, similar to "matching" double-stranded DNA. For example, if several pI variants are engineered into monomer A (e.g., to increase the pI), a similarly available "charge-paired" steric variant will not interfere with the pI variant; for example, the pI-increasing charge variant can be placed in the same "strand" or "monomer," retaining both functionalities. Similarly, for "skew" variants that result in paired sets, as outlined in more detail below, one skilled in the art will consider the pI when determining which strand or monomer one set of the pair will reside in; thus, pI separation is also maximized using the skewed pI.
[0192] As used herein, "target cell" means a cell that expresses a target antigen.
[0193] By "host cell" herein in the context of producing a bispecific antibody according to the invention is meant a cell that contains exogenous nucleic acid encoding the components of the bispecific antibody and that is capable of expressing the bispecific antibody under suitable conditions. Suitable host cells are described below.
[0194] As used herein, "wild-type or WT" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein is one that has not been intentionally modified. It has an amino acid sequence or a nucleotide sequence that does not exist.
[0195] Provided herein are several antibody domains (e.g., Fc domains) that share sequence identity with human antibody domains. Sequence identity between two similar sequences (e.g., antibody variable domains) can be determined using the local homology algorithm described by Smith, TF & Waterman, MS (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482; Needleman, SB & 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. (USA) 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, default parameters (window length, gap penalty, etc.) are used. In one embodiment, sequence identity is measured using the BLAST algorithm using default parameters.
[0196] Antibodies of the present invention are generally isolated or recombinant. "Isolated," as used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. "Recombinant" means that the antibody is produced using recombinant nucleic acid technology in an exogenous host cell, and may also be isolated.
[0197] "Specific binding" to a particular antigen or epitope, or "specifically binds to" or "specific for" a particular antigen or epitope, means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
[0198] Specific binding to a particular antigen or epitope is, for example, 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 A specific binding activity can be exhibited by an antibody having a KD of M or greater, where KD refers to the off-rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more greater than that of a control molecule relative to the antigen or epitope.
[0199] Specific binding to a particular antigen or epitope is also indicated by, for example, a K A or K A for the antigen or epitope that is at least 20-fold or 50-fold higher than that of a control. , 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more, greater antibody affinity, and K A or K A refers to the association rate of a particular antibody-antigen interaction. Binding affinity is typically measured using Biacore, SPR, or BLI assays.
[0200] IV. CD28 and B7H3 antigen-binding domains Provided herein are antigen binding domains (ABDs) and ABD compositions that bind to either B7H3 or CD28. In some embodiments, one or more of the ABDs are included in an antibody format described herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0201] A. CD28 Antigen-Binding Domains and Antibodies In one aspect, provided herein are CD28 antigen binding domains (ABDs) that bind to human CD28, and compositions comprising such CD28 antigen binding domains (e.g., antibodies, including heterodimeric antibodies provided herein). In some embodiments, the CD28 antigen binding domains described herein are agonistic CD28 ABDs that advantageously provide costimulatory activity. Thus, such CD28 ABDs provided herein are useful for enhancing immune responses, e.g., when used as monotherapy or in combination with other therapies (e.g., anti-cancer therapeutics for the treatment of certain cancers).
[0202] As will be appreciated by those of skill in the art, a suitable CD28 binding domain can comprise the set of six CDRs as shown in the sequence listing and figures, as identified using underlined or, if a different numbering scheme is used as described herein and shown in Table 2, other alignments within the variable heavy (VH) and variable light (VL) domain sequences as shown in Figures 18-21 and 23 and the sequence listing. A suitable CD28 ABD can also comprise the entire VH and VL sequences as shown in these sequences and figures, used as an scFv or Fab.
[0203] In one embodiment, the CD28 antigen-binding domain comprises six CDRs (i.e., vhCDR1-3 and vlCDR1-3) of any of the CD28 binding domains described herein, including the figures and sequence listing. In some embodiments, the CD28 antigen-binding domain binds to human CD28. ABD is one of the following: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28] _H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In an exemplary embodiment, the CD28 ABD is CD28 ABD: 1A7[CD28]_H1L1 or 1A7[CD28]_H1.14L1.
[0204] In addition to the parent CDR set disclosed in the Figures and Sequence Listing that form the ABD for CD28, provided herein are variant CD28 ABDs having CDRs that include at least one modification of a CD28 ABD CDR disclosed herein (e.g., Figures 18-21 and 23, and the Sequence Listing). In one embodiment, a CD28 ABD includes a set of six CDRs that have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications compared to the six CDRs of the CD28 ABDs described herein, including the Figures and Sequence Listing. In an exemplary embodiment, a CD28 ABD includes the six CDRs of one of the following CD28 ABDs: Contains a set of six CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid modifications compared to: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H 1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In an exemplary embodiment, the CD28 ABD is CD28 ABD: 1A7[CD28]_H1L1 or 1A7[CD28]_H1.14L1.
[0205] In certain embodiments, the CD28 ABD is capable of binding to the CD28 antigen as measured by at least one of a Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assay, the latter of which finds particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to the human CD28 antigen (see Figure 1).
[0206] In some embodiments, the CD28 ABD comprises six CDRs that are at least 90, 95, 97, 98, or 99% identical to the six CDRs of the CD28 ABDs described herein, including the Figures and Sequence Listing. In exemplary embodiments, the CD28 ABD comprises six CDRs that are at least 90, 95, 97, 98, or 99% identical to the six CDRs of one of the following CD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_ H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of a Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., octet assay) assay, the latter finding particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to the human CD28 antigen (see Figure 1).
[0207] In another exemplary embodiment, the CD28 ABD comprises the variable heavy (VH) domain and variable light (VL) domain of any one of the CD28 ABDs described herein, including the figures and sequence listing. In an exemplary embodiment, the CD28 ABD is one of the following CD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0 L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and Sequence Listing).
[0208] In addition to the parent CD28 variable heavy and variable light domains disclosed herein, variable heavy domains that are variants of the CD28 ABD VH and VL domains disclosed herein are also included. Provided herein are CD28 ABDs comprising a variable light chain domain and / or a variable light chain domain. In one embodiment, the variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH and / or VL domain of a CD28 ABD described herein, including the Figures and Sequence Listing. In an exemplary embodiment, the variant VH domain and / or VL domain has the following CD28 ABD VH and / or VL domain: and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH and / or VL domain of one of the ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD 28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of a Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., octet assay) assay, the latter finding particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to the human CD28 antigen (see Figure 1).
[0209] In one embodiment, the variant VH and / or VL domains are at least 90, 95, 97, 98, or 99% identical to the VH and / or VL of a CD28 ABD described herein, including the Figures and Sequence Listing. In an exemplary embodiment, the variant VH and / or VL domains are at least 90, 95, 97, 98, or 99% identical to the VH and / or VL of one of the following CD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L 1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., octet assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., octet assay) assays. The ABD is capable of binding to the human CD28 antigen (see Figure 1).
[0210] In one embodiment, the CD28 antigen binding domain comprises a variable heavy domain (VH) having vhCDR1-3 (i.e., vhCDR1-3) of 1A7_H1.14 (Figure 19). In some embodiments, the CD28 antigen binding domain further comprises any of the CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 1A7_L1 (Figure 18) or a variant thereof. In certain embodiments, the CD28 ABD is capable of binding to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to human CD28 antigen (see Figure 1). Such CD28 binding domains include, for example, the "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies of the antibodies provided herein. It can be included in either one.
[0211] In one embodiment, the CD28 ABD comprises a variable heavy domain (VH) having vhCDR1-3 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to vhCDR1-3 of 1A7_H1.14 (Figure 19). In some embodiments, the CD28 antigen binding domain further comprises any of the CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 1A7_L1 (Figure 18) or a variant thereof. In certain embodiments, the CD28 ABD is capable of binding to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to human CD28 antigen (see Figure 1). Such a CD28-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0212] In some embodiments, the CD28 ABD comprises a variable heavy domain (VH) having vhCDR1-3 that is at least 90, 95, 97, 98, or 99% identical to the six vhCDR1-3 of 1A7_H1.14 (Figure 19). In some embodiments, the CD28 antigen binding domain further comprises any of the CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 1A7_L1 (Figure 18) or a variant thereof. In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to human CD28 antigen (see Figure 1). Such a CD28-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0213] In another exemplary embodiment, the CD28 ABD comprises the variable heavy (VH) domain 1A7_H1.14 (Figure 19). In some embodiments, the CD28 antigen binding domain further comprises any of the CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 1A7_L1 (Figure 18) or a variant thereof. In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to human CD28 antigen (see Figure 1). Such a CD28-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0214] In addition to the parent CD28 heavy chain variable domains disclosed herein, provided herein are CD28 ABDs that comprise a heavy chain variable domain that is a variant of 1A7_H1.14 (FIG. 16). In one embodiment, the variant VH domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from 1A7_H1.14 (FIG. 19). In some embodiments, the CD28 antigen binding domain further comprises any of the CD28 binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 1A7_L1 (FIG. 18) or a variant thereof. In certain embodiments, the CD28 The ABD can bind to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the CD28 ABD can bind to the human CD28 antigen (see FIG. 1). Such a CD28-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0215] In one embodiment, the variant VH domain is at least 90, 95, 97, 98, or 99% identical to 1A7_H1.14 (FIG. 19). In some embodiments, the CD28 antigen-binding domain further comprises any of the CD28 binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 1A7_L1 (FIG. 18) or a variant thereof. In certain embodiments, the CD28 ABD is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the CD28 ABD is capable of binding to human CD28 antigen (see FIG. 1). Such a CD28-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0216] Particular anti-CD28 ABDs of interest include those of SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO: 619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:1198 and SEQ ID NO:1199.
[0217] In other cases, the anti-CD28 VH domain is selected from the group consisting of SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO: 09, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO: and having an amino acid sequence selected from SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:1198, and SEQ ID NO:1199; and a VL domain selected from SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:66 0, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691 , SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722 , SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753,It has an amino acid sequence selected from the group consisting of SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 1200 and SEQ ID NO: 756.
[0218] In some cases, the anti-CD28 binding domain comprises a VH domain and a VL domain having an amino acid sequence selected from the pairs of a) SEQ ID NOs: 1 and 5, b) SEQ ID NOs: 9 and 13, c) SEQ ID NOs: 17 and 21, d) SEQ ID NOs: 25 and 29, e) SEQ ID NOs: 33 and 37, f) SEQ ID NOs: 41 and 45, g) SEQ ID NOs: 49 and 53, h) SEQ ID NOs: 57 and 61, i) SEQ ID NOs: 65 and 69, j) SEQ ID NOs: 73 and 77, and k) SEQ ID NOs: 81 and 85.
[0219] B. B7H3 antigen-binding domain In one aspect, provided herein are B7H3 antigen binding domains (ABDs) and compositions comprising such B7H3 antigen binding domains (ABDs), including anti-B7H3 antibodies. Such B7H3 binding domains and related antibodies (e.g., anti-B7H3 x anti-CD28 bispecific antibodies) are used, for example, to treat B7H3-associated cancers.
[0220] As will be appreciated by those of skill in the art, a suitable B7H3 binding domain can include a set of six CDRs as shown in the Sequence Listing and Figures 26-31, as identified using underlined or, if a different numbering scheme is used as described herein and shown in Table 2, other alignments within the variable heavy (VH) and variable light (VL) domain sequences as shown in Figures 26-31 and the Sequence Listing (see Table 2). A suitable B7H3 ABD can also include these sequences and the entire VH and VL sequences as shown in the figures, used as an scFv or Fab domain.
[0221] In one embodiment, the B7H3 antigen binding domain comprises the six CDRs (i.e., vhCDR1-3 and vlCDR1-3) of the B7H3 ABD described herein, including the Figures and Sequence Listing. One of the ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857 , m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26 to 31 and Sequence Listing).
[0222] In addition to the parent CDR set disclosed in the Figures and Sequence Listing that form the ABD for B7H3, provided herein are variant B7H3 ABDs having CDRs that comprise at least one modification of a B7H3 ABD CDR disclosed herein. In one embodiment, the B7H3 ABD comprises a set of six CDRs that have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications compared to the six CDRs of the B7H3 ABD described herein, including the Figures and Sequence Listing. In an exemplary embodiment, the B7H3 ABD comprises the following B7H3 ABDs: The set of six CDRs includes the following: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP2 65, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chA b7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26-31 and Sequence Listing). In certain embodiments, the variant B7H3 ABD is capable of binding to the B7H3 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 2).
[0223] In one embodiment, the B7H3 ABD comprises six CDRs that are at least 90, 95, 97, 98, or 99% identical to the six CDRs of the B7H3 ABDs described herein, including the Figures and Sequence Listing. In an exemplary embodiment, the B7H3 ABD comprises six CDRs that are at least 90, 95, 97, 98, or 99% identical to the six CDRs of one of the following B7H3 ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1 ]_L1, 6A1[B7H3]_H1L1, Ombrutamab, Enobrituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H 1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26-31 and Sequence Listing). In certain embodiments, the B7H3 ABD is capable of binding to the B7H3 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 2).
[0224] In another exemplary embodiment, the B7H3 ABD comprises the variable heavy (VH) domain and variable light (VL) domain of any one of the B7H3 ABDs described herein, including the figures and sequence listing. One of the ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L 58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26-31 and Sequence Listing). In exemplary embodiments, the B7H3 ABD is one of the following: B7H3 ABD:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, or 6A1[B7H3]_H1L1.
