CD28 Bispecific Antibodies for Targeted T Cell Activation

Abstract

The present invention relates to an antibody arm that agonistically binds to human CD28. The present invention relates to a bispecific antibody that binds to human carcinoembryonic antigen (CEA) and human CD28 using the agonistic anti-CD28 arm described herein. The present invention relates to a bispecific antibody that binds to human mesothelin (MSLN) and human CD28 using the agonistic anti-CD28 arm described herein. TIFF2025508066000015.tif125128

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 317,491, filed March 7, 2022, and U.S. Provisional Patent Application No. 63 / 323,893, filed March 25, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Incorporation by reference of sequence listing The contents of the electronic sequence listing (NOVI_050_001WO_SeqList_ST26.xml, size: 129,730 bytes, and creation date: March 6, 2023) are incorporated herein by reference in their entirety.

[0003] Field The present invention relates generally to agonistic fully human and kappa-lambda compatible CD28 binding domains of defined affinity useful for the generation of bispecific antibodies (bsAbs). Specifically, the CD28 binding domains are paired with anti-tumor associated antigen (TAA) binding domains for the generation of TAAxCD28 bsAbs, particularly CEAxCD28 and MSLNxCD28 bispecific antibodies. [Background technology]

[0004] 2. Background of the Invention In the past few years, new ways have been developed to stimulate the body's own immune cells to better attack and kill cancer cells. Examples of successful cancer immunotherapy are monoclonal antibodies that are able to block so-called immune checkpoints. Currently approved immune checkpoint inhibitors (ICIs) block CTLA-4 (e.g., Ipilimumab, sold under the trade name Yervoy), PD-1 (e.g., Pembrolizumab, sold under the trade names Keytruda and Cemiplimab, sold under the trade name Libtayo) and PD-L1 (e.g., Atezolizumab, sold under the trade name Tecentriq). Sustainable antitumor responses can be obtained in various cancer types using ICIs. Unfortunately, responses are limited to a subset of patients and many cancer types are known to be essentially resistant to ICI monotherapy.

[0005] Other approved cancer immunotherapies include T cell bispecific antibodies, which cross-link T cells to target cells expressing tumor-associated antigens (TAA) via the CD3 receptor on T cells and chimeric antigen receptor (CAR) T cells. Despite very good antitumor responses observed with treatments using T cell bispecific antibodies or CAR T cells in hematological malignancies, there have been no significant breakthroughs of these approaches to date in the context of solid tumors, leaving many cancer patients without treatment options.

[0006] Thus, despite the success of these immunotherapies in some cancer types, the majority of cancer patients lack effective treatment options, highlighting the need for new therapies.The use of molecules capable of activating the immune system by targeting costimulatory signals on T cells has not been thoroughly explored and may pave the way for novel treatment options for patients with solid tumors.

[0007] There is a need for compositions and methods for targeting T cell activation useful in the treatment of solid cancers. Provided herein are methods and compositions that address this need. Summary of the Invention

[0008] The present disclosure provides a bispecific antibody, the bispecific antibody comprising: a. a first antigen-binding domain that binds to CD28, the first antigen-binding domain having: i. a first heavy chain variable region having a complementarity determining region 1 (CDRH1) comprising the amino acid sequence of (SEQ ID NO:1), a complementarity determining region 2 (CDRH2) comprising the amino acid sequence of (SEQ ID NO:2), and a complementarity determining region 3 (CDRH3) comprising the amino acid sequence of (SEQ ID NO:3); and ii. a first heavy chain variable region having: 1. a CDRL1 comprising the amino acid sequence of SEQ ID NO:23, a CDRL2 comprising the amino acid sequence of SEQ ID NO:24, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:25. 1. CDRL1 comprising the amino acid sequence of SEQ ID NO:26, CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and CDRL3 comprising the amino acid sequence of SEQ ID NO:28; 2. CDRL1 comprising the amino acid sequence of SEQ ID NO:29, CDRL2 comprising the amino acid sequence of SEQ ID NO:30, and CDRL3 comprising the amino acid sequence of SEQ ID NO:31; 3. CDRL1 comprising the amino acid sequence of SEQ ID NO:32, CDRL2 comprising the amino acid sequence of SEQ ID NO:33, and CDRL3 comprising the amino acid sequence of SEQ ID NO:34; 4. CDRL1 comprising the amino acid sequence of SEQ ID NO:32, CDRL2 comprising the amino acid sequence of SEQ ID NO:33, and CDRL3 comprising the amino acid sequence of SEQ ID NO:34; 5. CDR comprising the amino acid sequence of SEQ ID NO:35 L1, CDRL2 comprising the amino acid sequence of SEQ ID NO:36, and CDRL3 comprising the amino acid sequence of SEQ ID NO:37; 6. CDRL1 comprising the amino acid sequence of SEQ ID NO:38, CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and CDRL3 comprising the amino acid sequence of SEQ ID NO:40; 7. CDRL1 comprising the amino acid sequence of SEQ ID NO:41, CDRL2 comprising the amino acid sequence of SEQ ID NO:42, and CDRL3 comprising the amino acid sequence of SEQ ID NO:43; 8. CDRL1 comprising the amino acid sequence of SEQ ID NO:44, CDRL2 comprising the amino acid sequence of SEQ ID NO:45, and and CDRL3 comprising the amino acid sequence of SEQ ID NO: 46; 9. CDRL1 comprising the amino acid sequence of SEQ ID NO: 47, CDRL2 comprising the amino acid sequence of SEQ ID NO: 48, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 49; 10. CDRL1 comprising the amino acid sequence of SEQ ID NO: 50, CDRL2 comprising the amino acid sequence of SEQ ID NO: 51, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 52; 11. CDRL1 comprising the amino acid sequence of SEQ ID NO: 53, CDRL2 comprising the amino acid sequence of SEQ ID NO: 54, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 55; 12.13. CDRL1 comprising the amino acid sequence of SEQ ID NO:59, CDRL2 comprising the amino acid sequence of SEQ ID NO:60, and CDRL3 comprising the amino acid sequence of SEQ ID NO:61; 14. CDRL1 comprising the amino acid sequence of SEQ ID NO:62, CDRL2 comprising the amino acid sequence of SEQ ID NO:63, and CDRL3 comprising the amino acid sequence of SEQ ID NO:64; 15. CDRL1 comprising the amino acid sequence of SEQ ID NO:65, CDRL2 comprising the amino acid sequence of SEQ ID NO:66, and CDRL3 comprising the amino acid sequence of SEQ ID NO:67; or 16. CDRL1 comprising the amino acid sequence of SEQ ID NO:68, CDRL2 comprising the amino acid sequence of SEQ ID NO:69, and CDRL3 comprising the amino acid sequence of SEQ ID NO:70; 17. a. a first antigen-binding domain comprising a first light chain variable region having CDRL1 comprising the amino acid sequence of SEQ ID NO: 1, CDRL2 comprising the amino acid sequence of SEQ ID NO: 72, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 73; 18. a first light chain variable region having CDRL1 comprising the amino acid sequence of SEQ ID NO: 74, CDRL2 comprising the amino acid sequence of SEQ ID NO: 75, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 76; or 19. a first light chain variable region having CDRL1 comprising the amino acid sequence of SEQ ID NO: 77, CDRL2 comprising the amino acid sequence of SEQ ID NO: 78, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 79; and b. a second antigen-binding domain that binds to a tumor-associated antigen (TAA), comprising i. a second heavy chain variable region having CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 3.

[0009] In some embodiments, the TAA is CEA. In some aspects, the TAA is mesothelin (MSLN).

[0010] In some embodiments, a. the first light chain variable region of part a.ii.1. comprises the amino acid sequence of SEQ ID NO: 80; b. the first light chain variable region of part a.ii.2. comprises the amino acid sequence of SEQ ID NO: 81; c. the first light chain variable region of part a.ii.3. comprises the amino acid sequence of SEQ ID NO: 82; d. the first light chain variable region of part a.ii.4. comprises the amino acid sequence of SEQ ID NO: 83; or e. the first light chain variable region of part a.ii.5. comprises the amino acid sequence of SEQ ID NO: or f. the first light chain variable region of part a.ii.6. comprises the amino acid sequence of SEQ ID NO: 85; g. the first light chain variable region of part a.ii.7. comprises the amino acid sequence of SEQ ID NO: 86; h. the first light chain variable region of part a.ii.8. comprises the amino acid sequence of SEQ ID NO: 87; i. the first light chain variable region of part a.ii.9. comprises the amino acid sequence of SEQ ID NO: 88; k. the first light chain variable region of part a.ii.11. comprises the amino acid sequence of SEQ ID NO: 90; l. the first light chain variable region of part a.ii.12. comprises the amino acid sequence of SEQ ID NO: 91; m. the first light chain variable region of part a.ii.13. comprises the amino acid sequence of SEQ ID NO: 92; n. the first light chain variable region of part a.ii.14. comprises the amino acid sequence of SEQ ID NO: 93; o. part a.ii.15. . the first light chain variable region of part a.ii.16. comprises the amino acid sequence of SEQ ID NO: 95; q. the first light chain variable region of part a.ii.17. comprises the amino acid sequence of SEQ ID NO: 96; r. the first light chain variable region of part a.ii.18. comprises the amino acid sequence of SEQ ID NO: 97; or s. the first light chain variable region of part a.ii.19. comprises the amino acid sequence of SEQ ID NO: 98.

[0011] In some embodiments, a. the first light chain of part a.ii.1. comprises the amino acid sequence of SEQ ID NO: 99; b. the first light chain of part a.ii.2. comprises the amino acid sequence of SEQ ID NO: 100; c. the first light chain of part a.ii.3. comprises the amino acid sequence of SEQ ID NO: 101; d. the first light chain of part a.ii.4. comprises the amino acid sequence of SEQ ID NO: 102; e. the first light chain of part a.ii.5. comprises the amino acid sequence of SEQ ID NO: 1 or f. the first light chain of part a.ii.6. comprises the amino acid sequence of SEQ ID NO: 104; g. the first light chain of part a.ii.7. comprises the amino acid sequence of SEQ ID NO: 105; h. the first light chain of part a.ii.8. comprises the amino acid sequence of SEQ ID NO: 106; i. the first light chain of part a.ii.9. comprises the amino acid sequence of SEQ ID NO: 107; or j. the first light chain of part a.ii.10. or k. the first light chain of part a.ii.11. comprises the amino acid sequence of SEQ ID NO: 109; or l. the first light chain of part a.ii.12. comprises the amino acid sequence of SEQ ID NO: 110; or m. the first light chain of part a.ii.13. comprises the amino acid sequence of SEQ ID NO: 111; or n. the first light chain of part a.ii.14. comprises the amino acid sequence of SEQ ID NO: 112; or o. part a.ii.1 5. the first light chain comprises the amino acid sequence of SEQ ID NO: 113; p. part a.ii.16. the first light chain comprises the amino acid sequence of SEQ ID NO: 114; q. part a.ii.17. the first light chain comprises the amino acid sequence of SEQ ID NO: 115; r. part a.ii.18. the first light chain comprises the amino acid sequence of SEQ ID NO: 116; or s. part a.ii.19. the first light chain comprises the amino acid sequence of SEQ ID NO: 117.

[0012] In some embodiments, the second antigen-binding domain comprises a second light chain variable region having ii.1. a CDR1 comprising the amino acid sequence of SEQ ID NO:8, a CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR3 comprising the amino acid sequence of SEQ ID NO:10; 2. a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13; or 3. a CDR1 comprising the amino acid sequence of SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0013] In some embodiments, a. the second light chain variable region of part b.ii.1. comprises the amino acid sequence of SEQ ID NO: 17; b. the second light chain variable region of part b.ii.2. comprises the amino acid sequence of SEQ ID NO: 18; or c. the second light chain variable region of part b.ii.3. comprises the amino acid sequence of SEQ ID NO: 19.

[0014] In some embodiments, the second light chain of a. part b.ii.1. comprises the amino acid sequence of SEQ ID NO: 20, the second light chain of b. part b.ii.2. comprises the amino acid sequence of SEQ ID NO: 21, or the second light chain of c. part b.ii.3. comprises the amino acid sequence of SEQ ID NO: 22.

[0015] In some embodiments, the second antigen-binding domain comprises a second light chain variable region having ii.1. a CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130; 2. a CDR1 comprising the amino acid sequence of SEQ ID NO: 131, a CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 133; 3. a CDR1 comprising the amino acid sequence of SEQ ID NO: 134, a CDR2 comprising the amino acid sequence of SEQ ID NO: 135, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 136; 4. a CDR1 comprising the amino acid sequence of SEQ ID NO: 137, a CDR2 comprising the amino acid sequence of SEQ ID NO: 138, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139, or 5. a CDR1 comprising the amino acid sequence of SEQ ID NO: 140, a CDR2 comprising the amino acid sequence of SEQ ID NO: 141, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 142.

[0016] In some embodiments, a. the second light chain variable region of part b.ii.1. comprises the amino acid sequence of SEQ ID NO: 143; b. the second light chain variable region of part b.ii.2. comprises the amino acid sequence of SEQ ID NO: 144; c. the second light chain variable region of part b.ii.3. comprises the amino acid sequence of SEQ ID NO: 145; d. the second light chain variable region of part b.ii.4. comprises the amino acid sequence of SEQ ID NO: 146; or e. the second light chain variable region of part b.ii.5. comprises the amino acid sequence of SEQ ID NO: 147.

[0017] In some embodiments, a. the second light chain of part b.ii.1. comprises the amino acid sequence of SEQ ID NO: 148; b. the second light chain of part b.ii.2. comprises the amino acid sequence of SEQ ID NO: 149; c. the second light chain of part b.ii.3. comprises the amino acid sequence of SEQ ID NO: 150; d. the second light chain of part b.ii.4. comprises the amino acid sequence of SEQ ID NO: 151; or e. the second light chain of part b.ii.5. comprises the amino acid sequence of SEQ ID NO: 152.

[0018] In some embodiments, the first heavy chain variable region and the second heavy chain variable region comprise the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first heavy chain and the second heavy chain comprise the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

[0019] In some embodiments, the first light chain is kappa and the second light chain is lambda, hi some embodiments, the first light chain is lambda and the second light chain is kappa.

[0020] In some embodiments, the bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce binding to an activating Fc receptor and / or reduce effector function. In some embodiments, the amino acid substitutions comprise L234A and L235A substitutions. In some embodiments, the amino acid substitutions comprise P329A, P329G, or P329R substitutions. In some embodiments, the antibody has an IgG isotype. In some embodiments, the antibody is human.

[0021] The present disclosure provides a composition comprising any one of the bispecific antibodies of the present disclosure. In some embodiments, the composition comprising the bispecific antibody enables tumor-specific T cell activation.

[0022] The present disclosure provides methods of reducing the proliferation of and / or killing tumor cells comprising contacting the cells with any one of the compositions of the present disclosure.

[0023] The present disclosure provides a method of treating cancer in a subject comprising administering to the subject any one of the compositions of the present disclosure.