[0225] In addition to the parent B7H3 variable heavy and variable light domains disclosed herein, provided herein are B7H3 ABDs that comprise variable heavy and / or variable light domains that are variants of the B7H3 ABD VH and VL domains disclosed herein. In one embodiment, the variant VH and / or VL domains have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH and / or VL domains of a B7H3 ABD described herein, including the Figures and Sequence Listing. In exemplary embodiments, the variant VH and / or VL domains have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH and / or VL domain of one of the following B7H3 ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1. ]_L1, 6A1[B7H3]_H1L1, Ombrutamab, Enobrituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H 1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26-31 and Sequence Listing). In certain embodiments, the B7H3 ABD is capable of binding to B7H3 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter finding particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 2).
[0226] In one embodiment, the variant VH and / or VL domains are at least 90, 95, 97, 98, or 99% identical to the VH and / or VL of the B7H3 ABDs described herein, including the Figures and Sequence Listing. At least 90, 95, 97, 98, or 99% identical to the VH and / or VL of one of the ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50 L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26-31 and Sequence Listing). In certain embodiments, the B7H3 ABD is capable of binding to B7H3 as measured by at least one of a Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assay, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 2).
[0227] In one embodiment, the B7H3 antigen binding domain comprises a variable heavy domain (VH) having vhCDR1-3 (i.e., vhCDR1-3) of 2E4A3.189_H1.22 (Figure 27). In some embodiments, the B7H3 antigen binding domain further comprises any of the B7H3 or CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 2E4A3.189_L1 (Figure 26), 1A7_L1 (Figure 18), or a variant thereof. In certain embodiments, the B7H3 ABD is capable of binding to the B7H3 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 2). Such B7H3-binding domains may be used, for example, in the "1+1 Fa" domain. The fragment may be included in any of the antibodies provided herein, including "b-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0228] In one embodiment, the B7H3 ABD comprises a variable heavy domain (VH) having vhCDR1-3 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to vhCDR1-3 of 2E4A3.189_H1.22 (Figure 27). In some embodiments, the B7H3 antigen binding domain further comprises any of the B7H3 or CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 2E4A3.189_L1 (Figure 26), 1A7_L1 (Figure 18), or a variant thereof. In certain embodiments, the B7H3 ABD is capable of binding to the B7H3 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD can bind to the human B7H3 antigen (see Figure 2). Such a B7H3-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0229] In some embodiments, the B7H3 ABD comprises a variable heavy domain (VH) having vhCDR1-3 that are at least 90, 95, 97, 98, or 99% identical to the six vhCDR1-3 of 2E4A3.189_H1.22 (Figure 27). In some embodiments, the B7H3 antigen binding domain further comprises any of the B7H3 or CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 2E4A3.189_L1 (Figure 26), 1A7_L1 (Figure 18), or a variant thereof. In certain embodiments, the B7H3 ABD is capable of binding to B7H3 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., the Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD can bind to the human B7H3 antigen (see Figure 2). Such a B7H3-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0230] In another exemplary embodiment, the B7H3 ABD comprises the variable heavy (VH) domain 2E4A3.189_H1.22 (Figure 27). In some embodiments, the B7H3 antigen binding domain further comprises any of the B7H3 or CD28 binding domain variable light domains provided herein. In exemplary embodiments, the variable light domain is 2E4A3.189_L1 (Figure 26), 1A7_L1 (Figure 18), or a variant thereof. In certain embodiments, the B7H3 ABD is capable of binding to B7H3 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 1). Such a B7H3-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0231] In addition to the parent B7H3 heavy chain variable domain disclosed herein, B7H3 includes a heavy chain variable domain that is a variant of the variable heavy chain (VH) domain 2E4A3.189_H1.22. ABDs are provided herein (Figure 27). In one embodiment, the variant VH domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the variable heavy (VH) domain 2E4A3.189_H1.22 (Figure 27). In some embodiments, the B7H3 antigen binding domain further comprises any of the B7H3 or CD28 binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 2E4A3.189_L1 (Figure 26), 1A7_L1 (Figure 18), or a variant thereof. In certain embodiments, the B7H3 ABD is capable of binding to B7H3 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., octet assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD can bind to the human B7H3 antigen (see Figure 2). Such a B7H3-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0232] In one embodiment, the variant VH domain is at least 90, 95, 97, 98, or 99% identical to 2E4A3.189_H1.22 (Figure 27). In some embodiments, the B7H3 antigen-binding domain further comprises any of the B7H3 or CD28-binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 2E4A3.189_L1 (Figure 26), 1A7_L1 (Figure 18), or a variant thereof. In certain embodiments, the B7H3 ABD is capable of binding to B7H3 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet Assay) assays, the latter of which finds particular use in many embodiments. In certain embodiments, the B7H3 ABD is capable of binding to the human B7H3 antigen (see Figure 2). Such a B7H3-binding domain can be included in any of the antibodies provided herein, including, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies.
[0233] In some embodiments, the anti-B7H3 ABD is a) SEQ ID NOs: 89 and 93 from ombramab, b) SEQ ID NOs: 97 and 101 from enoblituzumab, c) SEQ ID NOs: 105 and 109 from BRCA84D, d) SEQ ID NOs: 113 and 117 from BRCA69D, e) SEQ ID NOs: 121 and 125 from PRCA157, f) SEQ ID NOs: 129 and 133 from huPRCA157, g) SEQ ID NOs: 137 and 141 from Mab-D, h) SEQ ID NOs: 145 and 149 from humAb-D, i) m30 j) SEQ ID NOs: 161 and 165 from M30-H1-L4, k) SEQ ID NOs: 169 and 173 from SP265, l) SEQ ID NOs: 177 and 181 from S10-H50L58, m) SEQ ID NOs: 185 and 189 from 8H9, n) SEQ ID NOs: 193 and 197 from m852, o) SEQ ID NOs: 201 and 205 from m857, p) SEQ ID NOs: 209 and 213 from m8524, q) SEQ ID NOs: 217 and 221 from 1-1, r) SEQ ID NOs: 1- 2) SEQ ID NOs: 225 and 229 from chAb1, s) SEQ ID NOs: 233 and 237 from 1-4, t) SEQ ID NOs: 241 and 245 from 1-5, u) SEQ ID NOs: 249 and 253 from 1-7, v) SEQ ID NOs: 257 and 261 from 2-5, w) SEQ ID NOs: 265 and 269 from 2-8, x) SEQ ID NOs: 273 and 277 from chAb2, y) SEQ ID NOs: 281 and 285 from chAb3, z) SEQ ID NOs: 289 and 293 from chAb4, aa) SEQ ID NOs: 291 and 293 from chAb5, 297 and 301, bb) SEQ ID NOs: 305 and 309 from chAb13, cc) SEQ ID NOs: 313 and 317 from chAb12, dd) SEQ ID NOs: 321 and 325 from chAb14, ee) SEQ ID NOs: 329 and 333 from chAb6, ff) SEQ ID NOs: 337 and 341 from chAb11, gg) SEQ ID NOs: 345 and 349 from chAb16, hh) SEQ ID NOs: 353 and 357 from chAb10, ii) SEQ ID NOs: 361 and 365 from ChAb7 , jj) SEQ ID NOs: 369 and 373 from chAb8, kk) SEQ ID NOs: 377 and 381 from chAb17, ll) SEQ ID NOs: 385 and 389 from chAb5, mm) SEQ ID NOs: 393 and 397 from huAb3v2.5, nn) SEQ ID NOs: 401 and 405 from huAb3v2.6, pp) SEQ ID NOs: 409 and 413 from huAb13v1, qq) SEQ ID NOs: 417 and 421 from TPP-5706, rr) SEQ ID NOs: 425 and 429 from TPP-6642, ss) SEQ ID NOs: 430 and 431 from TPP-6850 tt) SEQ ID NOs: 433 and 437 from TPP-3803, uu) SEQ ID NOs: 449 and 453 from TRL4542, vv) SEQ ID NOs: 457 and 461 from h1702, ww) SEQ ID NOs: 465 and 469 from h1703, xx) SEQ ID NOs: 473 and 477 from huA3, yy) SEQ ID NOs: 481 and 485 from huA9, and zz) SEQ ID NOs: 489 and 493 from m1704. See Figure 17 of USSN 63 / 092,272.
[0234] In some embodiments, the anti-B7H3 ABD has a VH domain having the amino acid sequence of SEQ ID NO: 942 (2E4A3.189_H1.22) and a VL domain having the amino acid sequence of SEQ ID NO: 874 (1A7[CD28]_L1, which is the common light chain of both B7H3 and CD28).
[0235] V. Antibodies In one aspect, provided herein are anti-CD28 antibodies and anti-B7H3 antibodies. The antibodies provided herein can comprise any of the B7H3 and / or CD28 binding domains provided herein (e.g., "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" antibodies).
[0236] The antibodies provided herein comprise different antibody domains. As described herein and known in the art, the heterodimeric antibodies described herein comprise different, and potentially overlapping, domains within the heavy and light chains. These domains include, but are not limited to, an Fc domain, a CH1 domain, a CH2 domain, a CH3 domain, a hinge domain, a heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), a variable heavy chain domain, a variable light chain domain, a light chain constant domain, a Fab domain, and an scFv domain.
[0237] As provided herein, there are several suitable linkers (used as either domain linkers or scFv linkers) that can be used to covalently link (including traditional peptide bonds produced by recombinant techniques) the listed domains (e.g., scFv, Fab, Fc domains, etc.). Exemplary linkers for attaching the domains of an antibody of interest to one another are shown in Figure 7. In some embodiments, the linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be long enough to join the two molecules in a manner that allows them to assume the correct conformation relative to one another so as to retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used, with about 5 to about 10 amino acids being used in some embodiments. Useful linkers include, for example, glycine-serine polymers, including (GS), (GSGGS), (GGGGS), and (GGGS), where n is at least an integer (generally 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, various non-proteinaceous polymers may be useful as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
[0238] Other linker sequences include any sequence of the CL / CH1 domain of any length, but may not include all residues of the CL / CH1 domain, for example, the first 5 to 12 amino acid residues of the CL / CH1 domain. Linkers can be derived from immunoglobulin light chains, such as Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other naturally occurring sequences from other proteins.
[0239] In some embodiments, the linker is a "domain linker" used to link together any two domains outlined herein. For example, in a 2+1 Fab2-scFv-Fc format, there may be a domain linker connecting the C-terminus of the CH1 domain of the Fab to the N-terminus of the scFv, and another optional domain linker connecting the C-terminus of the scFv to the CH2 domain (although in many embodiments, a hinge is used as this domain linker). While any suitable linker can be used, many embodiments utilize glycine-serine polymers as domain linkers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1 (generally 3-4-5), as well as any peptide sequence that allows for the recombinant attachment of two domains with sufficient length and flexibility so that each domain retains its biological function. In some cases, charged domain linkers can be used, as used in some embodiments of scFv linkers, with attention to "strandedness," as outlined below. Exemplary useful domain linkers are shown in FIG.
[0240] In some embodiments, the linker is an "scFv linker" used to covalently link the VH and VL domains discussed herein. In many cases, the scFv linker is a charged scFv linker, some of which are shown in Figure 6. Thus, provided herein are charged scFv linkers to facilitate pI separation between a first and second monomer. That is, by incorporating either a positively or negatively charged scFv linker (or both, in the case of scaffolds that use scFvs on different monomers), this allows for altering the pI of the monomer containing the charged linker without further altering the Fc domain. These charged linkers can be substituted into any scFv containing standard linkers. Again, as will be understood by those skilled in the art, the charged scFv linker is used on the correct "chain" or monomer depending on the desired change in pI. For example, as discussed herein, to generate a 1+1 Fab-scFv-Fc format heterodimeric antibody, the original pI of the Fv region for each of the desired antigen-binding domains is calculated and one is selected to generate the scFv, and depending on the pI, either a positive or negative linker is selected.
[0241] Charged domain linkers can also be used to increase the pI separation of the monomers of the present invention, and thus those included in Figure 6 can be used in any embodiment herein where a linker is utilized.
[0242] The provided B7H3-binding domain and CD28-binding domain can be included in any useful antibody format, including, for example, standard immunoglobulins, as well as the "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" formats provided herein (see, e.g., Figure 25). Other useful antibody formats include "mAb-Fv," "mAb-scFv," "scFv," and "scFv," as disclosed in US20180127501A1, which is incorporated herein by reference, particularly the relevant portions relating to antibody formats (see, e.g., Figure 2). Examples of antibodies include, but are not limited to, "central-Fv," "one-arm scFv-mAb," "scFv-mAb," "dual scFv," and "Trident" format antibodies.
[0243] In some embodiments, the subject antibodies comprise one or more of the B7H3 ABDs provided herein. In some embodiments, the antibodies comprise one B7H3 ABD. In other embodiments, the antibodies comprise two B7H3 ABDs. In exemplary embodiments, the B7H3 ABD is one of the following B7H3 ABDs: The variable heavy and light domains of one of the ABDs are: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9 , m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26 to 31 and Sequence Listing).In some embodiments, the B7H3 ABD is one of the following B7H3 ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m85 7, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704 (Figures 26 to 31 and Sequence Listing).