[0024] The present disclosure provides an antibody comprising an antigen-binding domain that binds to CD28, the antigen-binding domain being selected from the group consisting of: 1. CDRL1 comprising the amino acid sequence of SEQ ID NO:23, CDRL2 comprising the amino acid sequence of SEQ ID NO:24, and CDRL3 comprising the amino acid sequence of SEQ ID NO:25; 2. CDRL1 comprising the amino acid sequence of SEQ ID NO:26, CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and CDRL3 comprising the amino acid sequence of SEQ ID NO:28; 3. CDRL1 comprising the amino acid sequence of SEQ ID NO:29, CDRL2 comprising the amino acid sequence of SEQ ID NO:30, and CDRL3 comprising the amino acid sequence of SEQ ID NO:31; 4. CDRL1 comprising the amino acid sequence of SEQ ID NO:32, CDRL2 comprising the amino acid sequence of SEQ ID NO:33, and CDRL3 comprising the amino acid sequence of SEQ ID NO:34; 5. CDRL1 comprising the amino acid sequence of SEQ ID NO:35, CDRL2 comprising the amino acid sequence of SEQ ID NO:36, and CDRL3 comprising the amino acid sequence of SEQ ID NO:37; 6. CDRL1 comprising the amino acid sequence of SEQ ID NO:38, CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and CDRL3 comprising the amino acid sequence of SEQ ID NO:40; 7. CDRL1 comprising the amino acid sequence of SEQ ID NO:41. 8. CDRL1 comprising the amino acid sequence of SEQ ID NO: 44, CDRL2 comprising the amino acid sequence of SEQ ID NO: 45, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 46; 9. CDRL1 comprising the amino acid sequence of SEQ ID NO: 47, CDRL2 comprising the amino acid sequence of SEQ ID NO: 48, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 49; 10. CDRL1 comprising the amino acid sequence of SEQ ID NO: 50, CDRL2 comprising the amino acid sequence of SEQ ID NO: 51, and and CDRL3 comprising the amino acid sequence of SEQ ID NO:52; 10. CDRL1 comprising the amino acid sequence of SEQ ID NO:53, CDRL2 comprising the amino acid sequence of SEQ ID NO:54, and CDRL3 comprising the amino acid sequence of SEQ ID NO:55; 12. CDRL1 comprising the amino acid sequence of SEQ ID NO:56, CDRL2 comprising the amino acid sequence of SEQ ID NO:57, and CDRL3 comprising the amino acid sequence of SEQ ID NO:58; 13. CDRL1 comprising the amino acid sequence of SEQ ID NO:59, CDRL2 comprising the amino acid sequence of SEQ ID NO:60, and CDRL3 comprising the amino acid sequence of SEQ ID NO:61; 14.CDRL1 comprising the amino acid sequence of SEQ ID NO:62, CDRL2 comprising the amino acid sequence of SEQ ID NO:63, and CDRL3 comprising the amino acid sequence of SEQ ID NO:64; 15. CDRL1 comprising the amino acid sequence of SEQ ID NO:65, CDRL2 comprising the amino acid sequence of SEQ ID NO:66, and CDRL3 comprising the amino acid sequence of SEQ ID NO:67; or 16. CDRL1 comprising the amino acid sequence of SEQ ID NO:68, CDRL2 comprising the amino acid sequence of SEQ ID NO:69, and CDRL3 comprising the amino acid sequence of SEQ ID NO:70; 17. CDRL1 comprising the amino acid sequence of SEQ ID NO:71, CDRL2 comprising the amino acid sequence of SEQ ID NO:72, and CDRL3 comprising the amino acid sequence of SEQ ID NO:73; 18. CDRL1 comprising the amino acid sequence of SEQ ID NO:74, CDRL2 comprising the amino acid sequence of SEQ ID NO:75, and CDRL3 comprising the amino acid sequence of SEQ ID NO:76; or 19. CDRL1 comprising the amino acid sequence of SEQ ID NO:77, CDRL2 comprising the amino acid sequence of SEQ ID NO:78, and CDRL3 comprising the amino acid sequence of SEQ ID NO:79.

[0025] In some embodiments, the antibody is a F(ab) fragment, a F(ab')2 fragment, and an Fv fragment or a single chain Fv fragment. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is monovalent.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used to implement the present invention, but suitable methods and materials are described below.All publications, patent applications, patents, and other references described herein are expressly incorporated by reference in their entirety.In case of discrepancy, the present specification, including definitions, will prevail.In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0027] Other features and advantages of the present invention will be apparent from, and are encompassed by, the following detailed description and claims. [Brief description of the drawings]

[0028] [Figure 1] Figures 1A-1C are a series of graphs showing concentration-dependent binding of the anti-CD28 arm of the present invention to Jurkat cells expressing CD28. The anti-CD28 arm was tested as a bivalent monoclonal antibody (hIgG1). hIgG1: isotype control, TGN1412 (hIgG4): positive control. Figures 1A, 1B and 1C show candidates identified during successive phage display selection campaigns. [Diagram 2] Figures 2A-2D are a series of graphs showing concentration-dependent binding of CEAxCD28 bispecific antibodies of the invention to CD28 expressing Jurkat cells (Figures 2A and 2B), CEA expressing LS174T cells (Figure 2C) and CD28 and CEA double negative TIB153 cells (Figure 2D). A panel of CEAxCD28 bsAbs was generated using the high affinity anti-CEA arm AC84, IgG1 Fc with the three mutations LALAPA, and various anti-CD28 arms of different affinities. hIgG1: isotype control, TGN1412: positive control, 1a28 / AC84 / N, CEA_CD28_V8 and CEA_CD28_V15: CEAxCD28 reference bispecific antibodies. [Figure 3A]Figures 3A-3D are a series of graphs showing the proliferation of human CD4+ and CD8+ T cells (Figures 3A and 3C, and 3B and 3D, respectively) from healthy donor PBMCs mediated by the CEAxCD28 bispecific antibodies of the invention after so-called wet coating (Figures 3A and 3C) or dry coating (Figures 3B and 3D) of the antibodies on the test plate. A panel of anti-CD28 arms was tested for superagonist activity in the bispecific format. None of the anti-CD28 arms of the invention, except for "AI19", showed superagonist activity in this type of assay. PBS: vehicle control, TGN1412: positive control, 1a28 / AC84 / N, CEA_CD28_V8 and CEA_CD28_V15: CEAxC28 reference bispecific antibodies, mAb 14226P2, mAb V8 and mAb V15: reference monoclonal antibodies. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4] 1 is a series of graphs showing concentration-dependent IL-2 reporter cell activation of the CEAxCD28 bispecific antibody of the invention as measured by luminescence readings in the presence of MKN-45 cells expressing CEA and a fixed concentration (1 nM) of CEAxCD3. Different levels of CD28 costimulation induced by the CEAxCD28 bsAb of the invention are observed, reflecting the distinct binding capacities of these anti-CD28 arms. DR=dose response, no target: CD3 and CD28 bsAb are incubated in the presence of effector / reporter cells but in the absence of TAA-positive target cells. [Diagram 5]Figures 5A-5D are a series of graphs showing the killing / lysis of T cell retargeted LS174T cells by the CEAxCD28 bispecific antibody of the invention when combined with a CEAxCD3 bsAb. Effector cells are PBMCs from two different healthy donors (Figures 5A and 5B). The CEAxCD28 bsAb synergizes with CEAxCD3 to kill CEA positive target cells. No killing is induced in the absence of signal 1 (Figures 5C and 5D, data obtained with the same PBMC donor used in Figures 5A and 5B, respectively). Y4L3-1 / N: non-targeted CD3 monovalent antibody, 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody. CD28 bsAb concentration used: 1 μg / mL. [Figure 6] Figures 6A-6B are a series of graphs showing the killing / lysis of T cell retargeted KATO-III (Figure 6A) and HT-29 (Figure 6B) cells by the CEAxCD28 bispecific antibody of the invention when combined with CEAxCD3 bsAb. Synergy between CEAxCD3 and CEAxCD28 bsAb in killing CEA positive target cells is also observed in these low expressing cell lines. 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody. CD28 bsAb concentration used: 2.5 μg / mL. [Figure 7] 7A-7B are a series of graphs showing upregulation of the T cell activation marker CD25 on human CD4+ and CD8+ T cells (FIGS. 7A and 7B, respectively). PBMCs were co-cultured with CEA-positive LS174 T cells in the presence of either CEA×CD3 alone (to trigger T cell activation signal 1) or in combination with the CEA×CD28 bispecific antibody of the invention (to provide T cell activation signal 2). The combination of CEA×CD3 and CEA×CD28 bsAb induces much stronger activation of both CD4+ and CD8+ T cells than CEA×CD3 monotherapy. 1a28 / AC84 / N: CEA×CD28 reference bispecific antibody. [Figure 8]8A-8B are a series of graphs showing the proliferation of human CD4+ and CD8+ T cells (FIGS. 8A and 8B, respectively). PBMCs were co-cultured with CEA-positive LS174 T cells in the presence of either CEA×CD3 alone (to trigger T cell activation signal 1) or in combination with the CEA×CD28 bispecific antibody of the invention (to provide T cell activation signal 2). The combination of CEA×CD3 and CEA×CD28 bsAb strongly increases the percentage of proliferating CD4+ and CD8+ T cells compared to CEA×CD3 monotherapy. 1a28 / AC84 / N: CEA×CD28 reference bispecific antibody. [Figure 9] Figures 9A-9F are a series of graphs showing the secretion of Granzyme B (Figure 9A), IFN-γ (Figure 9B), TNF-α (Figure 9C), IL-2 (Figure 9D), IL-6 (Figure 9E), IL-10 (Figure 9F) mediated by the CEAxCD28 bispecific antibody of the invention in combination with CEAxCD3 bsAb following co-culture of PBMC with CEA positive LS174T tumor cells. Cytokine induction by CEAxCD3 is increased when T cells are simultaneously co-stimulated with CEAxCD28 bsAbs. 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody. [Figure 10] FIG. 12 is a schematic diagram of the experimental design of the in vivo efficacy study shown in FIG. 11, in which CEA×CD28 bispecific antibodies AI3AC84 / N and AI5AC84 / N were tested in combination with CEA×CD3 in the LS174T:PBMC co-transplant model in NOG mice. [Figure 11] Figures 11A-11B are a series of graphs showing the in vivo anti-tumor efficacy of CEAxCD28 bispecific antibodies AI3AC84 / N and AI5AC84 / N in the LS174T:PBMC co-graft model described in Figure 10. The mean tumor volume (Figure 11A) and growth (Figure 11B) of tumors in individual mice are shown for all treatment groups. The in vivo anti-tumor activity of CEAxCD3 is enhanced by both CEAxCD28 bsAbs. [Figure 12]FIG. 14 is a schematic diagram of the experimental design of the in vivo efficacy study shown in FIG. 13, in which CEA×CD28 bispecific antibodies AI3AC84 / N (within the dose range) and AI10AC84 / N were tested in combination with CEA×CD3 in the LS174T:PBMC co-transplant model in NOG mice. [Figure 13] Figures 13A-13B are a series of graphs showing the in vivo antitumor efficacy of CEAxCD28 bispecific antibodies AI3AC84 / N (within the dose range) and AI10AC84 / N in the LS174T:PBMC co-graft model described in Figure 12. The mean tumor volume (Figure 13A) and growth (Figure 13B) of tumors in individual mice are shown for all treatment groups. The in vivo antitumor activity of CEAxCD3 is enhanced by both CEAxCD28 bsAbs. However, at comparable therapeutic doses, antibody AI3AC84 / N resulted in better tumor control than AI10AC84 / N. [Figure 14] Figures 14A-14D are a series of graphs showing concentration-dependent binding of MSLNxCD28 bispecific antibodies of the invention to MSLN-expressing H226 (Figure 14A) and OVCAR-3 (Figure 14B) cells, CD28-expressing Jurkat cells (Figure 14C), and CD28 and MSLN double negative TIB153 cells (Figure 14D). A panel of huIgG1-LALAPA MSLNxCD28 bsAbs was generated using high affinity anti-MSLN arms O30, O35 and O41 coupled to either the "AI5" or "AI10" anti-CD28 arms. hIgG1: isotype control. [Figure 15]Figures 15A-15F are a series of graphs showing the effect of MSLNxCD3 and MSLNxCD28 bispecific antibody combinations on target cell killing (Figures 15A and 15D), T cell activation (Figures 15B and 15E) and T cell proliferation (Figures 15C and 15F). MSLNxCD28bsAb synergizes with HPN536 analog to kill MSLN-positive target cells and induce T cell activation and proliferation. When HPN536 analog is replaced by Y4L3-1 / N, a non-targeted CD3 monovalent antibody that cannot provide T cell activation signal 1, neither killing nor T cell activation nor T cell proliferation is induced. [Figure 16] 16A-16F are a series of graphs showing secretion of Granzyme B (FIG. 16A), IFN-γ (FIG. 16B), TNF-α (FIG. 16C), IL-2 (FIG. 16D), IL-6 (FIG. 16E), and IL-10 (FIG. 16F) mediated by the MSLN×CD28 bispecific antibody of the invention when combined with HPN536 analog following co-culture of PBMCs with MSLN-positive H226 tumor cells. Cytokine induction by HPN536 analog is importantly increased when T cells are simultaneously co-stimulated with MSLN×CD28 bsAb. [Figure 17] Figures 17A-17D are a series of graphs showing concentration-dependent binding of exemplary MSLNxCD28 bispecific antibodies of the invention to MSLN-expressing H226 (Figure 17A) and OVCAR-3 (Figure 17B) cells, CD28-expressing Jurkat cells (Figure 17C), and CD28 and MSLN double negative TIB153 cells (Figure 17D). Shown are exemplary MSLNxCD28 bsAbs generated using high affinity anti-MSLN arms O35 and O41 bound to the AI3 anti-CD28 arm. hIgG1: isotype control. [Figure 18]18A-18D are a series of graphs showing the killing / lysis of T cell-retargeted OVCAR3 cells by an exemplary MSLNxCD28 bispecific antibody of the invention when combined with the HPN536 analog MSLNxCD3 bsAb in the presence of different E:T ratios (10:1 (FIG. 18A), 3:1 (FIG. 18B), 1:1 (FIG. 18C) and 1:3 (FIG. 18D)). MSLNxCD28 bsAb cooperates with MSLNxCD3 to kill MSLN-positive target cells. Synergy is especially visible at unfavorable E:T ratios, where the combination compensates for the loss of efficacy of MSLNxCD3 bsAb alone. In the absence of signal 1, no killing is induced (CD28 bsAb alone). CD28 bsAb concentration used: 2.5 μg / mL. [Figure 19] Figures 19A-19D are a series of graphs showing killing / lysis of T cell retargeted CEA / MSLN double positive HPAC cells by an exemplary CD28 bispecific antibody of the invention when combined with CEAxCD3 in the presence of different E:T ratios (10:1 (Figure 19A), 3:1 (Figure 19B), 1:1 (Figure 19C) and 1:3 (Figure 19D)). Both CEAxCD28 and MSLNxCD28 bsAb cooperate with CEAxCD3 to kill the target cells. Synergy is especially visible at unfavorable E:T ratios where the combination compensates for the loss of efficacy of CEAxCD3 bsAb alone. In the absence of signal 1, no killing is induced (CD28 bsAb alone). CD28 bsAb concentration used: 2.5 μg / mL. [Figure 20-1]Figures 20A-20D are a series of graphs showing upregulation of the T cell activation marker CD25 on human CD4+ and CD8+ T cells (Figures 20A and 20B, respectively) and proliferation of human CD4+ and CD8+ T cells (Figures 20C and 20D, respectively). PBMCs were co-cultured with CEA / MSLN double positive HPAC cells in the presence of either CEAxCD3 alone (to trigger T cell activation signal 1) or in combination with CEAxCD28 or MSLNxCD28 bispecific antibodies of the invention (to provide T cell activation signal 2). The combination of CEAxCD3 and CD28 bsAb induced much stronger activation of both CD4+ and CD8+ T cells than CEAxCD3 monotherapy at the different E:T ratios tested. Similarly, the combination of CEAxCD3 and CD28 bsAb also resulted in higher percentages of proliferative CD4+ and CD8+ T cells compared to CEAxCD3 monotherapy at all E:T ratios tested. [Figure 20-2] See description of Figure 20-1. [Figure 21] FIG. 23 is a schematic diagram of the experimental design of the in vivo efficacy study shown in FIG. 22, in which the CEA×CD28 bispecific antibody AI3AC84 / N was tested in combination with CEA×CD3 in 67 mice subcutaneously implanted with HPAF-II cells. [Figure 22] Figures 22A-22B are a series of graphs showing the in vivo antitumor efficacy of the CEAxCD28 bispecific antibody AI3AC84 / N in the mouse model described in Figure 21. Survival rates 56 days after implantation (Figure 22A) and tumor growth in individual mice (Figure 22B) are shown for all treatment groups. The in vivo antitumor activity of CEAxCD3 is enhanced by AI3AC84 / N. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The present invention is based in part on bispecific antibodies (bsAbs) capable of tumor-dependent T cell activation and tumor cell killing. Specifically, the present invention is based on bsAbs co-binding of tumor-associated antigens (TAA) expressed on the surface of target cells to mediate CD28 clustering and thus tumor-specific T cell activation. The present invention also provides CD28 antigen-binding domains, antigen-binding fragments and antibodies.

[0030] The bsAbs of the invention are characterized by a single agonist CD28 antigen-binding domain for monovalent costimulation of CD28, and a second antigen-binding domain capable of specifically and monovalently binding a tumor-associated antigen for targeted delivery of the bsAb to the tumor microenvironment. The bsAbs of the invention are referred to herein as CD28xTAA bsAbs or TAAxCD28 bsAbs.