[0244] In exemplary embodiments, the antibody is a bispecific antibody comprising one or two B7H3 ABDs, including any of the B7H3 ABDs provided herein. Such bispecific antibodies comprising B7H3 ABDs include, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" bispecific format antibodies (Figure 25). In exemplary embodiments, the B7H3 ABD is one of the following B7H3 ABDs: One of the ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mA b-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3 v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704 (Figures 26-31 and Sequence Listing). In exemplary embodiments, the B7H3 binding domain is a Fab. In some embodiments, such bispecific antibodies are heterodimeric bispecific antibodies comprising any of the heterodimerization variants, pI variants, and / or deletion variants described herein. See Figure 8.
[0245] In some embodiments, the subject antibodies comprise one or more of the CD28 ABDs provided herein. In some embodiments, the antibodies comprise one CD28 ABD. In other embodiments, the antibodies comprise two CD28 ABDs. In exemplary embodiments, the antibodies comprise the variable heavy and variable light domains of one of the following CD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11 A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and sequence listing).
[0246] In exemplary embodiments, the antibody is a bispecific antibody comprising one or two CD28 ABDs, including any of the CD28 ABDs provided herein. Such bispecific antibodies comprising CD28 ABDs include, for example, "1+1 Fab-scFv-Fc," "2+1 Fab2-scFv-Fc," "1+1 common light chain," and "2+1 common light chain" bispecific format antibodies (Figure 25). In exemplary embodiments, the CD28 ABD is one of the following CD28 ABDs: One of the ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H 0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In an exemplary embodiment, the CD28 ABD is an anti-CD28 scFv contained in a "1+1 Fab-scFv-Fc" or "2+1 Fab2-scFv-Fc" bispecific format antibody (Figure 25). In some embodiments, such bispecific antibodies are heterodimeric bispecific antibodies comprising any of the heterodimerization variants, pI variants and / or deletion variants described herein. See Figure 8.
[0247] A. Chimeric and Humanized Antibodies In certain embodiments, the subject antibodies provided herein comprise a heavy chain variable region derived from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region derived from a particular germline light chain immunoglobulin gene. For example, such antibodies can comprise or consist of a human antibody comprising a heavy or light chain variable region that is the "product of" or "derived from" a particular germline sequence. A human antibody that is the "product of" or "derived from" a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin that is closest in sequence to the sequence of the human antibody (i.e., the highest % identity). Human antibodies that are "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, due, for example, to naturally occurring somatic mutations or the deliberate introduction of site-specific mutations. However, a humanized antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and includes amino acid residues that identify the antibody as derived from a human sequence when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence). In certain cases, a humanized antibody may be at least 95, 96, 97, 98, or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a germline immunoglobulin gene. Typically, a humanized antibody derived from a particular human germline sequence will exhibit no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene (prior to the introduction of any scuba variants, pI variants, and deletion variants herein, i.e., before the introduction of the variants of the present invention, the number of variants will generally be small). In certain cases, a humanized antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene (again, prior to the introduction of any scuba variants, pI variants, and deletion variants herein, i.e., before the introduction of the variants of the present invention, the number of variants will generally be small).
[0248] In one embodiment, the parent antibody has been affinity matured as known in the art. Structure-based methods can be used for humanization and affinity maturation, for example, as described in USSN 11 / 004,590. Selection-based methods may be used to humanize and / or affinity mature antibody variable regions, including, but not limited to, those described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are incorporated by reference in their entirety. Other humanization methods may involve grafting only portions of the CDRs, including, but not limited to, those described in USSN 09 / 810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all of which are incorporated by reference in their entirety.
[0249] B. Anti-CD28 x anti-tumor-associated antigen (TAA) antibody In another aspect, provided herein are anti-CD28× anti-TAA antibodies. In some embodiments, the anti-CD28× anti-TAA antibodies comprise a CD28-binding domain and one or more binding domains that bind to a tumor-associated antigen. In some embodiments, the CD28-binding domain of the antibody is an agonist CD28-binding domain that provides costimulatory function by binding to CD28 on T cells. Thus, the anti-CD28× anti-TAA antibodies provided herein selectively enhance immune responses at tumor sites that express a specific TAA (e.g., B7H3). In some embodiments, the anti-CD28× anti-TAA antibodies are bispecific antibodies. In some embodiments, the anti-CD28× anti-TAA antibodies are trispecific antibodies. In some embodiments, the anti-CD28× anti-TAA antibodies are bivalent antibodies. In some embodiments, the anti-CD28× anti-TAA antibodies are trivalent antibodies. In some embodiments, the anti-CD28× anti-TAA antibodies are bispecific bivalent antibodies. In an exemplary embodiment, the anti-CD28× anti-TAA antibodies are bispecific trivalent antibodies.
[0250] As outlined in more detail herein, anti-CD28 x anti-TAA antibodies can be in a variety of formats, as outlined below. Exemplary formats include the "1 + 1 Fab-scFv-Fc," "2 + 1 Fab2-scFv-Fc," "1 + 1 common light chain," and "2 + 1 common light chain" formats provided herein (see, e.g., Figure 25). Other useful antibody formats include, but are not limited to, antibodies in "mAb-Fv," "mAb-scFv," "central-Fv," "one-arm scFv-mAb," "scFv-mAb," "dual-scFv," and "trident" formats, as disclosed in US20180127501A1, particularly the relevant portions relating to antibody formats (see, e.g., Figure 2), which is incorporated herein by reference.
[0251] The anti-CD28x anti-TAA antibody can comprise any suitable CD28 ABD, including those described herein. In some embodiments, the CD28 ABD is an agonist ABD that provides costimulatory function upon binding to CD28. In some embodiments, the anti-CD28x anti-TAA antibody comprises a CD28 binding domain that includes the variable heavy and variable light domains of one of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28 .3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and the Sequence Listing) or variants thereof.
[0252] The anti-CD28x anti-TAA antibody provided herein can contain one or more TAA binding domains. In some embodiments, the anti-CD28x anti-TAA antibody contains one TAA binding domain. In certain embodiments, the anti-CD28x anti-TAA antibody contains two TAA binding domains. Depending on the tumor selected for targeting, any suitable TAA binding domain can be included in the subject anti-CD28x anti-TAA antibody. TAAs that can be targeted by the anti-CD28x anti-TAA antibodies provided herein include, but are not limited to, B7H, CD20, CD38, CD123; ROR1, ROR2, BCMA; PSMA; SSTR2; SSTR5, CD19, FLT3, CD33, PSCA, ADAM 17, CEA, Her2, EGFR, EGFR-vIII, CD30, FOLR1, GD-2, CA-IX, Trop-2, CD70, CD38, mesothelin, EphA2, CD22, CD79b, GPNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON, ERBB2, and EGFR. Additional TAAs are described, for example, in US2016 / 0355608 and US2017 / 0209492, which are incorporated herein in relevant portions pertaining to tumor-associated antigens. Suitable TAA binding domains that can be included in the subject anti-CD28 x anti-TAA antibodies are disclosed, for example, in US2019 / 0248898A1 (SSTR2), US2020 / 0165356A1 (FAP), US2017 / 0320947A1 (PSMA), all of which are incorporated by reference in relevant portions relating to TAA binding domains.
[0253] In certain embodiments, the anti-CD28×anti-TAA antibody comprises a B7H3 binding domain. In some embodiments, such an anti-CD28×anti-B7H3 (also referred to herein as "αB7H3×αCD28" or "αCD28×αB7H3") bispecific antibody comprises at least one B7H3 ABD and at least one CD28 binding domain. In exemplary embodiments, the anti-CD28×anti-B7H3 bispecific antibody comprises two B7H3 binding domains. In some embodiments, the CD28 binding domain of the bispecific antibody is The agonistic CD28-binding domain provides costimulatory function by binding to CD28 on T cells. Thus, the bispecific αB7H3×αCD28 provided herein selectively enhances immune responses at tumor sites that express B7H3.
[0254] Anti-CD28xanti-B7H3 bispecific antibodies can comprise any suitable CD28 ABD and B7H3 ABD, including those described herein. In some embodiments, anti-CD28xanti-B7H3 bispecific antibodies comprise a CD28 binding domain that includes the variable heavy and variable light domains of one of the following CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, h CD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and the sequence listing) or variants thereof.In some embodiments, the B7H3 ABD is The variable heavy and light domains of one of the ABDs are: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10- H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26 to 31 and Sequence Listing) or variants thereof.
[0255] Note that unless specified herein, the order of the named antigen listing does not confer structure, i.e., an anti-B7H3 x anti-CD28 1+1 Fab-scFv-Fc antibody may allow the scFv to bind to either B7H3 or CD28, but in some cases the structure is designated in the order indicated.
[0256] Additionally, in embodiments in which the subject antibodies comprise scFvs, the scFvs can be in an N- to C-terminal orientation of VH-scFv linker-VL or VL-scFv linker-VH. In some formats, one or more of the ABDs is generally a Fab, comprising a VH domain (generally as a component of a heavy chain) on one protein chain and a VL (generally as a component of a light chain) on another protein chain.
[0257] As will be appreciated by those skilled in the art, any set of six CDRs or VH and VL domains can be made into scFv or Fab format, which are then added to heavy and light chain constant domains, where the heavy chain constant domains contain mutations (including within the CH1 and Fc domains). The scFv sequences included in the sequence listing utilize specific charged linkers, however, uncharged or other charged linkers can be used, as outlined herein, including those shown in Figure 6.
[0258] In addition, as noted above, the numbering used in the sequence listing for identifying the CDRs is Kabat, but different numbering can be used, which would change the amino acid sequence of the CDRs as shown in Table 2.
[0259] Further variants can be generated for all variable heavy and light chain domains described herein. As outlined herein, in some embodiments, the set of six CDRs can have 0, 1, 2, 3, 4, or 5 amino acid modifications (depending on the particular amino acid substitutions used), as well as variations in the framework regions of the variable heavy and light chain domains, so long as the framework (excluding the CDRs) retains at least about 80, 85, or 90% identity with a human germline sequence selected from those listed in Figure 1 of U.S. Patent No. 7,657,380, which figure and legend are incorporated herein by reference in their entirety. Thus, for example, the same CDRs described herein can be combined with different framework sequences derived from human germline sequences, so long as the framework regions retain at least 80, 85, or 90% identity with a human germline sequence selected from those listed in Figure 1 of U.S. Patent No. 7,657,380. Alternatively, the CDRs can have amino acid modifications (e.g., 1, 2, 3, 4, or 5 amino acid modifications in the set of CDRs (i.e., the CDRs can have any combination of CDR modifications, e.g., 1 modification in vlCDR1, 2 modifications in vhCDR2, no modifications in vhCDR3, etc.), as long as the total number of modifications in the set of 6 CDRs is less than 6 amino acid modifications), as well as modifications in the framework regions, as long as the framework regions retain at least 80, 85, or 90% identity to a human germline sequence selected from those listed in Figure 1 of U.S. Pat. No. 7,657,380.
[0260] C. Heterodimeric Antibodies In exemplary embodiments, the anti-CD28 x anti-TAA (e.g., anti-CD28 x anti-B7H3) antibodies provided herein are heterodimeric bispecific antibodies comprising two variant Fc domain sequences, where such variant Fc domains comprise amino acid modifications to facilitate self-assembly and / or purification of the heterodimeric antibody.
[0261] A continuing challenge in antibody technology is the need for "bispecific" antibodies that can simultaneously bind to two different antigens, generally bringing the different antigens into close proximity and providing new functions and new therapeutic approaches. These antibodies are typically generated by including genes for each heavy and light chain in a host cell. This typically results in the formation of two homodimers (AA and BB (not including the issue of light chain heterodimers)) as well as the desired heterodimer (AB). However, a major obstacle in bispecific antibody formation is the difficulty of biasing heterodimer formation over homodimer formation and / or purifying heterodimeric antibodies away from homodimers.
[0262] There are many mechanisms that can be used to generate heterodimeric antibodies of interest. In addition, as will be appreciated by those skilled in the art, these different mechanisms can be combined to ensure high heterodimerization. Amino acid modifications that facilitate heterodimer generation and purification are collectively referred to as "heterodimerization variants." As discussed below, heterodimerization variants include "skew" variants (e.g., "knob-and-hole" and "charge-pair" variants, discussed below) and "pI variants" that allow for purification of heterodimers from homodimers. Mechanisms useful for heterodimerization are described generally in U.S. Pat. No. 9,605,084, which is incorporated herein by reference in its entirety, and specifically as follows for a discussion of heterodimerization variants. Mechanisms include "knob and hole" ("KIH") as described in U.S. Pat. No. 9,605,084, "electrostatic steering" or "charge pair" as described in U.S. Pat. No. 9,605,084, pI variants as described in U.S. Pat. No. 9,605,084, and further Fc variants in general as outlined in U.S. Pat. No. 9,605,084 and below.
[0263] Heterodimerization variants useful for forming and purifying the subject heterodimeric antibodies (e.g., bispecific antibodies) are discussed in more detail below.
[0264] 1. Scuba liant In some embodiments, the heterodimeric antibody comprises a scuba variant, which is one or more amino acid modifications in the first Fc domain (A) and / or the second Fc domain (B), that promote the formation of Fc homodimers (Fc dimers comprising two of the first Fc domains or two of the second Fc domains; AA or BB) over Fc dimers (Fc dimers comprising two of the first Fc domains or two of the second Fc domains; AA or BB). Suitable scuba variants are included in Figure 29 of U.S. Patent Application Publication No. 2016 / 0355608, which is incorporated herein by reference in its entirety and in particular for its disclosure of scuba variants and in Figures 3 and 9.
[0265] One particular type of scubariant is commonly referred to in the art as "knob-and-hole" and refers to amino acid manipulations that create steric effects that favor heterodimer formation and discourage homodimer formation, as described in USSN 61 / 596,846; Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; and U.S. Patent No. 8,216,805 (all of which are incorporated herein by reference in their entirety, particularly for their disclosure of "knob-and-hole" mutations). This is sometimes referred to herein as a "stereovariant." These figures identify several "monomer A-monomer B" pairs that rely on "knob-and-hole" mutations. Additionally, these "knob-and-hole" mutations can be combined with disulfide bonds to further promote the formation of Fc heterodimers, as described by Merchant et al., Nature Biotech. 16:677 (1998).
[0266] Another method used to generate heterodimers is sometimes called "electrostatic steering," as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), which is incorporated herein by reference in its entirety. This is sometimes referred to herein as "charge pairing." In this embodiment, electrostatics are used to skew formation toward heterodimerization. As will be appreciated by those skilled in the art, these may also affect pI, i.e., purification, and therefore may in some cases be considered pI variants. However, because these were generated to force heterodimerization and were not used as a purification tool, they are classified as "scubariant." These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (e.g., these are a "matched set of monomers"), and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.
[0267] In some embodiments, scubariants advantageously and simultaneously promote heterodimerization based on both "knob-and-hole" and "electrostatic steering" mechanisms. In some embodiments, heterodimeric antibodies comprise one or more sets of such heterodimerizing scubariants. These variants are "pairs" of "sets," i.e., pairs from one set are incorporated into a first monomer, and pairs from the other set are incorporated into a second monomer. It should be noted that these sets do not necessarily behave as "knob-and-hole" variants, but rather have a one-to-one correspondence between residues in one monomer and those in the other. That is, these pairs of sets may instead form an interface between the two monomers that promotes heterodimer formation and not homodimer formation, such that the percentage of heterodimers that form spontaneously under biological conditions exceeds 90% rather than the expected 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B). Exemplary heterodimerization "skew" variants are shown in Figure 4. In exemplary embodiments, the heterodimeric antibody comprises a "skewed" variant amino acid substitution set of S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; or T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C). In an exemplary embodiment, the heterodimeric antibody comprises the "S364K / E357Q:L368D / K370S" amino acid substitution set. In terms of nomenclature, the "S364K / E357Q:L368D / K370S" pair means that one of the monomers comprises an Fc domain comprising the amino acid substitutions S364K and E357Q, and the other monomer comprises an Fc domain comprising the amino acid substitutions L368D and K370S. As noted above, the "twistiness" of these pairs depends on the starting pI.
[0268] In some embodiments, the scubariants provided herein are independently incorporated into one or both of the first and second Fc domains of a heterodimeric antibody, along with other modifications, including, but not limited to, other scubariants (see, e.g., Figure 37 of U.S. Patent Application Publication No. 2012 / 0149876, particularly Figure 37, which is incorporated herein by reference for its disclosure of scubariants), pI variants, isotype variants, FcRn variants, deletion variants, etc. Furthermore, individual modifications can also independently and optionally include or exclude the heterodimeric antibody.
[0269] In some embodiments, the scuba variants outlined herein can optionally and independently incorporate any pI variants (or other variants such as Fc variants, FcRn variants, etc.) into one or both heavy chain monomers, which can independently and optionally be included or excluded from the proteins of the invention.
[0270] 2. Heterodimer pI (isoelectric point) variants In some embodiments, the heterodimeric antibody comprises a purification variant that advantageously allows for separation of the heterodimeric antibody (eg, an anti-B7H3 x anti-CD28 bispecific antibody) from homodimeric proteins.
[0271] There are several basic mechanisms that can facilitate the purification of heterodimeric antibodies. For example, modifications to one or both of the antibody heavy chain monomers A and B so that each monomer has a different pI allow for isoelectric purification of the heterodimeric AB antibody from the monomeric AA and BB proteins. Alternatively, several scaffold formats, such as the "1+1 Fab-scFv-Fc" format, the "2+1 Fab2-scFv-Fc" format, and the "2+1 CLC" format, allow for size-based separation. As noted above, it is also possible to "skew" heterodimer formation over homodimers using scubariants. Thus, the combination of heterodimerization scubariants and pI variants finds particular use in the heterodimeric antibodies provided herein.
[0272] Additionally, depending on the heterodimeric antibody format, pI variants may be contained within the constant regions and / or Fc domains of the monomers, as outlined more fully below. Domain linkers can be used. In some embodiments, the heterodimeric antibody contains additional modifications for alternative functions, such as Fc, FcRn, and KO variants, which can also create pI changes.
[0273] In some embodiments, the subject heterodimeric antibodies provided herein include at least one monomer having one or more modifications that alter the pI of the monomer (i.e., a "pI variant"). In general, as will be understood by those of skill in the art, there are two general categories of pI variants: those that increase the pI of a protein (a basic change) and those that decrease the pI of a protein (an acidic change). As described herein, all combinations of these variants can be made: one monomer can be wild-type or a variant that does not exhibit a pI that is significantly different from wild-type, and the other can be either more basic or more acidic. Alternatively, each monomer can be altered, one to be more basic and one to be more acidic.
[0274] Depending on the heterodimeric antibody format, pI mutations can be included within the constant and / or Fc domains of the monomers, or either charged linkers, domain linkers, or scFv linkers can be used. That is, antibody formats utilizing scFvs, such as "1+1 Fab-scFv-Fc," can include a charged scFv linker (either positive or negative) that provides an additional pI boost for purification purposes. As will be appreciated by those skilled in the art, the present specification also provides pI variants and / or charged domain linkers in one or both of the monomers, although some 1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fc formats are useful with only a charged scFv linker, without additional pI adjustment. Additionally, further amino acid engineering for alternative functionality can also confer pI changes, such as Fc, FcRn, and KO variants.
[0275] In subject heterodimeric antibodies that utilize pI as a separation mechanism to enable purification of the heterodimeric protein, amino acid variants are introduced into one or both of the monomer polypeptides. That is, the pI of one of the monomers (referred to herein for simplicity as simply "monomer A") can be engineered away from monomer B, or the pI of both monomers A and B can be varied, increasing the pI of monomer A and decreasing the pI of monomer B. As outlined more fully below, pI changes in either or both monomers can be achieved by removing or adding a charged residue (e.g., substituting a neutral amino acid with a positively or negatively charged amino acid residue, e.g., glycine to glutamic acid), by changing a charged residue from positive or negative to the opposite charge (e.g., aspartic acid to lysine), or by changing a charged residue to a neutral residue (e.g., eliminating the charge, lysine to serine). Some of these variants are shown in Figures 3 and 4.
[0276] Thus, in some embodiments, the subject heterodimeric antibodies comprise an amino acid modification in the constant region that alters the isoelectric point (pI) of at least one, if not both, of the monomers of the dimeric protein by incorporating an amino acid substitution (a "pI variant" or "pI substitution") into one or both of the monomers. As demonstrated herein, separation of the heterodimer from the two homodimers can be achieved when the pIs of the two monomers differ by as little as 0.1 pH units, with differences of 0.2, 0.3, 0.4, and 0.5 or more being of use in the present invention.
[0277] As will be appreciated by those skilled in the art, the number of pI mutations to be included in each or both monomers to obtain good separation will depend in part on the starting pIs of the components, e.g., the starting pIs of the scFvs (1+1 Fab-scFv-Fc, 2+1 Fab2-scFv-Fc) and Fabs of interest in 1+1 Fab-scFv-Fc, 2+1 Fab2-scFv-Fc, 1+1 CLC, and 2+1 CLC formats. That is, the starting pIs of the Fabs and scFvs will be used to determine which monomers to engineer or in which "direction" (e.g., more positive or more negative). To achieve this, the Fv sequences of the two target antigens are calculated and a determination is made therefrom. As is known in the art, different Fvs will have different starting pIs for use in the present invention. Generally, as outlined herein, the pI is engineered to result in a total pI difference for each monomer of at least about 0.1 log, with 0.2-0.5 being preferred, as outlined herein.
[0278] The use of heavy chain constant regions provides a more modular approach to designing and purifying bispecific proteins, including antibodies, when pI variants are used to achieve heterodimerization. Thus, in some embodiments, heterodimerization mutations (including skew and pI heterodimerization variants) are not included in the variable region, and therefore each individual antibody must be engineered. Additionally, in some embodiments, the potential for immunogenicity resulting from pI variants is significantly reduced by incorporating pI variants from different IgG isotypes to alter the pI without introducing significant immunogenicity. Therefore, a further problem to be solved is the elucidation of low pI constant domains with high human sequence content, e.g., minimizing or avoiding non-human residues at any specific positions. Alternatively or in addition to isotype substitutions, the potential for immunogenicity resulting from pI variants is significantly reduced by utilizing isosteric substitutions (e.g., Asn to Asp and Gln to Glu).
[0279] As discussed below, potential collateral benefits of this pI engineering are also increased serum half-life and increased FcRn ligation. That is, as described in U.S. Patent Application Publication No. 2012 / 0028304 (incorporated herein by reference in its entirety), lowering the pI of antibody constant domains (including those found in antibodies and Fc fusions) can result in longer serum retention in vivo. These pI variants for extending serum half-life also facilitate pI changes for purification.
[0280] Additionally, pI variants offer additional benefits to the analytical and quality control process of bispecific antibodies, as they are notable for their ability to eliminate, minimize, and distinguish when homodimers are present. Similarly, the ability to reliably test the reproducibility of heterodimeric antibody production is important.
[0281] Generally, the particular embodiment used relies on a set of variants including a scuba variant, which, in combination with a pI variant that increases the pI difference between the two monomers, favors heterodimer formation in preference to homodimer formation, facilitating purification of the heterodimer by removal of the homodimer.
[0282] Exemplary combinations of pI variants are shown in Figures 4 and 5 and Figure 30 of U.S. Patent Application Publication No. 2016 / 0355608, all of which are incorporated herein by reference in their entirety, particularly for their disclosure of pI variants. Preferred combinations of pI variants are shown in Figures 3 and 4. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be modified in this manner, as can isotype hybrids. R133E and R133Q can also be used when the heavy chain constant domain is derived from IgG2-4.
[0283] In one embodiment, a preferred combination of pI variants has one monomer (negative Fab side) comprising the 208D / 295E / 384D / 418E / 421D variants (N208D / Q295E / N384D / Q418E / N421D for human IgG1) and a second monomer (positive scFv side) comprising a positively charged scFv linker comprising (GKPGS)4 (SEQ ID NO: 796). However, as will be appreciated by those skilled in the art, the first monomer comprises a CH1 domain comprising position 208. Thus, constructs that do not comprise a CH1 domain (e.g., in the case of antibodies that do not utilize a CH1 domain in one of their domains) may be used. In this case, a preferred negative pI mutated Fc set comprises 295E / 384D / 418E / 421D mutations (Q295E / N384D / Q418E / N421D when compared to human IgG1).
[0284] Thus, in some embodiments, one monomer has a set of substitutions from Figure 4 and the other monomer has a charged linker (which can be selected from those shown in Figure 6, either in the format of an scFv or a charged scFv linker, as that monomer comprises a charged domain linker, as shown in the format).
[0285] In some embodiments, modifications occur in the hinge of the Fc domain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, and 230, based on EU numbering. Thus, pI variants, particularly substitutions, can be made at one or more of positions 216-230, with 1, 2, 3, 4, or 5 mutations finding use. Similarly, all possible combinations are contemplated, alone or in conjunction with other pI variants in other domains.
[0286] Particular substitutions used to reduce the pI of the hinge domain include, but are not limited to, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, a deletion at position 225, a deletion at position 235, and a deletion or non-native alanine at position 236. In some cases, only pI substitutions are made in the hinge domain, while in other instances, these substitutions are added in any combination to other pI variants in other domains.
[0287] In some embodiments, mutations can be made within the CH2 region, including positions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327, 334, and 339, based on EU numbering. Note that changes at 233-236 can be made to increase effector function (along with 327A) in an IgG2 backbone. Similarly, all possible combinations of these 14 positions can be made; for example, an anti-CD28 or anti-B7H3 antibody provided herein can comprise a variant Fc domain with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CH2 pI substitutions.
[0288] Specific substitutions used to reduce the pI of the CH2 domain include, but are not limited to, unnatural glutamine or glutamic acid at position 274, unnatural phenylalanine at position 296, unnatural phenylalanine at position 300, unnatural valine at position 309, unnatural glutamic acid at position 320, unnatural glutamic acid at position 322, unnatural glutamic acid at position 326, unnatural glycine at position 327, unnatural glutamic acid at position 334, unnatural threonine at position 339, and all possible combinations within CH2 and with other domains.
[0289] In this embodiment, modifications may be independently and optionally selected from positions 355, 359, 362, 384, 389, 392, 397, 418, 419, 444, and 447 (EU numbering) in the CH3 region. Specific substitutions used to reduce the pI of the CH3 domain include, but are not limited to, an unnatural glutamine or glutamic acid at position 355, an unnatural serine at position 384, an unnatural asparagine or glutamic acid at position 392, an unnatural methionine at position 397, an unnatural glutamic acid at position 419, an unnatural glutamic acid at position 359, an unnatural glutamic acid at position 362, an unnatural glutamic acid at position 389, an unnatural glutamic acid at position 418, an unnatural glutamic acid at position 444, and a deletion or unnatural aspartic acid at position 447.