[0031] CD28 is an important costimulatory receptor expressed on the surface of T lymphocytes. It belongs to a subfamily of costimulatory molecules characterized by an extracellular variable immunoglobulin-like domain. Other members of the family of molecules include CTLA-4, ICOS, PD-1 and BTLA.

[0032] In humans, CD28 is expressed on the cell surface of T lymphocytes as a disulfide-linked homodimer and is found on approximately 80% of human CD4+ T cells and 50% of CD8+ T cells.

[0033] Despite lacking intrinsic enzymatic activity, binding of CD28 by its ligands leads to specific phosphorylation and transcriptional signaling, ultimately resulting in metabolic changes and the production of important cytokines, chemokines, and survival signals that are essential for the long-term expansion and differentiation of T cells.

[0034] The primary ligands for CD28 are CD80 (B7.1) and CD86 (B7.2), which are primarily expressed on the surface of professional antigen-presenting cells (APCs). CD80 and CD86 diverge in their expression patterns, multimeric state, and functionality. CD28 and CTLA-4 are highly homologous and therefore compete for the same ligands. However, CTLA-4 binds to these ligands with higher affinity than CD28, so CTLA-4 competes with CD28 for the ligands and ultimately suppresses T cell responses.

[0035] Several anti-CD28 monoclonal antibodies have been proposed for therapeutic targeting of CD28. Identified fragments of anti-CD28 antibodies, called superagonist (SA) antibodies, were found to induce full activation of primary resting T cells, even in the absence of TCR ligation (signal 1), through clustering of CD28 on the surface of T cells. However, a first-in-human study of one such SA anti-CD28 antibody, TGN1412, led to severe inflammatory responses as well as chronic organ failure in all treated healthy volunteers. A cytokine storm, which was not predicted in either in vivo or in vitro preclinical safety studies, was responsible for these adverse events.

[0036] To avoid the safety issues associated with SA antibodies or systemic CD28 costimulation, tumor-targeting CD28 bispecific antibodies can be designed to costimulate T cells specifically within the tumor microenvironment. By pairing an agonist anti-CD28 binding domain with an anti-TAA binding domain, a molecule capable of cross-linking T cells to malignant cells expressing a selected TAA is generated. Because CD28 bsAb can only bind to CD28 monovalently, CD28 cannot cluster in the absence of TAA-positive target cells, thus preventing systemic T cell activation.

[0037] Moreover, even in the presence of TAA-positive cancer cells that allow clustering of CD28 on the surface of T cells, the full cytotoxic potential of T cells can only be unleashed by TAA × CD28 bispecific antibodies in the presence of primary T cell stimulation via the TCR. This is in contrast to the bivalent superagonist CD28 monoclonal antibodies mentioned above. Preclinical studies have shown the benefit of adding a costimulatory TAA × CD28 bsAb for the treatment of solid tumors, boosting the efficacy of bispecific T cell engagers or PD-(L)1 checkpoint inhibitors. Examples of agonistic TAAxCD28 bsAbs are described in WO2019246514, WO2020198009, WO2020132066, WO2020132024, WO2020127618, WO2021259890, WO2021155071 and WO2022040482, some of these molecules are currently being tested in clinical trials (ClinicalTrials.gov Identifiers: NCT04590326, NCT03972657, NCT04626635).

[0038] TAAs are well known in the art and include, for example, glioma associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, glypican 3 (GPC3), cMet, IL-IIRa, IL-13Ra, EGFR, FAP, FcRH5, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, HER3, β-human chorionic gonadotropin, alpha fetoprotein (AFP), ALK, CD19, CD123, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAXS, SART3, CLL-1, Fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGSS, polysialic acid, PLAC1, RUI, RU2(AS), intestinal carboxylesterase, Lewis Y, sLe, LY6K, mut hsp70-2, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-Ia, LMP2, NCAM, Ras mutant, gpIOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, legumain, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, FAP, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, CLDN18.2, TSHR, UPK2, and mesothelin (MSLN).

[0039] In certain embodiments, the TAA is against CEA, PSMA, EpCAM, GPC3, MSLN, HER2, HER3, or BCMA. In some embodiments, the TAA is CEA. In some embodiments, the TAA is MSLN.

[0040] Carcinoembryonic antigen Carcinoembryonic antigen (CEA, CEACAM5, CD66e) is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. CEACAM is involved in a variety of biological processes, including, but not limited to, cell adhesion, intracellular and intercellular signaling, cancer progression, angiogenesis, and metastasis.

[0041] CEA was initially discovered as a specific carcinoembryonic antigen in the human gastrointestinal (GI) system. It was found to be highly expressed by GI tissues during fetal development, but its expression is dramatically reduced prenatally, and in healthy individuals, CEA is normally found to be expressed at low levels only on the apical side of epithelial cells in the gastrointestinal epithelium and other mucosal epithelia such as the nasopharynx, lung, and urogenital tract. This polarized and restricted expression is lost during tumorigenesis, and CEA is overexpressed in a variety of solid tumors of epithelial origin, including colorectal, pancreatic, gastric, non-small cell lung, and breast cancers.

[0042] CEA represents a promising TAA for the development of antibody-based therapies due to the differential expression patterns and levels observed between cancer cells and normal epithelial cells, the latter of which express CEA exclusively at the apical surface of the epithelium, an area where therapeutic antibodies are excluded due to tight binding.

[0043] Several monoclonal antibodies targeting CEA have been developed for research purposes, as diagnostic tools, and for therapeutic purposes (see, for example, WO2012117002). Over the past few years, several bispecific antibodies carrying an anti-CEA arm as a targeting arm have been described, including the CEA x CD3 bispecific antibody sibisatamab (Bacac et al., 2016) and US20140242079), MEDI-565 (Oberst et al., 2014) and WO2016036678A1) or those described in WO2021053587, and more recently, CEA x CD28 bsAb (e.g., WO2020127628A1 and WO2020127618) and CEA x CD47 bsAb (e.g., WO2021110647).

[0044] Anti-CEA antibody Exemplary anti-CEA antibodies of the disclosure (e.g., "AC22", "AC61", "AC84") are described below. Table 1 shows the amino acid sequences of the antibodies described herein.

[0045] In some embodiments, the AC22 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:8, a CDRL2 comprising the amino acid sequence of SEQ ID NO:9, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:10.

[0046] In some embodiments, the AC22 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:17.

[0047] In some embodiments, the AC22 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:20.

[0048] In some embodiments, the AC61 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:11, a CDRL2 comprising the amino acid sequence of SEQ ID NO:12, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:13.

[0049] In some embodiments, the AC61 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:18.

[0050] In some embodiments, the AC61 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:21.

[0051] In some embodiments, the AC84 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0052] In some embodiments, the AC84 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0053] In some embodiments, the AC84 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:22.

[0054] Mesothelin Mesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that is expressed at relatively low levels in normal tissues. The mesothelin gene encodes a 628 amino acid precursor protein that is addressed to the cell membrane, where it is processed to a 40 kDa membrane-bound mature form and releases a 31 kDa fragment called megakaryocyte potentiating factor (MPF).

[0055] Mesothelin has been found to be overexpressed in many solid tumors, including mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, triple-negative breast cancer, and prostate cancer. Mesothelin has been suggested to play a role in cancer cell proliferation, local invasion, and tumor metastasis, and its expression on tumor cells correlates with increased tumor aggressiveness and poor clinical outcome.

[0056] Due to the observed differential expression between cancer cells and healthy tissues, mesothelin represents a preferred TAA for the development of mesothelin-targeting therapeutics, some of which are currently undergoing clinical trials.

[0057] Anti-MSLN antibody Exemplary anti-MSLN antibodies of the disclosure (e.g., "O30", "O25", "O35", "O38", "O41") are described below. Table 1 shows the amino acid sequences of the antibodies described herein.

[0058] In some embodiments, the O30 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:128, a CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0059] In some embodiments, the O30 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:143.

[0060] In some embodiments, the O30 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:148.

[0061] In some embodiments, the O25 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0062] In some embodiments, the O25 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0063] In some embodiments, the O25 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:149.

[0064] In some embodiments, the O35 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:134, a CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0065] In some embodiments, the O35 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:145.

[0066] In some embodiments, the O35 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:150.

[0067] In some embodiments, the O38 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:137, a CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0068] In some embodiments, the O38 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0069] In some embodiments, the O38 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:151.

[0070] In some embodiments, the O41 antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, and a light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:140, a CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0071] In some embodiments, the O41 antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:147.

[0072] In some embodiments, the O41 bispecific antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a light chain comprising the amino acid sequence of SEQ ID NO:152.

[0073] bispecific antibody The bsAbs antibodies according to the present invention may be generated de novo or engineered from existing monospecific CD28 and TAA antibodies. The bispecific antibodies of the present invention have one antigen-binding region specific for CD28 and a second antigen-binding region specific for a TAA. In some embodiments, the TAA is CEA. In some embodiments, the TAA is MSLN. In some aspects, the bispecific antibodies are monovalent for CD28, CEA and MSLN.

[0074] The bsAbs of the present invention may be based on any of the different antibody formats already described. Generally, IgG-like formats are preferred as they offer favorable properties such as long half-life and potentially reduced immunogenicity, but any other molecular bispecific format may also be used in the present invention. In some embodiments, the bispecific antibodies share a common heavy chain. In some embodiments, the heavy chain is a natural heavy chain (i.e., does not contain any mutations ("wild type")). In some embodiments, the heavy chain contains at least one mutation (i.e., has a "LALA" mutation or a "LALAPA" mutation). Optionally, the bispecific antibodies have different types of light chains. For example, one light chain is a kappa light chain and the other light chain is a lambda light chain (i.e., a kl body). The different light chains allow for easy purification of the bispecifics using kappa and lambda selective resins.

[0075] The heavy and light chain amino acid sequences of the antibodies are identified by their United States Adopted Names (USAN available, for example, through the American Medical Association at https: / / www.ama-assn.org / or through the CAS registry).

[0076] Monospecific CD28 and TAA binding variable domains may be selected de novo, for example, from a phage display library, where the phage are engineered to express human immunoglobulins or portions thereof, such as Fabs, single chain variable fragments (scFvs), or unpaired or paired antibody variable regions, and then engineered into a bispecific format. CD28 and TAA variable domains may be isolated, for example, from a phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage g3 protein (scFv-g3 fusion proteins).

[0077] The antibody library is screened for binding of CD28 antibody and TAA, and the obtained positive clones are further characterized.Such phage display method for isolating human antibody is established in the art.See, for example, U.S. Patent No. 5,223,409, U.S. Patent No. 5,403,484, and U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908, U.S. Patent No. 5,580,717, U.S. Patent No. 5,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,885,793, U.S. Patent No. 6,521,404, U.S. Patent No. 6,544,731, U.S. Patent No. 6,555,313, U.S. Patent No. 6,582,915 and U.S. Patent No. 6,593,081. The resulting novel variable region combinations are engineered into a bispecific format using methods known in the art and described herein.

[0078] Furthermore, the bispecific antibodies of the present invention can be produced using techniques including those disclosed in WO2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO2012 / 023053 produces bispecific antibodies that are structurally identical to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain and a second light chain variable region fused to a constant lambda domain. Each combination site exhibits a different antigen specificity with contributions from both the heavy and light chains. The light chain variable region may be of the lambda or kappa family, and is preferably fused to the lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide junctions.

[0079] However, it is also possible to obtain the bispecific antibodies of the invention by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO2012 / 023053 are called IgG κλ antibodies or "κλ bodies", a new fully human bispecific IgG format. This κλ body format has characteristics indistinguishable from standard monoclonal antibodies and therefore allows affinity purification of bispecific antibodies indistinguishable from standard IgG molecules, which is preferred compared to previous formats.

[0080] In addition to the above methods, bispecific antibodies of the present invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization by the method described in International Patent Publication No. WO2011 / 131746, in which a first monospecific bivalent antibody and a second monospecific bivalent antibody are engineered to have specific substitutions in the CH3 domains that promote heterodimer stability, and the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge regions to undergo disulfide bond isomerization, thereby generating the bispecific antibody by Fab arm exchange.

[0081] The antibodies of the present invention have two or more antigen-binding domains and are bispecific. The bispecific antibodies of the present invention include antibodies having a full-length antibody structure or a partial-length antibody structure such as Fab.

[0082] As used herein, a "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The full-length antibody heavy chain (HC) consists of the well-known heavy chain variable domain and constant domains VH, CH1, CH2, and CH3. The full-length antibody light chain (LC) consists of the well-known light chain variable domain and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine (K) in either one or both heavy chains.

[0083] The term "Fab arm" or "half molecule" refers to one heavy-light chain pair that specifically binds to an antigen.

[0084] The full-length bispecific antibodies of the invention can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules with distinct specificities in an in vitro cell-free environment, or by using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm may be engineered to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a distinct epitope.

[0085] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0086] As used herein, "heterodimerization" refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains having non-identical CH3 amino acid sequences.

[0087] A "knob-in-hole" strategy (see, e.g., PCT International Publication No. WO2006 / 028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain "hole" with the heavy chain "knob". Exemplary CH3 substitution pairs that form knobs and holes are (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0088] Other strategies may be used such as promoting heavy chain heterodimer formation using electrical interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface as described in U.S. Patent Application Publication No. US2010 / 0015133, U.S. Patent Application Publication No. US2009 / 0182127, U.S. Patent Application Publication No. US2010 / 028637 or U.S. Patent Application Publication No. US2011 / 0123532. In another strategy, heterodimerization is achieved by the following substitutions (represented as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_ ... It may be promoted by T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0089] Exemplary anti-cell surface antibodies that can be used to engineer bispecific molecules include, for example, anti-tumor associated antigen antibodies known in the art, such as pertuzumab and trastuzumab (HER-2), cetuximab, necitumumab, panitumumab and amivantamab (EGFR), labetuzumab and civisatamab (CEA), amatuximab (mesothelin), codrituzumab, (glypican 3), atezolizumab, avelumab and durvalumab (PD-L1), blinatumomab (CD19), brentuximab vedotin (CD30), daratumumab (CD38), gemtuzumab (CD33), tositumomab 9CD22) or obinutuzumab, ocrelizumab, ofatumumab, rituximab, and ibritumomab (CD20).

[0090] Exemplary CD28, CEA and MSLN antibodies that can be used to engineer bispecific molecules include the antibodies disclosed herein. Exemplary anti-CD28 antibodies from which the CD28 antigen-binding region may be derived include the "AI3", "AI5", "AI7", "AI8", "AI9", "AI10", "AI11", "AI12", "AI13", "AI14", "AI15", "AI16", "AI17", "AI18", "AI19", "AI20", "AI21", "AI22" or "AI23" antibodies. Exemplary anti-CEA antibodies from which the CEA antigen-binding region may be derived include the "AC22", "AC61" or "AC84" antibodies. Exemplary anti-MSLN antibodies from which the MSLN antigen-binding region may be derived include the "O30", "O25", "O35", "O38" or "O41" antibodies. Table 1 shows the amino acid sequences of the regions of the antibodies of the present disclosure.

[0091] Table 1: Exemplary amino acid sequences of the present disclosure TIFF2025508066000002.tif108149TIFF2025508066000003.tif215149TIFF2025508066000004.tif221149 TIFF2025508066000005.tif218149TIFF2025508066000006.tif214149TIFF2025508066000007.tif217149 TIFF2025508066000008.tif213149TIFF2025508066000009.tif213149TIFF2025508066000010.tif213149 TIFF2025508066000011.tif212149TIFF2025508066000012.tif216149TIFF2025508066000013.tif169149

[0092] Table 2 shows exemplary bispecific antibodies of the disclosure. The nomenclature of each antibody is shown, and the individual kappa light chain, lambda light chain and heavy regions of the κλ antibodies of the disclosure are described. "AI" indicates the anti-human CD28 antigen binding region, "AC" indicates the anti-human CEA antigen binding region, and "O" indicates the anti-human MSLN antigen binding region. / N indicates the heavy chain with the LALAPA mutation. Alternative nomenclature is shown in parentheses.

[0093] Table 2. Exemplary bispecific antibodies of the present disclosure TIFF2025508066000014.tif197146

[0094] In some embodiments, the AI3AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:23, a CDRL2 comprising the amino acid sequence of SEQ ID NO:24, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:25, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0095] In some embodiments, the AI3AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:80, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0096] In some embodiments, the AI3AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:99, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0097] In some embodiments, the AI5AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:26, a CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:28, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0098] In some embodiments, the AI5AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0099] In some embodiments, the AI5AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:100, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0100] In some embodiments, the AI8AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:32, a CDRL2 comprising the amino acid sequence of SEQ ID NO:33, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:34, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0101] In some embodiments, the AI8AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:83, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0102] In some embodiments, the AI8AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:102, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0103] In some embodiments, the AI9AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:35, a CDRL2 comprising the amino acid sequence of SEQ ID NO:36, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:37, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0104] In some embodiments, the AI9AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:84, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0105] In some embodiments, the AI9AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:103, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0106] In some embodiments, the AI10AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0107] In some embodiments, the AI10AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0108] In some embodiments, the AI10AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:104, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0109] In some embodiments, the AI11AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:41, a CDRL2 comprising the amino acid sequence of SEQ ID NO:42, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:43, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0110] In some embodiments, the AI11AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:86, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0111] In some embodiments, the AI11AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:105, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0112] In some embodiments, the AI12AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:44, a CDRL2 comprising the amino acid sequence of SEQ ID NO:45, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:46, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0113] In some embodiments, the AI12AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:87, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0114] In some embodiments, the AI12AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:106, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0115] In some embodiments, the AI13AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:47, a CDRL2 comprising the amino acid sequence of SEQ ID NO:48, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:49, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0116] In some embodiments, the AI13AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0117] In some embodiments, the AI13AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0118] In some embodiments, the AI14AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:50, a CDRL2 comprising the amino acid sequence of SEQ ID NO:51, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:52, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0119] In some embodiments, the AI14AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:89, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0120] In some embodiments, the AI14AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:108, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0121] In some embodiments, the AI15AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:53, a CDRL2 comprising the amino acid sequence of SEQ ID NO:54, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:55, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0122] In some embodiments, the AI15AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:90, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0123] In some embodiments, the AI15AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:109, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0124] In some embodiments, the AI16AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:56, a CDRL2 comprising the amino acid sequence of SEQ ID NO:57, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:58, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0125] In some embodiments, the AI16AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:91, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0126] In some embodiments, the AI16AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:110, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0127] In some embodiments, the AI17AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:59, a CDRL2 comprising the amino acid sequence of SEQ ID NO:60, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:61, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0128] In some embodiments, the AI17AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:92, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0129] In some embodiments, the AI17AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:111, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0130] In some embodiments, the AI18AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:62, a CDRL2 comprising the amino acid sequence of SEQ ID NO:63, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:64, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0131] In some embodiments, the AI18AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:93, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0132] In some embodiments, the AI18AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:112, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0133] In some embodiments, the AI19AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:65, a CDRL2 comprising the amino acid sequence of SEQ ID NO:66, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:67, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0134] In some embodiments, the AI19AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:94, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0135] In some embodiments, the AI19AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:113, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0136] In some embodiments, the AI20AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:68, a CDRL2 comprising the amino acid sequence of SEQ ID NO:69, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:70, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0137] In some embodiments, the AI20AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:95, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0138] In some embodiments, the AI20AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:114, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0139] In some embodiments, the AI21AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:71, a CDRL2 comprising the amino acid sequence of SEQ ID NO:72, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:73, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0140] In some embodiments, the AI21AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:96, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0141] In some embodiments, the AI21AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:115, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0142] In some embodiments, the AI22AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:74, a CDRL2 comprising the amino acid sequence of SEQ ID NO:75, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:76, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0143] In some embodiments, the AI22AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:97, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0144] In some embodiments, the AI22AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:116, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0145] In some embodiments, the AI23AC84 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:77, a CDRL2 comprising the amino acid sequence of SEQ ID NO:78, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:79, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:14, a CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0146] In some embodiments, the AI23AC84 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:98, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0147] In some embodiments, the AI23AC84 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:117, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:22.

[0148] In some embodiments, the AC61AI7 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:11, a CDRL2 comprising the amino acid sequence of SEQ ID NO:12, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:13, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:29, a CDRL2 comprising the amino acid sequence of SEQ ID NO:30, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:31.

[0149] In some embodiments, the AC61AI7 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:18, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:82.

[0150] In some embodiments, the AC61AI7 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:21, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:101.

[0151] In some embodiments, the AI3O30 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 25, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 128, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 129, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 130.

[0152] In some embodiments, the AI3O30 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:80, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:143.

[0153] In some embodiments, the AI3O30 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:99, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:148.

[0154] In some embodiments, the AI3O25 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:23, a CDRL2 comprising the amino acid sequence of SEQ ID NO:24, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:25, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0155] In some embodiments, the AI3O25 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:80, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0156] In some embodiments, the AI3O25 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:99, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:149.

[0157] In some embodiments, the AI3O35 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 25, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 134, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 135, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 136.

[0158] In some embodiments, the AI3O35 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:80, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:145.

[0159] In some embodiments, the AI3O35 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:99, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0160] In some embodiments, the AI3O38 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:23, a CDRL2 comprising the amino acid sequence of SEQ ID NO:24, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:25, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:137, a CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0161] In some embodiments, the AI3O38 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:80, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0162] In some embodiments, the AI3O38 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:99, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:151.

[0163] In some embodiments, the AI3O41 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 25, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 140, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 141, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 142.

[0164] In some embodiments, the AI3O41 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:80, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:147.

[0165] In some embodiments, the AI3O41 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:99, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:152.

[0166] In some embodiments, the AI5O30 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:26, a CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:28, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:128, a CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0167] In some embodiments, the AI5O30 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:143.

[0168] In some embodiments, the AI5O30 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:100, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:148.

[0169] In some embodiments, the AI5O25 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:26, a CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:28, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0170] In some embodiments, the AI5O25 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0171] In some embodiments, the AI5O25 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:100, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:149.

[0172] In some embodiments, the AI5O35 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:26, a CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:28, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:134, a CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0173] In some embodiments, the AI5O35 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:145.

[0174] In some embodiments, the AI5O35 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:100, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0175] In some embodiments, the AI5O38 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:26, a CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:28, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:137, a CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0176] In some embodiments, the AI5O38 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0177] In some embodiments, the AI5O38 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:100, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:151.

[0178] In some embodiments, the AI5O41 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:26, a CDRL2 comprising the amino acid sequence of SEQ ID NO:27, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:28, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:140, a CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0179] In some embodiments, the AI5O41 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:81, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:147.

[0180] In some embodiments, the AI5O41 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:100, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:152.

[0181] In some embodiments, the AI10O30 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:140, a CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0182] In some embodiments, the AI10O30 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:143.

[0183] In some embodiments, the AI10O30 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:104, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:148.

[0184] In some embodiments, the AI10O25 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0185] In some embodiments, the AI10O25 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0186] In some embodiments, the AI10O25 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:104, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:149.

[0187] In some embodiments, the AI10O35 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:134, a CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0188] In some embodiments, the AI10O35 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:145.

[0189] In some embodiments, the AI10O35 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:104, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0190] In some embodiments, the AI10O38 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:137, a CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0191] In some embodiments, the AI10O38 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0192] In some embodiments, the AI10O38 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:104, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:151.

[0193] In some embodiments, the AI10O41 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:140, a CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0194] In some embodiments, the AI10O41 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:147.

[0195] In some embodiments, the AI10O41 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:104, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:152.

[0196] In some embodiments, the AI13O30 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:38, a CDRL2 comprising the amino acid sequence of SEQ ID NO:39, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:40, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:128, a CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0197] In some embodiments, the AI13O30 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:143.

[0198] In some embodiments, the AI13O30 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:148.

[0199] In some embodiments, the AI13O25 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:47, a CDRL2 comprising the amino acid sequence of SEQ ID NO:48, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:49, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:131, a CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0200] In some embodiments, the AI13O25 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0201] In some embodiments, the AI13O25 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:149.

[0202] In some embodiments, the AI13O35 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:47, a CDRL2 comprising the amino acid sequence of SEQ ID NO:48, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:49, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:134, a CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0203] In some embodiments, the AI13O35 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:145.

[0204] In some embodiments, the AI13O35 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:150.

[0205] In some embodiments, the AI13O38 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:47, a CDRL2 comprising the amino acid sequence of SEQ ID NO:48, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:49, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:137, a CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0206] In some embodiments, the AI13O38 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0207] In some embodiments, the AI13O38 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:151.

[0208] In some embodiments, the AI13O41 / N bispecific antibody has a heavy chain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO:1, a CDRH2 comprising the amino acid sequence of SEQ ID NO:2, a CDRH3 comprising the amino acid sequence of SEQ ID NO:3, a kappa light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:47, a CDRL2 comprising the amino acid sequence of SEQ ID NO:48, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:49, and a lambda light chain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO:140, a CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0209] In some embodiments, the AI13O41 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:147.

[0210] In some embodiments, the AI13O41 / N bispecific antibody has a heavy chain variable and constant region comprising the amino acid sequence of SEQ ID NO:7, a kappa light chain comprising the amino acid sequence of SEQ ID NO:107, and a lambda light chain comprising the amino acid sequence of SEQ ID NO:152.

[0211] How to use The therapeutic formulations of the invention comprising the bsAbs of the invention are used to treat or alleviate symptoms associated with cancers such as, by way of non-limiting example, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral and pharyngeal cancer, uterine cancer, and / or melanoma. The invention also provides methods of treating or alleviating symptoms associated with cancer. Treatment regimens are made by identifying a subject, e.g., a human patient suffering from (or at risk of developing) cancer, using standard methods.

[0212] Efficacious treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody provides a clinical benefit.

[0213] Pharmaceutical Compositions The antibody of the present invention (also referred to herein as "active compound"), as well as its derivatives, fragments, analogs and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include an antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0214] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. The solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, or phosphates, and agents for adjusting osmolality such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0215] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent in the composition, such as sugar, mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin, in the composition.

[0216] Sterile injection solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.For the preparation of sterile powder for sterile injection solution, the preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0217] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, active compounds can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, swallowed, expectorated, or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0218] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0219] Systemic administration may be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0220] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0221] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells with monoclonal antibodies against viral antigens, can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0222] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.As used herein, dosage unit form refers to a physically separate unit suitable as a unit dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the unique characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for individual treatment.

[0223] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0224] definition As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0225] As used herein, "mAb V8" refers to "CD28(SA_variant 8) in molecule 11U:huIgGl PG-LALA isotype (PG-LALA), CD28(SA_variant 8) antibody" described in WO2020127618 (incorporated by reference in its entirety), including the amino acid sequences of SEQ ID NO:377 and SEQ ID NO:378 of WO2020127618.

[0226] As used herein, "mAb V15" refers to "Molecule 11W:CD28(SA_variant 15) in huIgGl PG-LALA isotype (PG-LALA), CD28(SA_variant 15) antibody" described in WO2020127618 (incorporated by reference in its entirety), including the amino acid sequences of SEQ ID NO:381 and SEQ ID NO:382 of WO2020127618.

[0227] As used herein, "CEA_CD28_V8" refers to "molecule 11B: bispecific huIgGl PGLALA CrossFab molecule with CEA(A5H1EL1D)-CD28(SA variant 8) 1+1 format, charge modifications in the CD28(SA_variant 8) Fab fragment (knob) and VH / VL exchange in the CEA(A5H1EL1D) Fab fragment (hole)" as described in WO2020127618 (incorporated by reference in its entirety), comprising the amino acid sequences of SEQ ID NOs: 351, 352, 355 and 356 of WO2020127618.

[0228] As used herein, "CEA_CD28_V15" refers to "molecule 11C: bispecific huIgGl PGLALA CrossFab molecule with CEA(A5H1EL1D)-CD28(SA variant 15) 1+1 format, charge modifications in the CD28(SA_variant 15) Fab fragment (knob) and VH / VL exchange in the CEA(A5H1EL1D) Fab fragment (hole)" as described in WO2020127618 (incorporated by reference in its entirety), comprising the amino acid sequences of SEQ ID NOs: 351, 352, 357 and 358 of WO2020127618.

[0229] As used herein, "mAb 14226P2" refers to "mAb14226P2 (CD28 parent "A")," which is the anti-CD28 arm used to generate bs16429D (PSMAxCD28 "A") as described in WO2019246514 (incorporated by reference in its entirety), which comprises the amino acid sequences of SEQ ID NOs: 81 and 83 of WO2019246514.

[0230] As used herein, "1a28 / AC84 / N" refers to the benchmark CEAxCD28 bsAb generated by pairing the anti-CD28 arm of mAb 14226P2 with the anti-CEA arm AC84 using crossMAb / knobs-in-hole technology and comprising the amino acid sequences of SEQ ID NOs: 122, 123, 124 and 125.

[0231] As used herein, "TGN1412" refers to a superagonistic (SA) anti-huCD28 antibody in the human IgG4 isotype described in WO2006050949, comprising the amino acid sequence of SEQ ID NOs:126 and 127.

[0232] As used herein, "HPN536 analog" refers to a trispecific MSLNxCD3xHSA molecule capable of redirecting T cells to MSLN-positive target cells described in WO2018209304 (incorporated by reference in its entirety), comprising the amino acid sequence of SEQ ID NO: 100 of WO2018209304.

[0233] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is determined by the dissociation constant (K D Affinity can be measured by common methods known in the art, including KinExA and Biacore.

[0234] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, and chimeric antibodies.

[0235] As used herein, unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments and individual antibody heavy or light chains, as well as individual heavy or light chain variable regions.

[0236] A "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0237] The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0238] A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain that also contains the VH and CH1 domains, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0239] An "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody. The "Fv region" contains the variable regions of both the heavy and light chains but lacks the constant region.

[0240] "Isolated antibody" refers to a purified state, and in such context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris and growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such materials, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with the experimental or therapeutic use of the binding compounds described herein.

[0241] As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences within the variable domains, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques such as those described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0242] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat, a rat cell, or a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0243] Generally, the basic "antibody" structural unit comprises a tetramer. In monospecific antibodies, each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain contains a "variable region" or "variable domain" of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.

[0244] Typically, human constant light chains are classified as kappa and lambda light chains. Furthermore, human constant heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. These IgG subtypes include, for example, IgG1 and IgG4.

[0245] As used herein, "variable region," "variable domain," "V region," or "V chain" refers to the segment of an IgG chain that is variable in sequence among different antibodies. An antibody "variable region" refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable region of the heavy chain is referred to as the "V H」 The light chain variable region may be referred to as "V L". Typically, both the heavy and light chain variable regions contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, which enable binding to a specific epitope. Generally, from N-terminus to C-terminus, both the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain generally follows the definition for the sequence of proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5th ed., NIH Publ. No. 91-3242 (1991), Kabat (1978) Adv. Prot. Chem. 32:1-75, Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia, et al., (1987) J Mol. Biol. 196:901-917, or Chothia, et al., (1989) Nature 342:878-883.

[0246] "CDR" is the antibody V H One of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the beta-sheet framework, or the antibody V LIt refers to one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of the beta-sheet framework. Thus, CDR is a variable region sequence interspersed in the framework region sequence. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable regions in antibody variable domains. CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved beta-sheet framework and therefore can fit into different structures. Both terms are well known in the art. CDR region sequences are also defined by AbM, Contact, and IMGT. The location of CDRs in standard antibody variable regions has been determined by comparison of multiple structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the numbers of residues in hypervariable regions vary in different antibodies, additional residues relative to standard positions are conventionally numbered with a, b, c, etc. next to the residue numbers in the standard variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those of skill in the art. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT specific numbering systems, is well known to those of skill in the art. In some embodiments, the CDRs are as defined by the Kabat numbering system. In other embodiments, the CDRs are as defined by the IMGT numbering system. In still other embodiments, the CDRs are as defined by the AbM numbering system. In yet other embodiments, the CDRs are as defined by the Chothia numbering system. In still other embodiments, the CDRs are as defined by the Contact numbering system.

[0247] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same in equivalent positions when the two sequences are optimally aligned.

[0248] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids which share similar properties and may be interchangeable are discussed above.

[0249] "Conservatively modified variant" or "conservative substitution" refers to the substitution of amino acids in a protein with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure and rigidity, etc.), so that changes can be frequently made without changing the biological activity of the protein.Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224 (4th Ed.)).In addition, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity.

[0250] As used herein, the term "epitope" refers to an area or region on an antigen to which an antibody or antigen-binding fragment binds. Binding of an antibody or antigen-binding fragment thereof disclosed herein to an epitope means that the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within the epitope.