[0290] 3. Isotype variants Additionally, many embodiments of the subject heterodimeric antibodies rely on the "import" of pI amino acids at specific positions from one IgG isotype to another, thus reducing or eliminating the possibility of introducing undesirable immunogenicity into the variant. Some of these are shown in Figure 21 of U.S. Patent Application Publication No. 2014 / 0370013, incorporated herein by reference. Specifically, 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 vs. 7.31). By introducing IgG2 residues into the IgG1 backbone at specific positions, the pI of the resulting monomer is lowered (or increased), additionally exhibiting a longer serum half-life. For example, IgG1 has a glycine (pI 5.97) at position 137, while IgG2 has a glutamic acid (pI 3.22), and the introduction of glutamic acid affects the pI of the resulting protein. As described below, several amino acid substitutions are generally required to significantly affect the pI of the variant antibody, however, it should be noted that even changes within the IgG2 molecule can increase serum half-life, as discussed below.
[0291] In other embodiments, non-isotypic amino acid changes are made to reduce the overall charge state of the resulting protein (e.g., by changing from a high pI amino acid to a low pI amino acid) or to allow for structural tuning, such as for stability, as described in more detail below.
[0292] Additionally, by pI engineering both the heavy and light constant domains, significant changes can be seen in each monomer of the heterodimer. As discussed herein, a difference in pI of at least 0.5 between the two monomers can allow for separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point.
[0293] 4. Calculate pI The pI of each monomer of the antibodies provided herein depends on the pI of the variant heavy chain constant domain and the pI of the entire monomer, which may include the variant heavy chain constant domain and fusion partner. Thus, in some embodiments, the change in pI is calculated based on the variant heavy chain constant domain using the chart in Figure 19 of U.S. Patent Application Publication No. 2014 / 0370013. As discussed herein, which monomers to engineer is generally determined by the inherent pI of the Fv and scaffold. Alternatively, the pI of each monomer can be compared.
[0294] 5. pI variants that also confer better in vivo binding to FcRn If the pI variant reduces the pI of the monomer, the pI variant may have the additional advantage of improving serum retention in vivo.
[0295] Although still under investigation, it is believed that Fc regions have a longer half-life in vivo because Fc is sequestered upon binding to FcRn at pH 6 within endosomes (Ghetie and Ward, 1997 Immunol Today. 18(12):592-598, incorporated by reference in its entirety). The endosomal compartment then recycles Fc to the cell surface. Once the compartment opens to the extracellular space, a higher pH of approximately 7.4 induces Fc release into the bloodstream. 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 half-lives identical to wild-type Fc (Dall'Acqua et al. 2002 J. Immunol. 169:5171-5180, incorporated by reference in its entirety). The increased affinity of Fc for FcRn at pH 7.4 is thought to prevent Fc release into the bloodstream. Therefore, Fc mutations that increase the half-life of Fc in vivo ideally inhibit FcRn binding at lower pH while still allowing Fc release 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 key positions in the Fc / FcRn complex.
[0296] Recently, it has been suggested that antibodies with variable regions with lower isoelectric points may also have longer serum half-lives (Igawa et al., 2010 PEDS. 23(5):385-392, incorporated herein by reference in its entirety). However, this mechanism is still poorly understood. Furthermore, variable regions vary from antibody to antibody. As described herein, constant region variants with reduced pI and extended half-lives may provide a more modular approach to improving the pharmacokinetic properties of antibodies.
[0297] D. Additional Fc variants for additional functionality In addition to the heterodimerization variants outlined above, there are several useful Fc amino acid modifications 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, as outlined below.
[0298] Thus, the antibodies (heterodimers, as well as homodimers) provided herein can contain such amino acid modifications with or without the heterodimerization variants (e.g., pI variants and conformational variants) outlined herein, each set of variants independently and optionally can be included or excluded from a particular heterodimeric protein.
[0299] 1. FcγR variants There are several useful Fc substitutions that can be made to alter binding to one or more FcγR receptors. In certain embodiments, the subject antibody contains modifications that alter binding to one or more FcγR receptors (i.e., "FcγR variants"). Substitutions that result in increased binding as well as decreased binding can be useful. For example, increased binding to FcγRIIIa is generally known to result in increased ADCC (antibody-dependent cell-mediated cytotoxicity, i.e., a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize ligated antibodies on target cells and subsequently cause lysis of the target cells). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can also be beneficial in some situations. Amino acid substitutions used in the subject antibodies include those listed in U.S. Pat. Nos. 8,188,321 (especially FIG. 41) and 8,084,582, and U.S. Patent Publication Nos. 2006 / 0235208 and 2007 / 0148170, all of which are expressly incorporated herein by reference in their entirety, particularly with respect to variants that affect Fcγ receptor binding disclosed therein. Particular variants of 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. Such modifications may be contained in one or both Fc domains of the subject antibody.
[0300] In some embodiments, the subject antibodies comprise one or more Fc modifications that increase serum half-life. Fc substitutions used to increase binding to the FcRn receptor and increase serum half-life include, but are not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, and 259I / 308F / 428L, as specifically disclosed in U.S. Pat. No. 12 / 341,769, incorporated herein by reference in its entirety. Such modifications may be included in one or both Fc domains of the subject antibodies.
[0301] 2. Elimination Variant In some embodiments, heterodimeric antibodies (e.g., anti-B7H3 x anti-CD28 bispecific antibodies) comprise one or more modifications that reduce or eliminate 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 an additional mechanism of action. Such modifications are referred to as "FcγR-ablated variants" or "Fc knockout (FcKO or KO)" variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or eliminate 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 an additional mechanism of action. Thus, for example, in many embodiments, particularly in the use of bispecific antibodies that monovalently bind to CD28, it is generally desirable to eliminate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. In some embodiments, at least one of the Fc domains of the subject antibodies described herein comprises one or more Fcγ receptor-depleted variants, hi some embodiments, both of the Fc domains of the subject antibodies described herein comprise one or more Fcγ receptor-depleted variants. These truncation variants are shown in Figure 5, and each can be independently optionally included or excluded in preferred embodiments using truncation 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. Note that the deletion variants referred to herein eliminate FcγR binding but do not eliminate FcRn binding.
[0302] As is known in the art, the Fc domain of human IgG1 has the highest binding to Fcγ receptors, therefore, when the constant domain (or Fc domain) of the scaffold of a heterodimeric antibody is IgG1, deletion variants can be used. Alternatively, or in addition to deletion variants of the IgG1 background, mutation at glycosylation position 297 (generally to A or S) can, for example, significantly eliminate binding to FcγRIIIa. Since human IgG2 and IgG4 have naturally reduced binding to Fcγ receptors, these scaffolds can be used with or without deletion variants.
[0303] E. Combination of Heterodimers and Fc Variants As will be appreciated by those skilled in the art, all of the listed heterodimerization variants (including scubariant and / or pI variants) can be optionally and independently combined, so long as they maintain their "strandedness" or "monomer split." Additionally, all of these variants can be combined in any of the heterodimerization formats.
[0304] In the case of pI variants, while a specifically used embodiment is shown in the figure, other combinations can be generated following the basic rule of varying the pI difference between the two monomers to facilitate purification.
[0305] Additionally, any of the heterodimerization variants, skews, and pIs can be independently and optionally combined with Fc-depleted variants, Fc variants, FcRn variants, as generally outlined herein.
[0306] Heterodimeric 1+1 Fab-scFv-Fc, 2+1 Fab2-scFv-Fc, 1+1 CLC, and 2+1 CLC format antibodies include those in some embodiments. Exemplary combinations of variants are included in Figure 8. In some embodiments, the heterodimeric antibody comprises a combination of variants as shown in Figure 8. In certain embodiments, the antibody is a heterodimeric 11+1 Fab-scFv-Fc, 2+1 Fab2-scFv-Fc, 1+1 CLC, or 2+1 CLC format antibody.
[0307] F. Useful Antibody Formats As will be appreciated by those of skill in the art and as described in more detail below, the bispecific heterodimeric antibodies provided herein can be in several different configurations, generally as shown in Figures 33 and 34.
[0308] As will be appreciated by those skilled in the art, the heterodimeric formats of the present invention may have different valencies and may be bispecific. That is, the heterodimeric antibodies of the present invention may be bivalent and bispecific, or trivalent and bispecific, with a first antigen bound by two binding domains and a second antigen bound by a second binding domain. As outlined herein, when CD28 is one of the target antigens, it is preferred that CD28 binds only monovalently.
[0309] The present invention utilizes a CD28 antigen-binding domain in combination with a B7H3-binding domain. As will be appreciated by those skilled in the art, any collection of anti-CD28 CDRs, anti-CD28 variable light and heavy domains, Fabs, and scFvs as shown in any of the figures (see particularly Figures 16-21) can be used. Similarly, any of the anti-B7H3 antigen-binding domains can be used, and any of the CDRs, variable light and heavy domains, Fabs, and scFvs as shown in any of the figures (e.g., Figures 29-31) can be used, optionally and independently, in any combination.
[0310] 1.1+1 Fab-scFv-Fc format ("bottle opener") One heterodimeric antibody format of particular use in the bispecific antibodies (e.g., anti-CD28 x anti-B7H3 antibodies) of the subject matter provided herein is the "1+1 Fab-scFv-Fc" or "bottle opener" format, as shown in Figure 33A. A 1+1 Fab-scFv-Fc format antibody comprises a first monomer that is a "conventional" heavy chain (VH1-CH1-hinge-CH2-CH3), where VH1 is the first variable heavy chain domain and CH2-CH3 are the first Fc domain. The 1+1 Fab-scFv-Fc also comprises a light chain that comprises a first variable light chain domain VL1 and a constant light chain domain CL. The light chain interacts with VH1-CH1 of the first monomer to form a first antigen-binding domain, which is a Fab. The second monomer of the antibody comprises a second binding domain, which is a single-chain Fv ("scFv" as defined below), and a second Fc domain. The scFv comprises a second variable heavy domain (VH2) and a second variable light domain (VL2), with VH2 linked to VL2 using a chargeable scFv linker (see, e.g., Figure 6). The scFv is linked to the heavy chain using a domain linker (see, e.g., Figure 7). The two monomers are held together by the use of amino acid variants (e.g., heterodimerization variants described above) in the constant regions (e.g., Fc domain, CH1 domain, and / or hinge region) that promote the formation of heterodimeric antibodies, as described in more detail below. This structure is sometimes referred to herein as the "bottle opener" (format) due to its rough visual resemblance to a bottle opener. In some embodiments, 1+1 Fab-scFv-Fc format antibodies are bivalent antibodies.
[0311] The current "1+1 Fab-scFv-Fc" format has several distinct advantages: as is known in the art, antibody analogs that rely on two scFv constructs often have stability and aggregation issues, and in the present invention, "conventional" heavy and light chains are used. Additionally, in contrast to formats that rely on two heavy chains and two light chains, there is no problem with mispairing of heavy and light chains (e.g., pairing of heavy chain 1 with light chain 2).
[0312] In some embodiments of a 1+1 Fab-scFv-Fc format antibody, one of the first or second antigen-binding domains is a CD28-binding domain, and the other binding domain is a tumor-associated antigen (TAA)-binding domain. In some embodiments where the 1+1 Fab-scFv-Fc comprises a CD28-binding domain and a tumor-associated antigen (TAA)-binding domain, it is the scFv that binds to CD28 and the Fab that binds to the TAA. In some embodiments, the TAA is B7H3. An exemplary anti-B7H3 x anti-CD28 bispecific antibody in the 1+1 Fab-scFv-Fc format is shown in Figure 35A.
[0313] In some embodiments, the first and second Fc domains of the 1+1 Fab-scFv-Fc format antibody are variant Fc domains comprising heterodimerization variants (e.g., the sets of amino acid substitutions shown in Figures 3 and 9). Particularly useful heterodimerizing scubariants include S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W, and T366S / L368A / Y407V / Y349C:T366W / S354C (EU numbering). In exemplary embodiments, one of the first or second variant Fc domains comprises heterodimerized scubariant L368D / K370S and the other of the first or second variant Fc domains comprises heterodimerized scubariant S364K / E357Q, where numbering is according to EU numbering. In exemplary embodiments, the first variant Fc domain comprises heterodimerized scubariant L368D / K370S and the second variant Fc domain comprises heterodimerized scubariant S364K / E357Q, where numbering is according to EU numbering.
[0314] In some embodiments, the variant Fc domain comprises a deletion variant, including those shown in Figure 5. In some embodiments, each of the first and second variant Fc domains comprises the deletion variants E233P / L234V / L235A / G236_ / S267K, where numbering is according to EU numbering.
[0315] In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises a pI variant, including those shown in Figure 4. In an exemplary embodiment, the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0316] In an exemplary embodiment, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0317] In some embodiments, the scFv of the 1+1 Fab-scFv-Fc format antibodies provided herein comprises a charged scFv linker (including those shown in Figure 6). In some embodiments, the 1+1 Fab-scFv-Fc format antibodies provided herein comprise the FcRn variant M428L / N434S, numbering of which is in accordance with EU numbering. Follow the numbering.
[0318] In exemplary embodiments, the first variant Fc domain comprises heterodimerizing scubariant L368D / K370S, the second variant Fc domain comprises heterodimerizing scubariant S364K / E357Q, each of the first and second variant Fc domains comprises deletion variants E233P / L234V / L235A / G236 / S267K, and the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises pI variants N208D / Q295E / N384D / Q418E / N421D, where numbering is according to EU numbering. In some embodiments, the scFv of the 1+1 Fab-scFv-Fc format antibodies provided herein comprises a (GKPGS)4 charged scFv linker. In some embodiments, the 1+1 Fab-scFv-Fc format antibodies provided herein comprise the FcRn variant M428L / N434S, where the numbering is according to EU numbering.
[0319] In some embodiments, one of the first binding domain or the second binding domain binds to CD28, and the other binding domain binds to a tumor-associated antigen (TAA) (see Figure 34A). Any suitable CD28 binding domain, including any of the CD28 binding domains provided herein, can be included in the subject 1+1 Fab-scFv-Fc format antibodies. In some embodiments, the CD28 binding domain is one of the following CD28 binding domains or a variant thereof: 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, TG N1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and sequence listing).
[0320] In some embodiments of the mAb-scFv format, the anti-CD28 ABD is selected from the group consisting of SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO: 636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 and SEQ ID NO:672 and a VH domain having an amino acid sequence as set forth in SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675. 5, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718 and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.
[0321] In some embodiments, one of the first binding domain or the second binding domain of a 1+1 Fab-scFv-Fc format antibody binds to a tumor-associated antigen (TAA). Suitable TAAs include any of the TAAs disclosed herein. In an exemplary embodiment, the TAA is B7H3. Any suitable B7H3 binding domain, including any of the B7H3 binding domains provided herein, can be included in a subject 1+1 Fab-scFv-Fc format antibody. In some embodiments, the B7H3 binding domain is one of the following B7H3 binding domains or variants thereof: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704 (Figures 26 to 31 and Sequence Listing).
[0322] In some embodiments, the anti-B7H3 ABD is SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ 21, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584, and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:874 and SEQ ID NO:932.
[0323] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:946 and a variable light domain having the amino acid sequence of SEQ ID NO:950.
[0324] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:956 and a variable light domain having the amino acid sequence of SEQ ID NO:960.
[0325] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:964 and a variable light domain having the amino acid sequence of SEQ ID NO:968.
[0326] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:972 and a variable light domain having the amino acid sequence of SEQ ID NO:976.
[0327] In some embodiments, the 1+1 Fab-scFv-Fc format antibody comprises a first binding domain that binds CD28 and a second binding domain that binds B7H3. In some embodiments, the CD28 binding domain is one of the following CD28 binding domains or variants thereof: 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, TG N1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and sequence listing).
[0328] In some embodiments, the B7H3 binding domain is one of the following B7H3 binding domains or variants thereof: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb -D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb1 3, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6 850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, and m1704 (Figures 26-31 and Sequence Listing).
[0329] In some embodiments, the anti-B7H3 ABD is selected from a) SEQ ID NOs: 89 and 93 from ombramab, b) SEQ ID NOs: 97 and 101 from enoblituzumab, c) SEQ ID NOs: 105 and 109 from BRCA84D, d) SEQ ID NOs: 113 and 117 from BRCA69D, e) SEQ ID NOs: 121 and 125 from PRCA157, f) SEQ ID NOs: 129 and 133 from huPRCA157, g) SEQ ID NOs: 137 and 141 from Mab-D, h) SEQ ID NOs: 145 and 149 from humAb-D, i) SEQ ID NOs: 153 and 157 from m30, j) SEQ ID NOs: 154 and 155 from M30-H1-L4 k) SEQ ID NOs: 169 and 173 from SP265, l) SEQ ID NOs: 177 and 181 from S10-H50L58, m) SEQ ID NOs: 185 and 189 from 8H9, n) SEQ ID NOs: 193 and 197 from m852, o) SEQ ID NOs: 201 and 205 from m857, p) SEQ ID NOs: 209 and 213 from m8524, q) SEQ ID NOs: 217 and 221 from 1-1, r) SEQ ID NOs: 225 and 229 from 1-2, s) SEQ ID NOs: 233 and 237 from 1-4, t) SEQ ID NOs: 241 and 24 from 1-5 5, u) SEQ ID NOs: 249 and 253 from 1-7, v) SEQ ID NOs: 257 and 261 from 2-5, w) SEQ ID NOs: 265 and 269 from 2-8, x) SEQ ID NOs: 273 and 277 from chAb2, y) SEQ ID NOs: 281 and 285 from chAb3, z) SEQ ID NOs: 289 and 293 from chAb4, aa) SEQ ID NOs: 297 and 301 from chAb18, bb) SEQ ID NOs: 305 and 309 from chAb13, cc) SEQ ID NOs: 313 and 317 from chAb12, dd) SEQ ID NOs: 321 and 325 from chAb14, e e) SEQ ID NOs: 329 and 333 from chAb6, ff) SEQ ID NOs: 337 and 341 from chAb11, gg) SEQ ID NOs: 345 and 349 from chAb16, hh) SEQ ID NOs: 353 and 357 from chAb10, ii) SEQ ID NOs: 361 and 365 from ChAb7, jj) SEQ ID NOs: 369 and 373 from chAb8, kk) SEQ ID NOs: 377 and 381 from chAb17, ll) SEQ ID NOs: 385 and 389 from chAb5, mm) SEQ ID NOs: 393 and 397 from huAb3v2.5, nn) huAb3v2.pp) SEQ ID NOs: 401 and 405 from TPP-5706, pp) SEQ ID NOs: 409 and 413 from huAb13v1, qq) SEQ ID NOs: 417 and 421 from TPP-5706, rr) SEQ ID NOs: 425 and 429 from TPP-6642, ss) SEQ ID NOs: 433 and 437 from TPP-6850, tt) SEQ ID NOs: 441 and 445 from TPP-3803, uu) SEQ ID NOs: 449 and 453 from TRL4542, vv) SEQ ID NOs: 457 and 461 from h1702, ww) SEQ ID NOs: 465 and 469 from h1703, xx) SEQ ID NOs: 473 and 477 from huA3, yy) SEQ ID NOs: 481 and 485 from huA9, and zz) SEQ ID NOs: 489 and 493 from m1704. See Figure 17 of USSN 63 / 092,272. Figure 10 shows some exemplary Fc domain sequences useful in 1+1 Fab-scFv-Fc format antibodies. The "monomer 1" sequence shown in Figure 10 typically refers to the Fc domain of the "Fab-Fc heavy chain," and the "monomer 2" sequence refers to the Fc domain of the "scFv-Fc heavy chain." In addition, Figures 12-15 show exemplary CH1-hinge domains, CH1 domains, and hinge domains that can be included in the first or second monomer of a 1+1 Fab-scFv-Fc format. Furthermore, Figure 16 shows convenient CL sequences that can be used in this format.
[0330] 2. 2+1 Fab2-scFv-Fc format (central-scFv format) One heterodimeric antibody format of particular use in the bispecific antibodies (e.g., anti-CD28 x anti-B7H3 antibodies) of the subject matter provided herein is the 2+1 Fab2-scFv-Fc format (also referred to as the "central-scFv format") shown in Figure 33B. This antibody format contains two Fab portions and a monomeric scFv. The 2+1 Fab2-scFv-Fc format antibody comprises three antigen-binding domains of scFv inserted between the VH-CH1 and CH2-CH3 regions of one of the Fab domains. In some embodiments of this format, each Fab portion binds to a tumor-associated antigen (TAA), and the "extra" scFv domain binds to CD28. In some embodiments, the 2+1 Fab2-scFv-Fc format antibody is a trivalent antibody.
[0331] In some embodiments of the 2+1 Fab2-scFv-Fc format, the first monomer comprises a standard heavy chain (i.e., VH1-CH1-hinge-CH2-CH3), where VH1 is the first variable heavy domain and CH2-CH3 is the first Fc domain. The second monomer comprises an scFv comprising another first variable heavy domain (VH1), a CH1 domain (and optional hinge), a second Fc domain, and an scFv variable light domain (VL2), an scFv linker, and an scFv variable heavy domain (VH2). The scFv is covalently linked between the C-terminus of the CH1 domain of the second monomer and the N-terminus of the Fc domain of the second monomer using an optional domain linker (VH1-CH1-[optional linker]-VH2-scFv linker-VH2-[optional linker]-CH2-CH3, or in the opposite orientation to the scFv, VH1-CH1-[optional linker]-VL2-scFv linker-VH2-[optional linker]-CH2-CH3). The optional linker can be any suitable peptide linker, including, for example, the domain linkers included in FIG. 7. This embodiment further utilizes a common light chain comprising a variable light domain (VL1) and a constant light domain (CL). The common light chain associates with the VH1-CH1 of the first and second monomers to form two identical Fabs. In some embodiments, each of the identical Fabs binds a tumor-associated antigen (e.g., B7H3). For many of the embodiments herein, these constructs may include scuba variants, pI variants, deletion variants, additional Fc variants, etc., as desired and described herein.
[0332] In some embodiments, the first and second Fc domains of the 2+1 Fab2-scFv-Fc format antibody are variant Fc domains comprising heterodimerization variants (e.g., the sets of amino acid substitutions shown in Figures 3 and 9). Particularly useful heterodimerizing scubariants include S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W, and T366S / L368A / Y407V / Y349C:T366W / S354C (EU numbering). In exemplary embodiments, one of the first or second variant Fc domains comprises heterodimerized scubariant L368D / K370S and the other of the first or second variant Fc domains comprises heterodimerized scubariant S364K / E357Q, where numbering is according to EU numbering. In exemplary embodiments, the first variant Fc domain comprises heterodimerized scubariant L368D / K370S and the second variant Fc domain comprises heterodimerized scubariant S364K / E357Q, where numbering is according to EU numbering.
[0333] In some embodiments, the variant Fc domain comprises a deletion variant, including those shown in Figure 5. In some embodiments, each of the first and second variant Fc domains comprises the deletion variants E233P / L234V / L235A / G236_ / S267K, where numbering is according to EU numbering.
[0334] In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises a pI variant (including those shown in Figure 4). In an exemplary embodiment, the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises a pI variant Includes N208D / Q295E / N384D / Q418E / N421D, numbering follows EU numbering.
[0335] In some embodiments, the scFv of the 2+1 Fab2-scFv-Fc format antibodies provided herein comprises a charged scFv linker, including those shown in Figure 6. In some embodiments, the 2+1 Fab2-scFv-Fc format antibodies provided herein comprise the FcRn variant M428L / N434S, where the numbering is according to EU numbering.
[0336] In exemplary embodiments, the first variant Fc domain comprises heterodimerizing scuba variants L368D / K370S, the second variant Fc domain comprises heterodimerizing scuba variants S364K / E357Q, each of the first and second variant Fc domains comprises deletion variants E233P / L234V / L235A / G236 / S267K, and the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises pI variants N208D / Q295E / N384D / Q418E / N421D, where numbering is according to EU numbering. In some embodiments, the scFv of the 2+1 Fab2-scFv-Fc format antibodies provided herein comprises a (GKPGS)4 charged scFv linker. In some embodiments, the 2+1 Fab2-scFv-Fc format antibodies provided herein comprise the FcRn variant M428L / N434S, where the numbering is according to EU numbering.
[0337] In some embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0338] In some embodiments, the scFv of the second monomer of a 2+1 Fab2-scFv-Fc format antibody is CD28-binding, and the VH1 of the first and second monomers and the VL1 of the common light chain each form a binding domain that binds to a tumor-associated antigen (TAA, e.g., B7H3) (see Figure 34B). Any suitable CD28 binding domain, including any of the CD28 binding domains provided herein, can be included in a subject 2+1 Fab2-scFv-Fc format antibody. In some embodiments, the CD28 binding domain is one of the following CD28 binding domains or variants thereof: 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, TG N1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18 to 21 and 23, and sequence listing). In some embodiments of the mAb-scFv format, the anti-CD28 ABD is selected from the group consisting of SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:65 a VH domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 874, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 670, SEQ ID NO: 671 and SEQ ID NO: 672; and a VH domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 874, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, Sequence number 667, Sequence number 668, Sequence number 669, Sequence number 670, Sequence number 671, Sequence number 672, Sequence number NO: 673, Sequence number 674, Sequence number 675, Sequence number 676, Sequence number 677, Sequence number 678, Sequence number 679, Sequence number 680, Sequence number 681, Sequence number 682, Sequence number 683, Sequence number 684, Sequence number 685, Sequence number 686, Sequence number 687, Sequence number 688, Sequence number 689, Sequence number 690, Sequence number 691, Sequence number 692, Sequence number 693, Sequence number 694, Sequence number 695, Sequence number 696, Sequence number 697 , SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728,and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.