[0251] An "isolated" nucleic acid molecule or polynucleotide refers to DNA or RNA, e.g., DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, in which the isolated polynucleotide is not associated with all or a portion of a polynucleotide found in nature, or associated with a polynucleotide with which it is not associated in nature. For purposes of this disclosure, it should be understood that a polynucleotide "comprising (or similar to)" a particular nucleotide sequence does not encompass an intact chromosome. An isolated polynucleotide "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include coding sequences for up to 10, or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0252] The term "control sequence" refers to a polynucleotide sequence necessary or useful for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers. In one embodiment of the present invention, the polynucleotide is operably linked to a promoter, such as a viral promoter, a CMV promoter, an SV40 promoter, or a non-viral promoter or an elongation factor (EF)-1 promoter, and / or an intron.

[0253] A nucleic acid is "operably linked" when it is placed into a functional relationship with another polynucleotide. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, but not necessarily, "operably linked" means that the polynucleotide sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0254] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transformations. It is also understood that not all progeny have exactly the same DNA content due to deliberate or accidental mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where separate designations are intended, it will be clear from the context.

[0255] Host cells include eukaryotic and prokaryotic host cells, including mammalian cells. Host cells include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells, among others. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophia, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindnen), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia piperi, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicansalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium spp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia spp., any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucnowens, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa. The invention includes any host cell (e.g., a CHO cell or a Pichia cell, e.g., Pichia pastoris) containing an anti-ILT4 antibody or antigen-binding fragment thereof, or containing a polynucleotide containing such an antibody or fragment, or containing a vector containing a polynucleotide.

[0256] "Treat" or "treating" refers to administering an antibody or antigen-binding fragment thereof of the present invention to a subject having one or more symptoms of a disease for which the antibody and antigen-binding fragment are effective, for example, in treating a subject having or suspected of having cancer or an infectious disease for which the agent has therapeutic activity. Typically, the antibody or fragment is administered in an "effective amount" or "effective dose" that will alleviate one or more symptoms (e.g., symptoms of cancer or infectious disease) in the subject or population being treated by inducing regression or elimination of such symptoms, or to any clinically measurable extent by inhibiting the progression of such symptoms, such as cancer symptoms, such as tumor growth or metastasis. The effective amount of the antibody or fragment may vary depending on factors such as the disease stage, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. EXAMPLES

[0257] Example 1: Phage display selection of CD28 Fv using a human scFv library containing fixed variable heavy chain domains The general procedure for constructing and handling a human scFv library displayed on M13 bacteriophage is described in Vaughan et al., (Nat. Biotech. 1996, 14:309-314), which is incorporated herein by reference in its entirety. The library for selection and screening encodes scFvs that all share the same VH domain and are diversified only in the VL domain. Methods for generating fixed VH libraries and their use for identifying and assembling bispecific antibodies are described in US2012 / 0184716 and WO2012 / 023053, each of which is incorporated herein by reference in its entirety. The procedure for identifying scFvs that bind to human CD28 (huCD28) is described below. Selections were performed in a solution containing biotinylated huCD28 protein and / or on cells expressing huCD28. The selection strategy included (i) up to four rounds of selection on the recombinant protein, (ii) up to four rounds of selection alternating between recombinant protein and cells, and (iii) two rounds on the recombinant protein followed by two rounds on cells.

[0258] Protein Selection Aliquots of scFv phage libraries were blocked with PBS containing 2% (w / v) skim milk. Blocked phages were first deselected on streptavidin / neutravidin magnetic beads (Dynabeads™ MyOne™ streptavidin T1 magnetic beads or Sera-Mag SpeedBeads Neutravidin™ coated magnetic particles) and then pre-incubated with 200 nM, 100 nM, 50 nM or 5 nM biotinylated recombinant human CD28 (CD8-H82E5, Acro Biosystems). Phage+antigen mixtures were then captured by blocked magnetic beads (the same type used for deselection) and washed five times with PBS / 0.1% Tween® 20 and twice with PBS only. Phages were eluted with 1 mg / mL trypsin and added directly to exponentially growing TG1 cells after adding AEBSF to block trypsin activity. An aliquot of infected TG1 was serially diluted to titer the selection output. The results were then rescued and used for the next round of selection.

[0259] Cell surface selection Phage-containing supernatants were blocked with PBS containing 10% FBS. Blocked phages were first deselected on CD28NEG TIB-153 cells (ATCC TIB 153) and then selected on CD28POS Jurkat cells (Jurkat clone E6-1, ATCC TIB 152). Cells were pelleted and washed five times with PBS containing 10% FBS, followed by one wash with PBS only. Phages were eluted with 1 mg / mL trypsin and added directly to exponentially growing TG1 cells after adding AEBSF to block trypsin activity. Aliquots of infected TG1 were serially diluted to titer the selection output. Results were then rescued and used for the next selection round.

[0260] Example 2: Screening for scFv binding / non-binding to human CD28 Screening of scFvs for binding to CD28 was tested either by ELISA using biotinylated huCD28-His (or biotinylated huCEA_ECD as a negative control) or by flow cytometry using CD28POS (Jurkat) and CD28NEG (TIB-153) cells.

[0261] For the binding ELISA, neutravidin-coated plates were blocked with a 1% casein solution in PBS. Biotinylated huCD28-His and biotinylated huCEA_ECD were captured at 5 nM. Dilutions of freshly prepared periplasmic extracts containing selected scFvs were applied to the plate and detected using a combination of mouse anti-c-myc and donkey anti-mouse IgG HRP antibodies. The OD at 450 nm generated after addition of TMB was measured using a spectrophotometer plate reader. Hits were classified as specific binders if they could not bind to an unrelated huCEA protein and if the OD450 on huCD28 was at least 3-fold higher than the background OD450.

[0262] For flow cytometry binding assays, cells were harvested, washed, and distributed into V-bottom 96-well plates at 150,000 or 200,000 cells / well. Dilutions of freshly prepared periplasmic extracts containing selected scFvs were preincubated with mouse anti-c-myc antibody and added to the cells. After incubation, cells were washed, incubated with goat anti-mouse IgG-APC detection antibody, and analyzed on an iQUE3 screening instrument (Sartorious). Hits were classified as positive and specific if at least 5% of the cells showed a binding signal on CD28POS Jurkat cells 3-fold higher than background GeoMFI and no such signal was observed on CD28NEG TIB-153 cells.

[0263] Positive and specific hits were sequenced after DNA extraction from single clones.

[0264] Example 3: Reformatting of anchored VH candidates into IgG and transient expression in mammalian cells After screening and sequencing, scFv candidates with the desired binding properties were reformatted into IgG and expressed by transient transfection into PEAK cells. The VH and VL sequences of the selected scFvs were amplified with specific oligonucleotides and cloned into an expression vector containing the heavy and light chain constant regions. The expression vector was verified by sequencing and transfected into mammalian cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 4 × 10 6 PEAK cells were cultured in 25 ml of culture medium containing fetal bovine serum in a T75 flask. The transfected cells were cultured at 37°C for 5-6 days and IgG production was quantified using an Octet RED96 instrument. The supernatant was harvested for IgG purification on FcXL affinity resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the supernatant from the transfected cells was incubated overnight at 4°C with an appropriate amount of FcXL resin. After washing the resin with PBS, the sample was loaded onto an Amicon Pro column and the resulting IgG was eluted with 50 mM glycine pH 3.5. The eluted IgG fraction was then dialyzed by Amicon 50 kDa against histidine NaCl pH 6.0 buffer and the IgG content was quantified by absorbance at 280 nm. Purity and IgG integrity were verified by electrophoresis using an Agilent Bioanalyzer 2100 according to the manufacturer's instructions (Agilent Technologies).

[0265] Example 4: Binding of anti-CD28 mAb to CD28-positive Jurkat cells The binding capacity of the anti-CD28 antibody arms of the invention tested as bivalent mAbs was assessed by flow cytometry using CD28 positive Jurkat cells (Jurkat clone E6-1, ATCC TIB 152).

[0266] Cells were harvested, checked for viability and counted. 200,000 cells were incubated for 15 min at 4° C. with increasing concentrations of antibodies diluted in FACS buffer (PBS 2% BSA, 0.1% NaN3). Cells were washed twice with cold FACS buffer and re-incubated with the appropriate anti-human IgG secondary antibody for another 15 min at 4° C. Cells were washed twice with cold FACS buffer and resuspended in 150 μl of FACS buffer with the appropriate viability marker. Binding of antibodies to live cells was measured by flow cytometry using a Cytoflex Platform (Beckman Coulter). Data was analyzed using FlowJo™ v10 software (BD Life Sciences) and dose-response binding curves were generated using GraphPad Prism 9 software.

[0267] The resulting binding profiles of a selection of κλ-matched anti-CD28 arms are shown in Figures 1A-1C. The superagonist anti-CD28 mAb TGN1412 and an irrelevant hIgG1 mAb served as positive and isotype controls, respectively, and were used in all experiments shown for comparison. The broad spectrum of binding curves shown on the three graphs, representing anti-CD28 candidates identified during successive phage display campaigns, highlights the fact that a panel of anti-CD28 antibody arms with different binding affinities for CD28 was identified by phage display.

[0268] Example 5: Expression and purification of a bispecific antibody carrying lambda and kappa light chains Co-expression of one heavy chain and two light chains in the same cell can result in the assembly of three different antibodies. Co-expression can be achieved in different ways, such as by transfection of multiple vectors expressing one of the co-expressed chains, or by using a vector driving the expression of multiple genes.

[0269] Here, the two light chains were cloned into the vector pNovi κHλ, which was previously generated to allow for the co-expression of one heavy chain, one kappa light chain and one lambda light chain, as described in US20120184716 and WO2012023053, each of which is incorporated herein by reference in its entirety. Expression of the three genes was driven by the human cytomegalovirus promoter (hCMV), and the vector also contains the glutamine synthetase gene (GS), which allows for the selection and establishment of stable cell lines. The common VH and VL genes of anti-CD28 IgG and anti-CEA IgG were cloned into the vector pNovi κHλ for transient expression in mammalian cells. Expi293 cells were cultured in suspension in an appropriate Erlenmeyer flask with the appropriate number of cells and culture medium volume. Plasmid DNA was transfected into Expi293 cells using PEI. Antibody concentrations in the supernatants of transfected cells were measured during production using Octet RED96. According to the antibody concentration, the supernatant was collected 5-7 days after transfection and clarified by filtration after the addition of diatomaceous earth (Sartorius). The purification was based on a three-step purification process. First, the CaptureSelect™ FcXL affinity substrate (Thermo Fisher Scientific) was washed with PBS and then added to the clarified supernatant. After overnight incubation at +4°C and 20 rpm, the supernatant was centrifuged at 2000 g for 10 min, the flow-through was saved and the resin was washed twice with PBS. The resin was then transferred to an Amicon Pro column and a solution containing 50 mM glycine at pH 3.5 was used for elution. Several elution fractions were generated, neutralized with Tris-HCl pH 7.4 and pooled. The pool containing total human IgG (bispecific and two monospecific antibodies) was quantified using a Nanodrop spectrophotometer (NanoDrop Technologies). A small aliquot was saved for further analysis and the remaining sample was incubated with an appropriate volume of CaptureSelect™ Copper XL Affinity Substrate (Thermo Fisher Scientific) for 30 minutes at room temperature at 20 rpm.Resin recovery and washing, elution and neutralization steps were performed as described above. The final affinity purification step was performed using CaptureSelect™ Lambda Fab affinity substrate (Thermo Fisher Scientific) applying the same process as the Kappa purification step. Alternatively, purification was based on a two-step purification process using only CaptureSelect™ Kappa XL affinity substrate and CaptureSelect™ Lambda Fab affinity substrate. All elution fractions were pooled and desalted against His-NaCl pH 6.0 formulation buffer using 50 kDa Amicon Ultra centrifugal filter units (Merck Millipore). The final product was quantified using Nanodrop.

[0270] The purified bispecific antibodies were analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer with the Protein 80 kit as described by the manufacturer (Agilent Technologies). Aggregate levels were determined by SEC-UPLC. All samples were tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories). Table 2 summarizes the CEA×CD28 and MSLN×CD28 κλ-bispecific antibodies generated.

[0271] Example 6: In vitro characterization of CEA x CD28 bispecific antibodies Binding of the CEAxCD28 bsAb to CD28-positive Jurkat cells, CEA-positive LS174T cells or CEA and CD28 double-negative TIB153 cells was tested. A series of flow cytometry-based experiments were performed to demonstrate binding of CD28xCEA κλ bodies to target cells.

[0272] Examples of cells that can be used include CEA positive cell lines such as colorectal adenocarcinoma cell line LS174T, CD28 positive cell lines such as leukemic Jurkat T cells, and CEA and CD28 double negative cell lines such as leukemic TIB153 cells. Cell staining and binding assessment were performed as described in Example 4.

[0273] Binding curves of the CEAxCD28 bsAb of the present invention obtained using Jurkat, LS174T and TIB153 cells are shown in Figures 2A, 2B, 2C and 2D, respectively. In addition to the κλ bodies, other antibodies were included as reference antibodies: hIgG1 denotes an irrelevant monoclonal antibody of hIgG1 Fc serving as an isotype control, 1a28 / AC84 / N denotes the LALAPA mutant huIgG1, which is a 1+1 bispecific molecule based on the anti-CD28 antibody arm mAb14226P2 described in WO2019246514A1, using as targeting arm the anti-CEA arm AC84 described in WO2021110647 comprising the amino acid sequences of SEQ ID NOs: 122, 123, 124 and 125, while CEA_CD28_V8 and CEA_CD28_V15 correspond to the CEA×CD28 bispecific antibodies described in WO2020127618, referred to in the text as molecule 11B and molecule 11C, respectively.

[0274] Binding on Jurkat cells highlights the range of binding affinities of selected anti-CD28 antibody arms of the invention to huCD28 in line with published anti-CD28 antibodies or lower binding affinities (Figures 2A and 2B). Binding on LS174T cells highlights how antibodies sharing the same anti-CEA arm bind similarly to cells, with AC84-containing antibodies (high affinity anti-CEA arm) showing comparable dose range profiles, followed by two CEA_CD28s (V8 and V15), both containing the same medium affinity anti-CEA arm, and finally the only molecule based on the low affinity anti-CEA arm AC61, AC61AI7 / N (Figure 2C). The absence of binding signal on TIB-153 suggests that all binding arms of the invention are specific to their designated targets (Figure 2D).

[0275] Example 7: Wet and dry plate coating T cell proliferation assay To exclude anti-CD28 arms with superagonist activity, the ability of CD28 bsAbs to induce T cell proliferation in the absence of signal 1 was assessed using wet and dry plate coating T cell proliferation assays.

[0276] 96-well polypropylene plates were coated with antibodies diluted to 10 μg / ml in PBS overnight either with 100 μl of antibody solution (wet coating) at 4°C or with 50 μL of antibody solution at room temperature, unsealed, and in a class II laminar flow cabinet (to allow evaporation of the buffer = dry coating). Following either coating procedure, plates were washed twice with PBS. In parallel, PBMCs isolated from buffy coats obtained from healthy donors were stained with the CellTrace Violet Cell Proliferation Kit (ThermoFischer Scientific) according to the manufacturer's instructions. 100,000 stained PBMC cells were added to the 96-well plate in a final volume of 200 uL / well and incubated at 37°C + 5% CO2 for 6 days. Cells were then harvested and stained for flow cytometry evaluation using anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (BioLegend, 301032) as detailed in Examples 4 and 6b. Proliferation rates of live CD4+ and CD8+ T cells were calculated by measuring the level of CellTrace Violet staining by flow cytometry using CytoFLEX (Beckman Coulter) and results were evaluated by FlowJo software for both coating techniques (Figures 3A-3D). CD28 SA antibody TGN1412 served as a positive control, while background proliferation rates of T cells were determined in the absence of any antibody (PBS only). The three CEAxCD28 bsAbs 1a28 / AC84 / N, CEA_CD28_V8 and CEA_CD28_V15 (described above) and their respective parental anti-CD28 monoclonal antibodies mAb 14226P2, mAb V8 and mAb V15 were included as reference antibodies.