[0339] In some embodiments, the VH1 of the first and second monomers and the VL1 of the common light chain of a 2+1 Fab2-scFv-Fc format antibody each form a binding domain that binds to a tumor-associated antigen (TAA) (see Figure 34B). Suitable TAAs include any of the TAAs disclosed herein. In an exemplary embodiment, the TAA is B7H3. Any suitable B7H3 binding domain, including any of the B7H3 binding domains provided herein, can be included in a subject 2+1 Fab2-scFv-Fc format antibody. In some embodiments, the B7H3 binding domain is one of the following B7H3 binding domains or variants thereof: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1 / 1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22 / 1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab, enoblitutuzumab, BRCA84D, BRCA6 9D, PRCA157, huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2 -5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5, huA b3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704 (Figures 26-31 and Sequence Listing). In some embodiments, anti-B7H3 ABDs are a) SEQ ID NOs: 89 and 93 from ombramab, b) SEQ ID NOs: 97 and 101 from enoblituzumab, c) SEQ ID NOs: 105 and 109 from BRCA84D, d) SEQ ID NOs: 113 and 117 from BRCA69D, e) SEQ ID NOs: 121 and 125 from PRCA157, f) SEQ ID NOs: 129 and 133 from huPRCA157, g) SEQ ID NOs: 137 and 141 from Mab-D, h) SEQ ID NOs: 145 and 149 from humAb-D, i) SEQ ID NOs: 153 and 154 from m30 157, j) SEQ ID NOs: 161 and 165 from M30-H1-L4, k) SP265 with SEQ ID NOs: 169 and 173, l) SEQ ID NOs: 177 and 181 from S10-H50L58, m) SEQ ID NOs: 185 and 189 from 8H9, n) SEQ ID NOs: 193 and 197 from m852, o) SEQ ID NOs: 201 and 205 from m857, p) SEQ ID NOs: 209 and 213 from m8524, q) SEQ ID NOs: 217 and 221 from 1-1, r) SEQ ID NOs: 225 and 229 from 1-2, s) SEQ ID NOs: 1-4 SEQ ID NOs: 233 and 237, t) SEQ ID NOs: 241 and 245 from 1-5, u) SEQ ID NOs: 249 and 253 from 1-7, v) SEQ ID NOs: 257 and 261 from 2-5, w) SEQ ID NOs: 265 and 269 from 2-8, x) SEQ ID NOs: 273 and 277 from chAb2, y) SEQ ID NOs: 281 and 285 from chAb3, z) SEQ ID NOs: 289 and 293 from chAb4, aa) SEQ ID NOs: 297 and 301 from chAb18, bb) SEQ ID NOs: 305 and 309 from chAb13, cc) c SEQ ID NOs: 313 and 317 from hAb12, dd) SEQ ID NOs: 321 and 325 from chAb14, ee) SEQ ID NOs: 329 and 333 from chAb6, ff) SEQ ID NOs: 337 and 341 from chAb11, gg) SEQ ID NOs: 345 and 349 from chAb16, hh) SEQ ID NOs: 353 and 357 from chAb10, ii) SEQ ID NOs: 361 and 365 from ChAb7, jj) SEQ ID NOs: 369 and 373 from chAb8, kk) SEQ ID NOs: 377 and 381 from chAb17,ll) SEQ ID NOs: 385 and 389 from chAb5, mm) SEQ ID NOs: 393 and 397 from huAb3v2.5, nn) SEQ ID NOs: 401 and 405 from huAb3v2.6, pp) SEQ ID NOs: 409 and 413 from huAb13v1, qq) SEQ ID NOs: 417 and 421 from TPP-5706, rr) SEQ ID NOs: 425 and 429 from TPP-6642, ss) SEQ ID NOs: 433 and 437 from TPP-6850, tt) TPP-3803 17) and 18) have a VH domain and a VL domain having an amino acid sequence selected from the pairs: SEQ ID NOs: 441 and 445 from TRL4542, uu) SEQ ID NOs: 449 and 453 from TRL4542, vv) SEQ ID NOs: 457 and 461 from h1702, ww) SEQ ID NOs: 465 and 469 from h1703, xx) SEQ ID NOs: 473 and 477 from huA3, yy) SEQ ID NOs: 481 and 485 from huA9, and zz) SEQ ID NOs: 489 and 493 from m1704. See Figure 17 of USSN 63 / 092,272.
[0340] In some embodiments, the anti-B7H3 ABD is SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ 26, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584, and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:874 and SEQ ID NO:932.
[0341] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:946 and a variable light domain having the amino acid sequence of SEQ ID NO:950.
[0342] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:956 and a variable light domain having the amino acid sequence of SEQ ID NO:960.
[0343] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:964 and a variable light domain having the amino acid sequence of SEQ ID NO:968.
[0344] In some embodiments, the anti-B7H3 ABD comprises a VH domain having the amino acid sequence of SEQ ID NO:972 and a variable light domain having the amino acid sequence of SEQ ID NO:976.
[0345] Figure 11 shows some exemplary Fc domain sequences useful in the 2+1 Fab2-scFv-Fc format. The "monomer 1" sequence shown in Figure 11 typically refers to the Fc domain of the "Fab-Fc heavy chain," and the "monomer 2" sequence refers to the Fc domain of the "Fab-scFv-Fc heavy chain." In addition, Figures 12-15 provide exemplary CH1-hinge domains, CH1 domains, and hinge domains that can be included in the first or second monomer of a 2+1 Fab2-scFv-Fc format. Furthermore, Figure 16 shows convenient CL sequences that can be used in this format. An exemplary anti-B7H3 x anti-CD28 bispecific antibody in a 2+1 Fab2-scFv-Fc format is shown in Figure 36.
[0346] 3.1+1 CLC format One heterodimeric antibody format of particular use in the subject bispecific antibodies provided herein (e.g., anti-CD28 x anti-B7H3 antibodies) is the "1+1 common light chain" or "1+1 CLC" format shown in Figure 33C. A 1+1 CLC format antibody comprises a first monomer comprising VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 is a first Fc domain; a second monomer comprising VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain; and a third monomer "common light chain" comprising VL-CL, where VL is a common variable light domain and CL is a constant light domain. In such embodiments, VL pairs with VH1 to form a first binding domain with a first antigen-binding specificity, and VL pairs with VH2 to form a second binding domain with a second antigen-binding specificity. In some embodiments, the 1+1 CLC format antibody is a bivalent antibody.
[0347] In some embodiments, the first and second Fc domains in a 1+1 CLC format is a variant Fc domain comprising a heterodimerizing scubariant (e.g., the set of amino acid substitutions shown in Figures 3 and 9). Particularly useful heterodimerizing scubariants include S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W, and T366S / L368A / Y407V / Y349C:T366W / S354C (EU numbering). In exemplary embodiments, one of the first or second variant Fc domains comprises heterodimerized scubariant L368D / K370S and the other of the first or second variant Fc domains comprises heterodimerized scubariant S364K / E357Q, where numbering is according to EU numbering. In exemplary embodiments, the first variant Fc domain comprises heterodimerized scubariant L368D / K370S and the second variant Fc domain comprises heterodimerized scubariant S364K / E357Q, where numbering is according to EU numbering.
[0348] In some embodiments, the variant Fc domain comprises a deletion variant, including those shown in Figure 5. In some embodiments, each of the first and second variant Fc domains comprises the deletion variants E233P / L234V / L235A / G236_ / S267K, where numbering is according to EU numbering.
[0349] In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of the first or second monomer comprises a pI variant, including those shown in Figure 4. In an exemplary embodiment, the constant domain (CH1-hinge-CH2-CH3) of the first or second monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, where the numbering is according to EU numbering.
[0350] In some embodiments, the 1+1 CLC format antibodies provided herein comprise the FcRn variant M428L / N434S, where the numbering is according to EU numbering.
[0351] In an exemplary embodiment, the first variant Fc domain comprises heterodimerizing scubariant L368D / K370S, the second variant Fc domain comprises heterodimerizing scubariant S364K / E357Q, each of the first and second variant Fc domains comprises deletion variants E233P / L234V / L235A / G236 / S267K, and the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises pI variants N208D / Q295E / N384D / Q418E / N421D, where numbering is according to EU numbering.
[0352] In some embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where numbering is according to EU numbering.
[0353] In some embodiments, the 1+1 CLC format antibodies provided herein further comprise the FcRn variant M428L / N434S, where the numbering is according to EU numbering.
[0354] In some embodiments, one of the first binding domain or the second binding domain binds to CD28, and the other binding domain binds to a tumor-associated antigen (TAA) (Figure 34C (See, e.g., ). Any suitable CD28 binding domain, including any of the CD28 binding domains provided herein, can be included in a subject 1 + 1 CLC format antibody. In some embodiments, the CD28 binding domain is one of the following CD28 binding domains or variants thereof: 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, TG N1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (Figures 18-21 and 23, and the Sequence Listing). In exemplary embodiments, the CD28 binding domain comprises a 1A7[CD28]_H1.14 variable heavy chain domain. In some embodiments, the CD28 binding domain comprises a 1A7[CD28]_H1.14 variable heavy chain domain or a variant thereof and a variable light chain domain of any of the CD28 binding domains provided herein. In exemplary embodiments, the CD28 binding domain is 1A7[CD28]_H1.14L1 or a variant thereof. In some embodiments of the mAb-scFv format, the anti-CD28 ABDs are SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627,SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO: a VH domain having an amino acid sequence selected from the group consisting of SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 and SEQ ID NO:672, and a VH domain having an amino acid sequence selected from the group consisting of SEQ ID NO:674, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, ...75, SEQ ID NO:676, SEQ ID No. 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705 05, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, and a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.
[0355] In some embodiments, one of the first binding domain or the second binding domain of the 1+1 CLC format antibody binds to a tumor-associated antigen (TAA). Suitable TAAs include any of the TAAs disc...
Claims
1. A heterodimeric antibody, a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-VH1-CH1-hinge-CH2-CH3, where VH1 is each a first variable heavy domain and CH2-CH3 are each a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain; c) a common light chain comprising, from N-terminus to C-terminus, VL-CL, where VL is a variable light chain domain and CL is a constant light chain domain; the common light chain separately pairs with each VH1-CH1 in the first monomer and with each VH2-CH1 in the second monomer; A heterodimeric antibody, wherein the VH1 and the VL together form a first antigen-binding domain (ABD), the VH2 and the VL together form a second ABD, one of the first and second ABDs binds to human CD28, and the other of the first and second ABDs binds to human B7H3.
2. The heterodimeric antibody of claim A1, wherein the first ABD binds to human CD28 and the second ABD binds to human B7H3.
3. The heterodimeric antibody of claim A1, wherein the first ABD binds to human B7H3 and the second ABD binds to human CD28.
4. the amino acid sequence of the VH1 domain is SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, 4. The heterodimeric antibody of claim 3, wherein the VL domain is selected from the group consisting of SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584, and wherein the amino acid sequence of the VL domain is SEQ ID NO:
874.
5. The amino acid sequence of the VH2 domain is SEQ ID NO:585, SEQ ID NO:870, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:6 06, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629 30, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, and SEQ ID NO:
651. The heterodimeric antibody of any one of claims 1 to 4, wherein the antibody is selected from the group consisting of:
6. The heterodimerized antibody according to any one of claims 1 to 5, wherein the first Fc domain and the second Fc domain are each a variant Fc domain.
7. 7. The heterodimeric antibody of claim 6, wherein the first and second Fc domains comprise a set of heterodimeric 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.
8. 8. The heterodimeric antibody of claim 7, wherein the first and second Fc domains comprise the heterodimeric variant S364K / E357Q:L368D / K370S.
9. The heterodimeric antibody of any one of claims 1 to 7, wherein the first and second Fc domains each comprise one or more deletion variants.
10. The heterodimeric antibody of claim 9, wherein the one or more deletion variants are E233P / L234V / L235A / G236del / S267K and the numbering is according to EU numbering.
11. The heterodimeric antibody of any one of claims 1 to 10, wherein one of the first or second monomers further comprises a pI variant.
12. The heterodimeric antibody of claim 11, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variant N208D / Q295E / N384D / Q418E / N421D, and the numbering is according to EU numbering.
13. the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K; the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K; A heterodimeric antibody according to any one of claims 1 to 12, wherein the numbering is according to EU numbering.
14. The heterodimeric antibody of any one of claims 1 to 13, wherein the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
15. The heterodimeric antibody of any one of claims 1 to 14, wherein the second monomer comprises the amino acid sequence of SEQ ID NO: 1019, the first monomer comprises the amino acid sequence of SEQ ID NO: 1020, and the light chain has the amino acid sequence of SEQ ID NO: 1021.
16. A heterodimeric antibody, a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where VH1 is a first variable heavy domain and CH2-CH3 are a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, where VH2 is a second variable heavy domain and CH2-CH3 is a second Fc domain; c) a common light chain comprising, from N-terminus to C-terminus, VL-CL, where VL is a variable light chain domain and CL is a constant light chain domain; a heterodimeric antibody, wherein the first VH domain and the VL domain together form a first ABD, and the second VH domain and the VL domain together form a second ABD, and one of the first and second ABDs binds to human CD28, and the other of the first and second ABDs binds to human B7H3.
17. the amino acid sequence of the VH1 domain is SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, 16. The heterodimeric antibody of claim 15, wherein the VL domain is selected from the group consisting of SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584, and wherein the amino acid sequence of the VL domain is SEQ ID NO:
874.
18. The amino acid sequence of the VH2 domain is SEQ ID NO:585, SEQ ID NO:870, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, Sequence number 609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, Sequence no.
18. The heterodimeric antibody of claim 16 or 17, selected from the group consisting of SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:
651.
19. The heterodimerized antibody of any one of claims 16 to 18, wherein the first Fc domain and the second Fc domain are each a variant Fc domain.
20. 20. The heterodimeric antibody of claim 19, wherein the first and second Fc domains comprise a set of heterodimeric 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.