[0277] With the exception of candidate AI19AC84 / N, which induced a mild but significant T cell proliferation, all other candidates tested resulted in CD4+ and CD8+ T cell proliferation consistent with that induced by the vehicle control (PBS) in both coating procedures, thereby ruling out undesirable superagonist properties for these anti-CD28 arms. Candidate AI19AC84 / N was precautionarily excluded from further analysis.

[0278] In contrast to all of the anti-CD28 arms of the present invention (except for the AI19 arm), the anti-CD28 arms V8 and V15 described in WO2020127618 show some superagonist activity, both as monoclonal antibodies (mAb V8 and mAb V15) and as bispecific CEAxCD28 antibodies (CEAxCD28 V8 and CEAxCD28 V15), as determined by induction of proliferation of resting CD4+ and CD8+ T cells under both experimental conditions (wet and dry coating). For anti-CD28 arm 14226P2, no superagonist activity was observed, either as a monoclonal antibody (mAb 14226P2) or as part of bsAb 1a28 / AC84 / N.

[0279] Example 8: T cell activation bioassay (IL-2 promoter) Excluding possible superagonist activity, the ability of selected anti-CD28 arms to stimulate the CD28 costimulatory receptor on the surface of T cells was assessed using an IL-2 reporter system in a bispecific format, i.e., CEA×CD28 bsAb.

[0280] The T cell activation bioassay (IL-2) kit developed by Promega (J1651) contains engineered Jurkat T cells expressing a luciferase reporter gene driven by the IL-2 promoter. In the presence of both CD3 and CD28 stimulation, receptor-mediated signaling induces activation of the IL-2 pathway, resulting in luminescence of engineered Jurkat T cells. Because CD28 bsAb requires both a T cell signal 1 provider and a TAA positive target cell line to costimulate T cells, the assay was performed in the presence of CEA positive MKN-45 cells and CEAxCD3 (CEAxCD3 bispecific kappa lambda body, originally described in WO2021053587, containing a common heavy chain HC of SEQ ID NO: 118, a kappa light chain of SEQ ID NO: 119, and a lambda light chain of SEQ ID NO: 120).

[0281] Briefly, 37,500 tumor target cells (MKN-45) were incubated with 75000 IL-2 reporter cells (E:T=2:1) ​​for 6 h at 37°C in the presence of a fixed dose (1 nM) of CEA×CD3 alone or in combination with increasing concentrations of CEA×CD28 bispecific antibody. Alternatively, CEA×CD3 was replaced by Y4L3-1 / N, a non-targeted anti-CD3 bsAb containing a common heavy chain HC of SEQ ID NO: 118, a kappa light chain of SEQ ID NO: 121, and a lambda light chain of SEQ ID NO: 120. Plates were equilibrated for 15 min at room temperature before measurements. 75 μl of substrate (Bio-Glo Reagent, Promega) was added to the cells and luminescence (relative luminescence units) was measured after 5-10 min of incubation at room temperature in the dark using a SpectraMax i3X luminometer (Molecular Devices).

[0282] In the presence of basal CD3 stimulation (1 nM CEAxCD3), a concentration-dependent increase in IL-2 promoter-mediated luminescence was observed with increasing CEAxCD28 bsAb concentrations, suggesting proper CD28 stimulation via the anti-CD28 arm of the bsAb, resulting in enhanced T cell activation. No T cell activation could be observed in the absence of target cells or when CEAxCD3 was replaced with Y4L3-1 / N, a non-targeted CD3 bsAb incapable of delivering T cell activation signal 1 (Figure 4).

[0283] This reporter assay confirms that the CEAxCD28 bispecific antibody of the invention is able to enhance T cell responses only in the presence of (1) CEA-positive target cells and (2) primary T cell activation (signal 1).

[0284] Example 9: CEA x CD28 bispecific antibody-mediated T cell-dependent cytotoxicity (TDCC) TDCC in CEA-positive and CEA-negative cell lines The T cell dependent cytotoxicity (TDCC) of different CEA positive and CEA negative tumor cell lines induced by the CEAxCD28 bispecific antibody of the invention was evaluated in combination with CEAxCD3 bsAb using either human PBMC or purified primary T cells as effector cells.

[0285] After washing twice with PBS, the target cells are detached with trypsin or cell dissociation solution. After a centrifugation step, the cells are resuspended in assay medium, adjusted to the required concentration, and seeded into 96-well plates.

[0286] Effector cells can be either human peripheral blood mononuclear cells (PBMCs) or purified T cells. PBMCs were isolated from buffy coats derived from healthy human donors using SepMate™ Tubes (Stemcell Technologies) containing Lymphoprep™ buffer (Stemcell Technologies). When purified T cells were used as effector cells, an additional purification step was performed in which T cells were negatively isolated from PBMCs using a T cell immunomagnetic negative selection kit (STEMCELL Technologies).

[0287] For TDCC assays, when PBMCs were used as effector cells, they were added to the target cells at a final E:T ratio of 10:1 (unless otherwise stated), and when purified T cells were used, a final E:T ratio of 5:1 was used. A range of doses of CEAxCD3 and fixed doses (2.5, 1.0, 0.5, or 0.1 μg / mL) of the CEAxCD28 antibody of the present invention were added to pre-seeded target and effector cells. Alternatively, a non-targeted CD3 bsAb (Y4L3-1 / N) can be used instead of CEAxCD3. Target cell killing is assessed after either 24 h, 48 h, 72 h, or 6 days of incubation at 37° C., 5% CO2 by quantifying LDH released into the medium by apoptotic / necrotic cells (Cytotoxicity Detection Kit PLUS (LDH), Roche) (unless otherwise stated). Maximal LDH release (=100% lysis) was obtained by incubating target cells with the lysis solution provided by the kit. Spontaneous LDH release (=0% lysis) refers to target cells co-incubated with effector cells without the addition of any antibody. TDCC curves (Figures 5A-5D and 6A-6B) were plotted using GraphPad Prism9. Alternatively, instead of LDH release, an assay based on ATP quantification was used (Promega, CellTiter-Glo® Viability Assay #G7571). In this case, the plate was shaken on a rocking platform to resuspend PBMCs in the supernatant. The plate was then washed twice with PBS to remove all PBMCs. Meanwhile, the kit's reagents were reconstituted and stabilized at room temperature for 10 min. Afterwards, 100 μL of reconstituted reagent was added to each well and incubated at room temperature for 10 min, protected from direct light. The luminescence signal coming from adenosine triphosphate (ATP) contained by the remaining live target cells in the wells was measured using a microplate reader in luminescence acquisition mode (Spectra i3Max). The percentage of specific lysis was calculated using the following formula: % specific lysis = [1 - (sample value) / (effector + target)] x 100, where effector + target corresponds to the baseline value in the absence of antibody.Specific lysis results were then analyzed using GraphPad Prism software.

[0288] CEAxCD28 bsAb synergizes with CEAxCD3 bsAb to kill CEA-positive LS174T target cells expressing 26'000 CEA / cell (Figures 5A-5D). Shown are two PBMC donor-induced killing in the presence of a dose range of CEAxCD3 and a fixed dose of CEAxCD28 antibody at 1 μg / mL. Synergy can be observed as a lower EC50 or higher overall killing at the highest concentration tested, or both. Importantly, when CEAxCD3 was replaced by Y4L3-1 / N, a non-targeted CD3 bsAb unable to deliver signal 1 to effector cells, there was no CEAxCD28 bsAb-induced killing, highlighting the importance of primary T cell stimulation (signal 1) for the activity of CD28 bsAb. BsAbs 1a28 / AC84 / N and CEA_CD28_V8 (above) were included as reference CEAxCD28 bsAbs and demonstrated comparable killing to the kappa lambda bodies tested, with the exception of candidate AI11AC84 / N, which consistently yielded weak activity in vitro.

[0289] The synergy between CEAxCD3 and CEAxCD28 bsAbs was not cell line specific, as KATO-III and HT-29 cells, which express 15,000 and approximately 1,000 CEA / cell, respectively, were also killed by the combination of CEAxCD3 (dose response) and CEAxCD28 (2.5 μg / mL) bsAbs more efficiently than CEAxCD3 bsAb alone (see Figures 6A-5B). BsAb 1a28 / AC84 / N (above) was included as a reference CEAxCD28 bsAb.

[0290] Upregulation of T cell activation markers during CEA-expressing tumor cell killing induced by the combination of CEA x CD3 and CEA x CD28 bsAbs CEA×CD3 bsAb-induced CEA-positive tumor cell killing is based on T cell activation. The activation state of T cells can be further increased by CD28 costimulation. Therefore, the ability of CEA×CD28 κλ bodies to enhance T cell activation in the presence of appropriate signals 1 was quantified by flow cytometry using antibodies that recognize specific T cell activation markers such as CD69 (early activation marker) or CD25 (late activation marker).

[0291] To assess the activation state of T cells at the end of the killing assay (detailed in Example 9a), the following procedure was followed: Floating cells (containing both CD4+ and CD8+ T cells) were transferred to a new V-bottom 96-well plate. The supernatant was removed by centrifugation and the cells were washed twice with cold FACS buffer (PBS 2% BSA, 0.1% NaN3) before being incubated with Fc Block Reagent (BD Biosciences) for 15 minutes at 4°C. After washing twice with FACS buffer, the cells were incubated with the following antibodies: anti-CD69-FITC (BioLegend), anti-CD8-PerCP-Cy5.5 (BioLegend), anti-CD25-PE (BioLegend) and anti-CD4-APC (ThermoFisher) for 15 minutes at 4°C. The cells were washed and analyzed by flow cytometry using a Cytoflex Platform (Beckman Coulter). Data was analyzed using FlowJo™ v10 software (BD Life Sciences). Results from experiments in which LS174T cells were co-cultured with PBMCs at an E:T ratio of 10:1 for 72 h in the presence of a dose range of CEAxCD3 and a fixed dose of CEAxCD28 bsAb at 1 μg / mL are shown in Figures 7A-7B.

[0292] T cell activation was measured by quantifying the late activation marker CD25 on the surface of both CD4+ and CD8+ T cells (Figures 7A and 7B, respectively). Compared to single treatment with CEAxCD3, the combination of CEAxCD3 and CEAxCD28 bsAbs activated both CD4+ and CD8+ T cells to a greater extent, with much brighter CD25 staining for the combined treatment. BsAb 1a28 / AC84 / N (above) was included as a reference CEAxCD28 bsAb.

[0293] Effect of CEA×CD28 bsAb-mediated CD28 costimulation on T cell proliferation in the presence of CEA×CD3 bsAb CEA×CD28 bsAb was analyzed for its ability to potentiate the effect of CEA×CD3 on induction of T cell proliferation in the presence of CEA-positive tumor target cells. Freshly isolated human PBMCs were stained with the CellTrace Violet Cell Proliferation Kit (ThermoFischer Scientific) according to the manufacturer's instructions, washed, and co-cultured with target cells for 5-6 days in the presence of a dose range of CEA×CD3 and the indicated fixed dose of CEA×CD28 bsAb at a final E:T ratio of 10:1 (unless otherwise stated). After co-culture, effector cells were harvested, washed, and stained with appropriate viability markers to remove dead cells as well as with anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (BioLegend, 301032) to identify the target population. The proliferation rate of T cells was calculated by measuring the level of CellTrace Violet staining intensity on live CD4+ or CD8+ T cells by flow cytometry using a CytoFLEX (Beckman Coulter). Data were evaluated by FlowJo software and plotted using GraphPad Prism (Figures 8A-8B).

[0294] The percentages of proliferating CD4+ and CD8+ T cells are shown in Figures 8A and 8B, respectively. The ability of CEAxCD3 bsAb to induce T cell proliferation was greatly enhanced by the addition of 2.5 μg / mL of CEAxCD28 bsAb, and both CD4+ and CD8+ T cells responded equally to the combination treatment.

[0295] Cytokines released into the supernatant during enhanced killing of CEA-expressing tumor cells by CD28 costimulation with CEA×CD28 bsAb The ability of the CD28 bispecific antibodies of the invention to enhance cytokine release by T cells during killing of CEA-expressing tumor cells in the presence of CEAxCD3 was assessed by quantifying selected cytokines in the supernatant at the end of the TDCC assay. After co-culture of CEA-positive target cells and T cell-containing PBMCs as described in Example 9, the culture supernatants were collected by centrifugation and stored frozen at -80°C until further analysis. Cytokines / enzymes such as granzyme B, IL2, IL6, IL10, TNFα and IFNγ were quantified using the Mesoscale Discovery Platform by using multiplex kits. The results of an experiment in which LS174T cells were co-cultured with PBMCs at a dose range of CEAxCD3 and a fixed dose of CEAxCD28 at 1 μg / mL at a 5:1 E:T ratio for 72 hours are shown in Figures 9A-9F. BsAb 1a28 / AC84 / N (above) was included as a reference CEAxCD28 bsAb.

[0296] Single agent treatment with CEAxCD3 resulted in modest cytokine release by T cells, whereas combination treatment of CEAxCD3 with any of the CEAxCD28 bsAbs tested substantially increased all cytokines measured, reflecting the better activation and proliferation of T cells observed previously.

[0297] Example 10: Evaluation of CD28 bsAb-mediated T cell costimulation on the antitumor activity of CEA x CD3 T cell retargeting bsAb in a humanized mouse tumor model Antitumor activity of AI3AC84 / N and AI5AC84 / N in PBMC-humanized mouse tumor models This efficacy study aims to evaluate the antitumor efficacy of two CEAxCD28 κλ bodies carrying anti-CD28 of intermediate affinity (AI3 and AI5) when combined with CEAxCD3.

[0298] The experimental design is summarized in Figure 10, which also shows the treatment groups and treatment regimens. 6 LS174T cells were cultured at 1 × 10 in 7- to 8-week-old NOG mice (NOD / Shi-scid / OL-2Rγ null mice, Taconic Biosciences). 6 PBMCs (E:T=1:1) were co-transplanted subcutaneously and treatment was initiated 7 days after randomization and engraftment. CEAxCD3 and CEAxCD28 bsAbs were co-injected at 10 mg / kg twice weekly for a total of 6 injections. Mice were monitored for tumor development 2-3 times weekly and were followed up until the experimental endpoint (tumor volume = 1500 mm 3 Tumors were measured by digital calipers until the onset of GvHD symptoms or the onset of sarcoidosis. Tumor volume was calculated using the formula (length × width). 2 )×0.5. Figures 11A-11B show tumor growth kinetics (mean + SEM) per group (Figure 11A) and mouse (Figure 11B).

[0299] In mice treated with CEA×CD3, we observed only a mild inhibition of tumor growth compared to the vehicle group. However, both AI3AC84 / N and AI5AC84 / N in combination with CEA×CD3 induced complete tumor regression, with tumors that were not palpable 28 days after implantation when treatment was completed. The experiment was stopped on day 41, and mice in the CEA×CD3+AI5AC84 / N group were still tumor-free, in contrast to the CEA×CD3+AI3AC84 / N group, with two mice relapsing and tumors growing slowly.

[0300] Antitumor activity of AI3AC84 / N (dose range tested) and AI10AC84 / N in PBMC humanized mouse tumor models This efficacy study aims to evaluate the antitumor efficacy of a dose-range CEAxCD28 κλ body carrying an intermediate affinity (AI3) anti-CD28 arm and to compare it with the antitumor efficacy of a CEAxCD28 κλ body carrying a lower affinity (AI10) anti-CD28 arm in combination with CEAxCD3.

[0301] The experimental design is summarized in Figure 12, which also shows the treatment groups and treatment regimens. 6 LS174T cells were cultured at 1 × 10 in 7- to 8-week-old NOG mice (NOD / Shi-scid / OL-2Rγ null mice, Taconic Biosciences). 6 PBMCs (E:T=1:1) were co-transplanted subcutaneously and treatment was initiated 7 days after randomization and engraftment. CEAxCD3 and CEAxCD28 bsAbs were co-injected at the indicated doses twice weekly for a total of six injections. Mice were monitored for tumor development 2-3 times weekly and were followed up until the experimental endpoint (tumor volume = 1500 mm 3 Tumors were measured by digital calipers until the onset of GvHD symptoms or the onset of sarcoidosis. Tumor volume was calculated using the formula (length × width). 2 )×0.5. Figures 13A-13B show tumor growth kinetics (mean + SEM) per group (Figure 13A) and mouse (Figure 13B).

[0302] In mice treated with CEA×CD3, only mild inhibition of tumor growth was observed compared to the vehicle group, whereas all combinations of CD28 bsAb with CEA×CD3 induced significant differential antitumor activity. More specifically, the highest dose of AI3AC84 / N (10 mg / kg) led to complete tumor regression in 6 / 8 mice, with the remaining 2 mice having palpable but non-measurable tumors, whereas the intermediate dose of AI3AC84 / N (2 mg / kg) led to robust tumor control, with no recurrent tumors, and all tumor volumes ultimately reaching <50 mm. 3The efficacy of 10 mg / kg AI10AC84 / N was reduced (complete tumor eradication was observed in only one mouse), and the lowest dose (0.4 mg / kg) resulted in variable tumor control, with half of the mice eradicating tumors and treatment only slowing tumor growth. Treatment with 10 mg / kg AI10AC84 / N, a CEAxCD28 bsAb containing a lower affinity (AI10) anti-CD28 arm, was overall less effective than treatment with AI3AC84 / N at a comparable dose, with only slowing of tumor growth observed in two mice and complete tumor eradication observed in only three mice.

[0303] Example 11: In vitro characterization of MSLNxCD28 bispecific antibodies Binding of MSLN × CD28 bsAb to CD28-positive Jurkat cells, MSLN-positive H226 and OVCAR-3 cells or MSLN and CD28 double-negative TIB153 cells.

[0304] To demonstrate binding of MSLNxCD28κλ bodies to target cells, a series of flow cytometry-based experiments were performed.

[0305] Examples of cells that can be used include MSLN-positive cell lines, such as mesothelioma cell line H226 or ovarian adenocarcinoma cell line OVCAR-3, CD28-positive cell lines, such as leukemia Jurkat T cells, and MSLN and CD28 double-negative cell lines, such as leukemia TIB153 cells. Mesothelin cell surface expression of H226 and OVCAR-3 cells was measured at 180,000 and 68,000 MSLN / cell, respectively. Cell staining and binding assessment were performed as described in Example 4.

[0306] Binding curves of MSLNxCD28bsAb of the invention obtained using H226, OVCAR-3, Jurkat and TIB153 cells are shown in Figures 14A, 14B, 14C and 14D, respectively. Binding curves of additional MSLNxCD28bsAb of the invention to H226, OVCAR-3, Jurkat and TIB153 cells are shown in Figures 17A, 17B, 17C and 17D, respectively. hIgG1 denotes an irrelevant monoclonal antibody of hIgG1 Fc that served as an isotype control.

[0307] Binding to H226 and OVCAR-3 cells highlights how antibodies sharing the same anti-MSLN arm bind similarly with high affinity to MSLN-positive cells (Figures 14A and 14B and Figures 17A and 17B). Binding to Jurkat cells reflects the difference in binding affinity with the AI5 arm versus the AI10 arm (Figure 14C), but highlights the similarity in binding affinity of the AI5 arm versus the AI3 arm, in line with data generated with the CEAxCD28 bsAb of the present invention (Figure 17C). The absence of binding signal on TIB-153 suggests that all binding arms of the present invention are specific to their designated targets (Figures 14D and 17D).

[0308] Example 12: MSLN x CD28 bispecific antibody-mediated T cell-dependent cytotoxicity (TDCC) TDCC of MSLN-positive cell lines The T-cell-dependent cytotoxicity (TDCC) of different MSLN-positive cell lines induced by the MSLNxCD28 bispecific antibody of the present invention was evaluated as described in Example 9. MSLNxCD28bsAb was tested in combination with HPN536 analog, a trispecific MSLNxCD3xHSA molecule that can redirect T cells to MSLN-positive target cells. TDCC curves obtained with H226 cells as targets, a range of doses of HPN536 analog and a fixed dose of 1 or 0.1 μg / mL of MSLNxCD28bsAb are shown in Figures 15A and 15D. TDCC curves obtained with OVCAR3 cells as targets, a range of doses of HPN536 analog and a fixed dose of 2.5 μg / mL of MSLNxCD28bsAb (at different E:T ratios) are shown in Figures 18A-18D.

[0309] MSLNxCD28 bsAb cooperated with HPN536 analogue to kill MSLN-positive H226 target cells. Synergy can be observed for most of the MSLNxCD28 bsAbs at either of the concentrations tested (1 μg / mL vs. 0.1 μg / mL), but is most pronounced for bsAbs carrying O35 or O41 as the targeting anti-MSLN arm. Nearly the same maximum specific lysis of 50-60% of tumor cells was obtained for AI5O35 and AI5O41 at 0.1 and 1 μg / ml, whereas the maximum lysis of the lower affinity AI10 arm achieved was 40-60%. Importantly, when the HPN536 analog was replaced by Y4L3-1 / N, a non-targeted CD3 bsAb unable to deliver signal 1 to effector cells, there was no MSLNxCD28 bsAb-induced killing, highlighting the importance of the primary T cell stimulus (signal 1) for the activity of CD28 bsAb.

[0310] Notably, lower E:T ratios (from 10:1 to 3:1, 1:1, and finally 1:3, Figures 18A-18D) reduced the killing potential of the HPN536 analogs. At both 10:1 and 3:1 E:T ratios, over 90% of the target cells were killed at the highest HPN536 analog dose range, while at E:T ratios equal to 1:1 and 1:3, only 70% and 26% of the target cells were killed at the highest MSLNxCD3 concentrations, respectively. Addition of the MSLNxCD28bsAb of the present invention compensated for this reduced activity, fully restoring the activity of the HPN536 analogs at E:T=1:1, and resulting in up to 60% killing of the target cells at E:T=1:3.

[0311] Combination of MSLNxCD3 and MSLNxCD28 bsAb induced upregulation of T cell activation markers during killing of MSLN-expressing tumor cells The ability of MSLNxCD28 κλ bodies to enhance T cell activation in the presence of the appropriate signal 1 was quantified by flow cytometry using antibodies that recognize specific T cell activation markers as described in Example 9. Results from experiments in which H226 cells were co-cultured with PBMCs at an E:T ratio of 10:1 for 48 hours in the presence of a dose range of MSLNxCD3 (HPN536 analog) and a fixed dose of CD28 bsAb at 1 μg / mL or 0.1 μg / mL are shown in Figures 15B and 15E.

[0312] Compared to single treatments with HPN536 analogs, the MSLNxCD3 and MSLNxCD28 bsAb combinations activated CD4+ T cells to a greater extent, with much brighter CD25 staining for the majority of combination treatments. We observed differences between T cell activation levels depending on the dose of CD28 bsAb tested, with 1 μg / mL resulting in better T cell activation than 0.1 μg / mL. We also demonstrated the importance of both the anti-MSLN and anti-CD28 arms, with O35 and O41 based bsAbs activating T cells better than O30 based bsAb, and the intermediate affinity AI5 arm inducing better T cell activation than the low affinity AI10 arm.

[0313] Effect of MSLN×CD28 bsAb-mediated CD28 costimulation on T cell proliferation in the presence of MSLN×CD3 As described in Example 9, MSLNxCD28 bsAb was analyzed for its ability to potentiate the effect of MSLNxCD3 in inducing T cell proliferation in the presence of MSLN-positive tumor target cells. The percentage of proliferating CD4+ T cells is shown in Figures 15C and 15F, respectively. The ability of HPN536 analogs to induce T cell proliferation was significantly enhanced by the addition of MSLNxCD28 bsAb, although the greatest effect was obtained with AI5-based bsAbs bearing either O35 or O41 as the targeting arm dosed at 1 μg / mL.

[0314] Cytokines released into the supernatant during enhanced killing of MSLN-expressing tumor cells by CD28 costimulation with MSLN×CD28 bsAb The ability of the MSLNxCD28 bispecific antibody of the invention to enhance cytokine release by T cells during killing of MSLN-expressing tumor cells in the presence of MSLNxCD3 (HPN536 analog) was assessed by quantifying selected cytokines in the supernatants at the end of the TDCC assay as described in Example 9. The results of an experiment in which H226 cells were co-cultured with PBMCs at a dose range of HPN536 analog and a fixed dose of MSLNxCD28 at 1 μg / mL or 0.1 μg / mL at an E:T ratio of 10:1 for 48 hours are shown in Figures 16A-16F.

[0315] Single agent treatment with HPN536 analogs resulted in modest cytokine release by T cells, whereas combination treatment of HPN536 analogs with any of the MSLNxCD28 bsAbs tested substantially increased all cytokines measured, particularly IL-2, reflecting the better activation and proliferation of T cells observed previously.

[0316] Example 13: T-cell dependent cytotoxicity (TDCC) mediated by TAAxCD3 and TAAxCD28 bispecific antibody combinations targeting different TAAs. TDCC in CEA / MSLN double positive cells The MSLNxCD28 bispecific antibody of the invention induced T cell dependent cytotoxicity (TDCC) of CEA / MSLN double positive cell lines (HPAC cells, 280,000 CEA / cell and 14,000 CEA / cell) was assessed as described in Example 9 using the CellTiter-Glo® viability assay as readout. CD28 bsAb was tested in combination with CEAxCD3. TDCC curves (at different E:T ratios) obtained using HPAC cells as targets, a dose range of CEAxCD3 and either a fixed dose of 2.5 μg / mL CEAxCD28 or MSLNxCD28 bsAb are shown in Figures 19A-19D.

[0317] Both CEAxCD28 and MSLNxCD28 bsAbs cooperated equally well with CEAxCD3 to kill CEA / MSLN double-positive HPAC target cells, suggesting that TAAxCD28 bsAbs can be successfully paired with TAAxCD3 bsAbs targeting different TAAs. Synergy can be observed at all tested E:T ratios, but is best accentuated at lower E:T ratios, with the addition of CD28 bsAb compensating for the lower activity of CEAxCD3.

[0318] Importantly, when tested alone (i.e., in the absence of signal 1), there was no CD28 bsAb induced killing, highlighting the importance of the primary T cell stimulus (signal 1) for the activity of CD28 bsAb.

[0319] Combination of CD3 and CD28 bsAbs targeting different TAAs induces T cell activation and proliferation The ability of CD28 κλ bodies to enhance T cell activation and proliferation in the presence of an appropriate signal 1 was quantified following TDCC experiments shown in Figure 19. T cell activation data is shown in Figures 20A and 20B for CD4+ and CD8+ T cells, respectively, while Figures 20C and 20D show T cell proliferation for CD4+ and CD8+ T cells, respectively. The different E:T ratios tested are shown and clearly marked at the top of the figures.

[0320] Compared to single treatment with CEA×CD3, the combination of CEA×CD3 with either CEA×CD28 or MSLN×CD28 bsAb activated CD4+ T cells to a greater extent, accompanied by more CD25-positive T cells for both combination treatments and across different E:T ratios. The synergy between CD3 and CD28 bsAb for CD8+ T cell activation was more evident at lower E:T ratios, and at E:T>1, CEA×CD3 bsAb successfully induced CD8+ T cell activation by itself. We observed similar levels of T cell activation with the two bsAbs, suggesting that TAA×CD28 bsAb may be successfully paired with TAA×CD3 bsAb targeting different TAAs.

[0321] The T cell proliferation potential reflected the higher T cell activation observed when using the combination, resulting in enhanced T cell proliferation induced by the combination across the four E:T ratios tested. Of note, CEA×CD3 alone only minimally induced T cell proliferation at E:T ratios of 1 or less, highlighting the advantage of the combination with CD28 bsAb to induce T cell proliferation even at unfavorable E:T ratios.

[0322] Example 13: Evaluation of CD28 bsAb-mediated T cell costimulation on the antitumor activity of CEA x CD3 T cell retargeting bsAb in BRGSF-HIS mice This efficacy study aimed to evaluate the antitumor efficacy of AI3AC84 / N in combination with CEA×CD3 in BRGSF-HIS mice implanted subcutaneously with CEA+HPAF-II tumors. The experimental design is summarized in FIG. 22 and treatment groups and treatment regimens are shown. Briefly, 1.5×10 6 HPAF-II cells were implanted subcutaneously. Treatment was initiated 7 days after implantation after randomization (mean tumor volume approximately 85 mm 3CEAxCD3 and CEAxCD28 bsAbs were co-injected at 10 mg / kg twice weekly for a total of six injections. Mice were monitored for tumor development 2-3 times weekly and were maintained until the end of the experiment (tumor volume = 1500 mm). 3 Tumors were measured by digital calipers until the tumor volume reached 100 mm. Tumor volume was calculated using the formula (length × width). 2 )×0.5. Figure 23A shows the percentage of survival 56 days after engraftment, while Figure 23B shows the tumor growth kinetics of individual mice in each treatment group.

[0323] Only a mild survival advantage was measured in mice treated with CEA×CD3 over the vehicle group. However, the combination of AI3AC84 / N with CEA×CD3 induced a slower tumor growth kinetic and a higher survival probability at day 56 (when the experiment was terminated), with 8 of 12 mice in the CEA×CD3+AI3AC84 / N group still not reaching the tumor endpoint, in contrast to 1 of 12 and 3 of 12 mice in the vehicle and CEA×CD3 groups, respectively.