21. 21. The heterodimeric antibody of claim 20, wherein the first and second Fc domains comprise the heterodimeric variant S364K / E357Q:L368D / K370S.
22. The heterodimeric antibody of any one of claims 16 to 21, wherein the first and second Fc domains each comprise one or more deletion variants.
23. 23. The heterodimeric antibody of claim 22, wherein the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, and the numbering is according to EU numbering.
24. A heterodimeric antibody according to any one of claims 16 to 23, wherein one of the first or second monomers further comprises a pI variant.
25. 25. The heterodimeric antibody of claim 24, wherein the CH1-hinge-CH2-CH3 of the first monomer comprises the pI variant N208D / Q295E / N384D / Q418E / N421D, and the numbering is according to EU numbering.
26. wherein the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K; the second Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K; A heterodimeric antibody according to any one of claims 16 to 25, wherein the numbering is according to EU numbering.
27. 29. The heterodimeric antibody of any one of claims 16 to 28, wherein the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
28. A heterodimeric antibody, a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-first domain linker-scFv-second domain linker-CH2-CH3; a first monomer, wherein VH1 is a first variable heavy domain, the scFv is an anti-CD28 scFv, and CH2-CH3 is a first Fc domain; b) a third amino acid sequence comprising, from N- to C-terminus, VH1-CH1-hinge-CH2-CH3; a second monomer of 2, wherein CH2-CH3 is a second Fc domain; and c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, where VL1 is a variable light chain domain and CL is a constant light chain domain; a heterodimeric antibody, wherein each of the VH1 domain and the first VL1 domain together form a first antigen-binding domain (ABD); the scFv comprises a second VH domain (VH2), an scFv linker, and a second VL domain (VL2), wherein the VH2 and the VL2 form a second ABD, and one of the first and second ABDs binds to human CD28, and the other of the first and second ABDs binds to a tumor target antigen (TTA).
29. 29. The heterodimeric antibody of claim 28, wherein the first ABD binds to human CD28 and the second ABD binds to TTA.
30. 29. The heterodimeric antibody of claim 28, wherein the first ABD binds to TTA and the second ABD binds to human CD28.
31. The heterodimeric antibody of any one of claims 28 to 30, wherein the scFv comprises, from the N-terminus to the C-terminus, VL2-scFv linker-VH2.
32. The heterodimeric antibody of any one of claims 28 to 30, wherein the scFv comprises, from the N-terminus to the C-terminus, VH2-scFv linker-VL2.
33. The amino acid sequence of the VH2 is SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO: No. 613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO: and the amino acid sequence of VL2 is selected from the group consisting of SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651, and the amino acid sequence of VL2 is selected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, S Sequence number 669, sequence number 670, sequence number 671, sequence number 672, sequence number NO: 673, sequence number 674, sequence number 675, sequence number 676, sequence number 677, sequence number 678, sequence number 679, sequence number 680, sequence number 681, sequence number 682, sequence number 683, sequence number 684, sequence number 685, sequence number 686, sequence number 687, sequence number 688, sequence number 689, sequence number 690, sequence number 691, sequence number 692, sequence number 693, sequence number 694, sequence number 695, sequence number 696, sequence number 697, sequence number 698, sequence number 699,SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, SEQ ID NO: 731, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736, SEQ ID NO: 737, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 748, SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752, SEQ ID NO: 753, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 1200 and SEQ ID NO:
756. The heterodimeric antibody of any one of claims 28 to 32, selected from the group consisting of:
34. The heterodimeric antibody of any one of claims 28 to 33, wherein the TTA is human B7H3.
35. The heterodimerized antibody of any one of claims 28 to 34, wherein the first Fc domain and the second Fc domain are each a variant Fc domain.
36. 36. The heterodimeric antibody of claim 35, wherein the first and second Fc domains comprise a set of heterodimeric 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.
37. 37. The heterodimeric antibody of claim 36, wherein the first and second Fc domains comprise the heterodimeric variant S364K / E357Q:L368D / K370S.
38. The heterodimeric antibody of any one of claims 28 to 37, wherein the first and second Fc domains each comprise one or more deletion variants.
39. 39. The heterodimeric antibody of claim 38, wherein the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, and the numbering is according to EU numbering.
40. The heterodimeric antibody of any one of claims 28 to 39, wherein one of the first or second monomers further comprises a pI variant.
41. The heterodimeric antibody of claim 40, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variant N208D / Q295E / N384D / Q418E / N421D, and the numbering is according to EU numbering.
42. the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K; the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K; A heterodimeric antibody according to any one of claims 28 to 41, wherein the numbering is according to EU numbering.
43. the first and second variant Fc domains each comprising the amino acid variant 428L A heterodimeric antibody according to any one of claims 28 to 42, comprising / 434S.
44. A heterodimeric antibody, a) a first monomer, i) an scFv comprising a first variable heavy domain, an scFv linker, and a first variable light domain; ii) a first Fc domain, wherein the scFv is covalently linked to the N-terminus of the first Fc domain using a domain linker; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, where VH is a first variable heavy domain and CH2-CH3 is a second Fc domain; c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, where VL1 is a variable light chain domain and CL is a constant light chain domain; a heterodimeric antibody, wherein the VH1 and the VL1 together form a first ABD, the scFv comprises a second VH domain (VH2), an scFv linker, and a second VL domain (VL2), the VH2 and VL2 together form a second ABD, one of the first ABD and the second ABD binds to CD28, and the other of the first ABD and the second ABD binds to TTA.
45. The heterodimeric antibody of claim 44, wherein the scFv comprises, from N-terminus to C-terminus, VL2-scFv linker-VH2.
46. The heterodimeric antibody of claim 44, wherein the scFv comprises, from the N-terminus to the C-terminus, VH2-scFv linker-VL2.
47. the second ABD binds to human CD28, and the amino acid sequence of the VH2 is 0, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639 9, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:651, and the amino acid sequence of the VL2 is selected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666 66, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696 697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:
47. The heterodimeric antibody of any one of claims 44 to 46, wherein the antibody is selected from the group consisting of SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:
756.
48. The heterodimerized antibody of any one of claims 44 to 47, wherein the first Fc domain and the second Fc domain are each a variant Fc domain.
49. 49. The heterodimeric antibody of claim 48, wherein the first and second Fc domains comprise a set of heterodimeric 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.
50. 50. The heterodimeric antibody of claim 49, wherein the first and second Fc domains comprise the heterodimeric variant S364K / E357Q:L368D / K370S.
51. The heterodimeric antibody of any one of claims 44 to 50, wherein the first and second Fc domains each comprise one or more deletion variants.
52. 52. The heterodimeric antibody of claim 51, wherein the one or more deletion variants are E233P / L234V / L235A / G236del / S267K, and the numbering is according to EU numbering.
53. The heterodimeric antibody of any one of claims 44 to 52, wherein one of the first or second monomers further comprises a pI variant.
54. 54. The heterodimeric antibody of claim 53, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variant N208D / Q295E / N384D / Q418E / N421D, and the numbering is according to EU numbering.
55. the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K; the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K; The heterodimeric antibody of any one of claims 44 to 54, wherein the numbering is according to EU numbering.
56. 56. The heterodimeric antibody of any one of claims 44 to 55, wherein the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
57. A heterodimeric antibody, a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3-domain linker-scFv, a first monomer, wherein VH1 is a first variable heavy domain, the scFv is an anti-CD28 scFv, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N- to C-terminus, VH1-CH1-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and c) a light chain comprising, from N-terminus to C-terminus, VL1-CL, where VL1 is a variable light chain domain and CL is a constant light chain domain; a heterodimeric antibody, wherein each of the VH1 domain and the first VL1 domain together form a first antigen-binding domain (ABD); the scFv comprises a second VH domain (VH2), an scFv linker, and a second VL domain (VL2), wherein the VH2 and the VL2 together form a second ABD, and one of the first and second ABDs binds to human CD28, and the other of the first and second ABDs binds to a tumor target antigen (TTA).
58. 58. The heterodimeric antibody of claim 57, wherein the first ABD binds to human CD28 and the second ABD binds to TTA.
59. 58. The heterodimeric antibody of claim 57, wherein the first ABD binds to TTA and the second ABD binds to human CD28.
60. The heterodimeric antibody of any one of claims 57 to 59, wherein the scFv comprises, from the N-terminus to the C-terminus, VL2-scFv linker-VH2.
61. The heterodimeric antibody of any one of claims 57 to 59, wherein the scFv comprises, from the N-terminus to the C-terminus, VH2-scFv linker-VL2.
62. The amino acid sequence of the VH2 is SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO: and the amino acid sequence of VL2 is selected from the group consisting of SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ ID NO:
651. 670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:7 16, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:
756. The heterodimeric antibody of any one of claims 57 to 61, selected from the group consisting of:
63. The heterodimeric antibody of any one of claims 57 to 62, wherein the TTA is human B7H3.
64. A heterodimerized antibody according to any one of claims 57 to 63, wherein the first Fc domain and the second Fc domain are each a variant Fc domain.
65. 65. The heterodimeric antibody of claim 64, wherein the first and second Fc domains comprise a set of heterodimeric 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.
66. 66. The heterodimeric antibody of claim 65, wherein the first and second Fc domains comprise the heterodimeric variant S364K / E357Q:L368D / K370S.
67. The heterodimeric antibody of any one of claims 57 to 66, wherein the first and second Fc domains each comprise one or more deletion variants.
68. 68. The heterodimeric antibody of claim 67, wherein the one or more deletion variants are E233P / L234V / L235A / G236del / S267K and the numbering is according to EU numbering.
69. The heterodimeric antibody of any one of claims 57 to 68, wherein one of the first or second monomers further comprises a pI variant.
70. 70. The heterodimeric antibody of claim 69, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pI variant N208D / Q295E / N384D / Q418E / N421D, and the numbering is according to EU numbering.
71. the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K; the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K; A heterodimeric antibody according to any one of claims 57 to 70, wherein the numbering is according to EU numbering.
72. 72. The heterodimeric antibody of any one of claims 57 to 71, wherein the first and second variant Fc domains each comprise the amino acid variants 428L / 434S.
73. a) a first nucleic acid encoding a first monomer according to any one of claims 1 to 72; b) a second nucleic acid encoding a second monomer according to any one of claims 1 to 72; and and c) a third nucleic acid encoding a light chain according to any one of claims 1 to 72.
74. a) a first expression vector comprising the first nucleic acid of claim 73; b) a second expression vector comprising the second nucleic acid of claim 73; and and c) a third expression vector comprising the third nucleic acid of claim 73.
75. 75. A host cell comprising the expression vector composition of claim 74.
76. 73. A method of making the dimeric antibody of any one of claims 1 to 72, comprising culturing the host cell of claim 75 under conditions in which the dimeric antibody is expressed, and recovering the dimeric antibody.
77. 73. A method of treating cancer in a patient in need thereof, comprising administering to said patient the heterodimeric antibody of any one of claims 1 to 72.
78. 1. A method of treating cancer in a patient in need thereof, comprising: a) the heterodimeric antibody of any one of claims 1 to 72, wherein said TTA is human B7H3; and b) administering to said patient a bispecific antibody that binds to CD3 and B7H3.
79. 1. A method of treating cancer in a patient in need thereof, comprising: a) the heterodimeric antibody of any one of claims 1 to 72, wherein said TTA is human B7H3; and b) administering to the patient a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody and an anti-PD-L1 antibody.
80. a) SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:6 23, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 and SEQ ID NO:672; b) SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:67 8, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706 , SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, and a variable light chain domain having an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:
756.
81. a) a heavy chain comprising VH-CH1-hinge-CH2-CH3; The composition of claim 80, which is an antibody comprising a light chain comprising VL-CL.
82. a) SEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, No. 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545, SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 549, SEQ ID NO: 550, SEQ ID NO: 551, SEQ ID NO: 552, SEQ ID NO: 553, SEQ ID NO: 554, SEQ ID NO: 555, SEQ ID NO: 556, SEQ ID NO: 557, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 56 a variable heavy chain domain having an amino acid sequence selected from the group consisting of SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583, and SEQ ID NO:584; b) a variable light chain domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 874 and SEQ ID NO:
932.
83. a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 946; b) a variable light chain domain having the amino acid sequence of SEQ ID NO:
950.
84. a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 956; b) a variable light chain domain having the amino acid sequence of SEQ ID NO:
960.
85. a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 964; b) a variable light chain domain having the amino acid sequence of SEQ ID NO:
968.
86. a) a variable heavy chain domain having the amino acid sequence of SEQ ID NO: 972; b) a variable light chain domain having the amino acid sequence of SEQ ID NO:
976.
87. a) a heavy chain comprising a VH linked to -CH1-hinge-CH2-CH3; b) a light chain comprising a VL linked to a CL; and b) a light chain comprising a VL linked to a CL.
88. a) a first nucleic acid encoding a VH of claim 80 or 82; and and b) a second nucleic acid encoding the VL of claim 80 or 82, respectively.
89. (a) a first expression vector comprising the first nucleic acid of claim 88; (b) a second expression vector comprising the second nucleic acid of claim 88.
90. 90. A host cell comprising the nucleic acid composition of claim 88 or the expression vector composition of claim 89.
91. 91. A method of producing a composition, comprising culturing the host cell of claim 90 under conditions in which the composition is expressed, and recovering the composition.
Citation Information
Patent Citations
Bispecific Anti-PSMA x Anti-CD28 antibodies and uses thereof
US20190389951A1