[0324] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0325] Additional embodiments of the present disclosure include the following. Embodiment 1. Bispecific antibodies, including: a. a first antigen-binding domain that binds to CD28, i. a first heavy chain variable region having a complementarity determining region 1 (CDRH1) comprising the amino acid sequence of (SEQ ID NO:1), a complementarity determining region 2 (CDRH2) comprising the amino acid sequence of (SEQ ID NO:2), and a complementarity determining region 3 (CDRH3) comprising the amino acid sequence of (SEQ ID NO:3); ii. a first light chain variable region, 1. CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 25, or 2. CDRL1 comprising the amino acid sequence of SEQ ID NO: 26, CDRL2 comprising the amino acid sequence of SEQ ID NO: 27, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 28, or 3. CDRL1 comprising the amino acid sequence of SEQ ID NO: 29, CDRL2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 31; or 4. CDRL1 comprising the amino acid sequence of SEQ ID NO: 32, CDRL2 comprising the amino acid sequence of SEQ ID NO: 33, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 34, or 5. CDRL1 comprising the amino acid sequence of SEQ ID NO: 35, CDRL2 comprising the amino acid sequence of SEQ ID NO: 36, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 37, 6. CDRL1 comprising the amino acid sequence of SEQ ID NO: 38, CDRL2 comprising the amino acid sequence of SEQ ID NO: 39, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 40, or 7. CDRL1 comprising the amino acid sequence of SEQ ID NO: 41, CDRL2 comprising the amino acid sequence of SEQ ID NO: 42, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 43, or 8. CDRL1 comprising the amino acid sequence of SEQ ID NO: 44, CDRL2 comprising the amino acid sequence of SEQ ID NO: 45, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 46, or 9. CDRL1 comprising the amino acid sequence of SEQ ID NO: 47, CDRL2 comprising the amino acid sequence of SEQ ID NO: 48, and CDRL3 comprising the amino acid sequence of SEQ ID NO:49, or 10. CDRL1 comprising the amino acid sequence of SEQ ID NO:50, CDRL2 comprising the amino acid sequence of SEQ ID NO:51, and CDRL3 comprising the amino acid sequence of SEQ ID NO:52, or 11. CDRL1 comprising the amino acid sequence of SEQ ID NO:53, CDRL2 comprising the amino acid sequence of SEQ ID NO:54, and CDRL3 comprising the amino acid sequence of SEQ ID NO:55; or 12. CDRL1 comprising the amino acid sequence of SEQ ID NO:56, a CDRL2 comprising the amino acid sequence of SEQ ID NO:57, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:58; or 13. CDRL1 comprising the amino acid sequence of SEQ ID NO:59, CDRL2 comprising the amino acid sequence of SEQ ID NO: 60, and CDRL3 comprising the amino acid sequence of SEQ ID NO:61, or 14. CDRL1 comprising the amino acid sequence of SEQ ID NO: 62, CDRL2 comprising the amino acid sequence of SEQ ID NO: 63, and CDRL3 comprising the amino acid sequence of SEQ ID NO:64, or 15. CDRL1 comprising the amino acid sequence of SEQ ID NO: 65. CDRL2 comprising the amino acid sequence of SEQ ID NO:66, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:67; or 16. CDRL1 comprising the amino acid sequence of SEQ ID NO: 68, CDRL2 comprising the amino acid sequence of SEQ ID NO:69, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 70; or 17. CDRL1 comprising the amino acid sequence of SEQ ID NO: 71, CDRL2 comprising the amino acid sequence of SEQ ID NO: 72, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 73; or 18. CDRL1 comprising the amino acid sequence of SEQ ID NO: 74, CDRL2 comprising the amino acid sequence of SEQ ID NO: 75, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 76, or 19. CDRL1 comprising the amino acid sequence of SEQ ID NO: 77, CDRL2 comprising the amino acid sequence of SEQ ID NO: 78, and CDRL3 comprising the amino acid sequence of SEQ ID NO:79 and a first light chain variable region having a first antigen-binding domain comprising: b. A second antigen-binding domain that binds to a tumor-associated antigen (TAA), i. a second heavy chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3. a second antigen-binding domain comprising: Embodiment 2. 2. The bispecific antibody of claim 1, wherein the TAA is CEA. Embodiment 3. The second antigen-binding domain comprises: c. A second light chain variable region, i. CDR1 comprising the amino acid sequence of SEQ ID NO:8, CDR2 comprising the amino acid sequence of SEQ ID NO:9, and CDR3 comprising the amino acid sequence of SEQ ID NO:10; ii. CDR1 comprising the amino acid sequence of SEQ ID NO: 11, CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR3 comprising the amino acid sequence of SEQ ID NO: 13; iii. CDR1 comprising the amino acid sequence of SEQ ID NO: 14, CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR3 comprising the amino acid sequence of SEQ ID NO: 16. A second light chain variable region having 3. The bispecific antibody of claim 2, comprising: Embodiment 4. The bispecific antibody according to any one of claims 1 to 3, wherein the first heavy chain variable region and the second heavy chain variable region comprise the amino acid sequence of SEQ ID NO:4. Embodiment 5. The bispecific antibody according to any one of claims 1 to 4, wherein the first heavy chain and the second heavy chain comprise the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7. Embodiment 6. d. the first light chain variable region of part ii(1) comprises the amino acid sequence of SEQ ID NO: 80; e. the first light chain variable region of part ii(2) comprises the amino acid sequence of SEQ ID NO: 81; f. the first light chain variable region of part ii(3) comprises the amino acid sequence of SEQ ID NO: 82; g. the first light chain variable region of part ii(4) comprises the amino acid sequence of SEQ ID NO: 83; h. the first light chain variable region of part ii(5) comprises the amino acid sequence of SEQ ID NO: 84; i. the first light chain variable region of part ii(6) comprises the amino acid sequence of SEQ ID NO: 85; j. the first light chain variable region of part ii(7) comprises the amino acid sequence of SEQ ID NO: 86; k. the first light chain variable region of part ii(8) comprises the amino acid sequence of SEQ ID NO: 87; l. the first light chain variable region of part ii(9) comprises the amino acid sequence of SEQ ID NO: 88; m. the first light chain variable region of part ii(10) comprises the amino acid sequence of SEQ ID NO: 89; n. the first light chain variable region of part ii (11) comprises the amino acid sequence of SEQ ID NO: 90; o. the first light chain variable region of part ii(12) comprises the amino acid sequence of SEQ ID NO: 91; p. the first light chain variable region of part ii(13) comprises the amino acid sequence of SEQ ID NO: 92; q. the first light chain variable region of part ii(14) comprises the amino acid sequence of SEQ ID NO:93; r. the first light chain variable region of part ii (15) comprises the amino acid sequence of SEQ ID NO: 94; s. the first light chain variable region of part ii (16) comprises the amino acid sequence of SEQ ID NO: 95; T. the first light chain variable region of part ii (17) comprises the amino acid sequence of SEQ ID NO: 96; u. the first light chain variable region of part ii (18) comprises the amino acid sequence of SEQ ID NO: 97; v. The first light chain variable region of part ii (19) comprises the amino acid sequence of SEQ ID NO: 98; 2. The bispecific antibody of claim 1. Embodiment 7. a. the first light chain of part ii(1) comprises the amino acid sequence of SEQ ID NO: 99; b. the first light chain of part ii(2) comprises the amino acid sequence of SEQ ID NO: 100; c. the first light chain of part ii(3) comprises the amino acid sequence of SEQ ID NO: 101; d. the first light chain of part ii(4) comprises the amino acid sequence of SEQ ID NO: 102; e. the first light chain of part ii(5) comprises the amino acid sequence of SEQ ID NO: 103; f. the first light chain of part ii(6) comprises the amino acid sequence of SEQ ID NO: 104; g. the first light chain of part ii(7) comprises the amino acid sequence of SEQ ID NO: 105; h. the first light chain of part ii(8) comprises the amino acid sequence of SEQ ID NO: 106; i. the first light chain of part ii(9) comprises the amino acid sequence of SEQ ID NO: 107; j. the first light chain of part ii(10) comprises the amino acid sequence of SEQ ID NO: 108; k. the first light chain of part ii(11) comprises the amino acid sequence of SEQ ID NO: 109; l. the first light chain of part ii (12) comprises the amino acid sequence of SEQ ID NO: 110; m. the first light chain of part ii (13) comprises the amino acid sequence of SEQ ID NO: 111; n. the first light chain of part ii (14) comprises the amino acid sequence of SEQ ID NO: 112; o. the first light chain of part ii (15) comprises the amino acid sequence of SEQ ID NO: 113; p. the first light chain of part ii (16) comprises the amino acid sequence of SEQ ID NO: 114; q. the first light chain of part ii (17) comprises the amino acid sequence of SEQ ID NO: 115; r. the first light chain of part ii (18) comprises the amino acid sequence of SEQ ID NO: 116; s. the first light chain of part ii (19) comprises the amino acid sequence of SEQ ID NO: 117; 2. The bispecific antibody of claim 1. Embodiment 8. a. the second light chain variable region of part a(i) comprises the amino acid sequence of SEQ ID NO: 17; b. the second light chain variable region of part a(ii) comprises the amino acid sequence of SEQ ID NO: 18; c. The second light chain variable region of part a(iii) comprises the amino acid sequence of SEQ ID NO: 19. The bispecific antibody of claim 3. EMBODIMENT 9. a. the second light chain of part a(i) comprises the amino acid sequence of SEQ ID NO: 20; b. the second light chain of part a(ii) comprises the amino acid sequence of SEQ ID NO: 21; c. The second light chain of part a(iii) comprises the amino acid sequence of SEQ ID NO: 22; The bispecific antibody of claim 3. Embodiment 10. 10. The bispecific antibody according to claim 1, wherein the first light chain is kappa and the second light chain is lambda. Embodiment 11. 11. The bispecific antibody according to claim 1, wherein the first light chain is lambda and the second light chain is kappa. Embodiment 12. 2. The bispecific antibody of claim 1, comprising an Fc domain comprising one or more amino acid substitutions that reduce binding to activating Fc receptors and / or reduce effector function. Embodiment 13. 13. The bispecific antibody of claim 12, wherein the amino acid substitutions include L234A and L235A substitutions. Embodiment 14. 13. The bispecific antibody of claim 12, wherein the amino acid substitution comprises a P329A, P329G, or P329R substitution. EMBODIMENT 15. The bispecific antibody according to any one of claims 1 to 14, wherein the antibody has an IgG isotype. EMBODIMENT 16. The bispecific antibody according to any one of claims 1 to 15, wherein the antibody is human. EMBODIMENT 17. 17. The bispecific antibody of claim 1, wherein the composition enables tumor-specific T cell activation. EMBODIMENT 18. A composition comprising the bispecific antibody according to any one of claims 1 to 17. EMBODIMENT 19. 19. The composition of claim 18, further comprising a CD3xCEA bispecific antibody. EMBODIMENT 20. The composition of claim 19, wherein the CD3xCEA bispecific antibody comprises two heavy chains comprising the amino acid sequence of SEQ ID NO: 118, a first light chain comprising the amino acid sequence of SEQ ID NO: 119, and a second light chain comprising the amino acid sequence of SEQ ID NO: 120. EMBODIMENT 21. 21. A method for reducing the proliferation of and / or killing tumor cells, comprising contacting a cell with a composition according to any one of claims 18 to 20. EMBODIMENT 22. A method for treating cancer in a subject, comprising administering to the subject a composition according to any one of claims 18 to 20. EMBODIMENT 23. An antibody comprising an antigen-binding domain that binds to CD28, The antigen-binding domain is i. A heavy chain variable region having a complementarity determining region 1 (CDRH1) comprising the amino acid sequence of (SEQ ID NO:1), a complementarity determining region 2 (CDRH2) comprising the amino acid sequence of (SEQ ID NO:2), and a complementarity determining region 3 (CDRH3) comprising the amino acid sequence of (SEQ ID NO:3); ii. A light chain variable region, 1. CDRL1 comprising the amino acid sequence of SEQ ID NO: 23, CDRL2 comprising the amino acid sequence of SEQ ID NO: 24, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 25, or 2. CDRL1 comprising the amino acid sequence of SEQ ID NO: 26, CDRL2 comprising the amino acid sequence of SEQ ID NO: 27, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 28, or 3. CDRL1 comprising the amino acid sequence of SEQ ID NO: 29, CDRL2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 31; or 4. CDRL1 comprising the amino acid sequence of SEQ ID NO: 32, CDRL2 comprising the amino acid sequence of SEQ ID NO: 33, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 34, or 5. CDRL1 comprising the amino acid sequence of SEQ ID NO: 35, CDRL2 comprising the amino acid sequence of SEQ ID NO: 36, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 37, 6. CDRL1 comprising the amino acid sequence of SEQ ID NO: 38, CDRL2 comprising the amino acid sequence of SEQ ID NO: 39, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 40, or 7. CDRL1 comprising the amino acid sequence of SEQ ID NO: 41, CDRL2 comprising the amino acid sequence of SEQ ID NO: 42, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 43, or 8. CDRL1 comprising the amino acid sequence of SEQ ID NO: 44, CDRL2 comprising the amino acid sequence of SEQ ID NO: 45, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 46, or 9. CDRL1 comprising the amino acid sequence of SEQ ID NO: 47, CDRL2 comprising the amino acid sequence of SEQ ID NO: 48, and CDRL3 comprising the amino acid sequence of SEQ ID NO:49, or 10. CDRL1 comprising the amino acid sequence of SEQ ID NO:50, CDRL2 comprising the amino acid sequence of SEQ ID NO:51, and CDRL3 comprising the amino acid sequence of SEQ ID NO:52, or 11. CDRL1 comprising the amino acid sequence of SEQ ID NO:53, CDRL2 comprising the amino acid sequence of SEQ ID NO:54, and CDRL3 comprising the amino acid sequence of SEQ ID NO:55; or 12. CDRL1 comprising the amino acid sequence of SEQ ID NO:56, CDRL2 comprising the amino acid sequence of SEQ ID NO:57, and CDRL3 comprising the amino acid sequence of SEQ ID NO:58; or 13. CDRL1 comprising the amino acid sequence of SEQ ID NO:59, CDRL2 comprising the amino acid sequence of SEQ ID NO: 60, and CDRL3 comprising the amino acid sequence of SEQ ID NO:61, or 14. CDRL1 comprising the amino acid sequence of SEQ ID NO: 62, CDRL2 comprising the amino acid sequence of SEQ ID NO: 63, and CDRL3 comprising the amino acid sequence of SEQ ID NO:64, or 15. CDRL1 comprising the amino acid sequence of SEQ ID NO: 65. CDRL2 comprising the amino acid sequence of SEQ ID NO:66, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:67; or 16. CDRL1 comprising the amino acid sequence of SEQ ID NO: 68, CDRL2 comprising the amino acid sequence of SEQ ID NO:69, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 70; or 17. CDRL1 comprising the amino acid sequence of SEQ ID NO: 71, CDRL2 comprising the amino acid sequence of SEQ ID NO: 72, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 73; or 18. CDRL1 comprising the amino acid sequence of SEQ ID NO: 74, CDRL2 comprising the amino acid sequence of SEQ ID NO: 75, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 76, or 19. CDRL1 comprising the amino acid sequence of SEQ ID NO: 77, CDRL2 comprising the amino acid sequence of SEQ ID NO: 78, and CDRL3 comprising the amino acid sequence of SEQ ID NO:79 and a light chain variable region having An antibody comprising: EMBODIMENT 24. 24. The antibody of claim 23, which is an F(ab) fragment, an F(ab')2 fragment, and an Fv fragment or a single-chain Fv fragment. EMBODIMENT 25. 24. The antibody of claim 23, which is monospecific. 26. 24. The antibody of claim 23, which is monovalent.

Claims

1. Bispecific antibodies including the following: a. The first antigen-binding domain that binds to CD28, i. A first heavy chain variable region having a heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence of SEQ ID NO: 2, and a heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence of SEQ ID NO: 3, ii. The first light chain variable region, 1. Light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence of SEQ ID NO: 23, Light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence of SEQ ID NO: 24, and Light chain complementarity determination region 3 (CDRL3) containing the amino acid sequence of SEQ ID NO: 25, 2. CDRL1 containing the amino acid sequence of SEQ ID NO: 26, CDRL2 containing the amino acid sequence of SEQ ID NO: 27, and CDRL3 containing the amino acid sequence of SEQ ID NO: 28, 3. CDRL1 containing the amino acid sequence of SEQ ID NO: 29, CDRL2 containing the amino acid sequence of SEQ ID NO: 30, and CDRL3 containing the amino acid sequence of SEQ ID NO: 31, 4. CDRL1 containing the amino acid sequence of SEQ ID NO: 32, CDRL2 containing the amino acid sequence of SEQ ID NO: 33, and CDRL3 containing the amino acid sequence of SEQ ID NO: 34, 5. CDRL1 containing the amino acid sequence of SEQ ID NO: 35, CDRL2 containing the amino acid sequence of SEQ ID NO: 36, and CDRL3 containing the amino acid sequence of SEQ ID NO: 37, 6. CDRL1 containing the amino acid sequence of SEQ ID NO: 38, CDRL2 containing the amino acid sequence of SEQ ID NO: 39, and CDRL3 containing the amino acid sequence of SEQ ID NO: 40, 7. CDRL1 containing the amino acid sequence of SEQ ID NO: 41, CDRL2 containing the amino acid sequence of SEQ ID NO: 42, and CDRL3 containing the amino acid sequence of SEQ ID NO: 43, 8. CDRL1 containing the amino acid sequence of SEQ ID NO: 44, CDRL2 containing the amino acid sequence of SEQ ID NO: 45, and CDRL3 containing the amino acid sequence of SEQ ID NO: 46, 9. CDRL1 containing the amino acid sequence of SEQ ID NO: 47, CDRL2 containing the amino acid sequence of SEQ ID NO: 48, and CDRL3 containing the amino acid sequence of SEQ ID NO: 49, 10. CDRL1 containing the amino acid sequence of SEQ ID NO: 50, CDRL2 containing the amino acid sequence of SEQ ID NO: 51, and CDRL3 containing the amino acid sequence of SEQ ID NO: 52, 11. CDRL1 containing the amino acid sequence of SEQ ID NO: 53, CDRL2 containing the amino acid sequence of SEQ ID NO: 54, and CDRL3 containing the amino acid sequence of SEQ ID NO: 55, 12. CDRL1 containing the amino acid sequence of SEQ ID NO: 56, CDRL2 containing the amino acid sequence of SEQ ID NO: 57, and CDRL3 containing the amino acid sequence of SEQ ID NO: 58, 13. CDRL1 containing the amino acid sequence of SEQ ID NO: 59, CDRL2 containing the amino acid sequence of SEQ ID NO: 60, and CDRL3 containing the amino acid sequence of SEQ ID NO: 61, 14. CDRL1 containing the amino acid sequence of SEQ ID NO: 62, CDRL2 containing the amino acid sequence of SEQ ID NO: 63, and CDRL3 containing the amino acid sequence of SEQ ID NO: 64, 15. CDRL1 containing the amino acid sequence of SEQ ID NO: 65, CDRL2 containing the amino acid sequence of SEQ ID NO: 66, and CDRL3 containing the amino acid sequence of SEQ ID NO: 67, 16. CDRL1 containing the amino acid sequence of SEQ ID NO: 68, CDRL2 containing the amino acid sequence of SEQ ID NO: 69, and CDRL3 containing the amino acid sequence of SEQ ID NO: 70, 17. CDRL1 containing the amino acid sequence of SEQ ID NO: 71, CDRL2 containing the amino acid sequence of SEQ ID NO: 72, and CDRL3 containing the amino acid sequence of SEQ ID NO: 73, 18. CDRL1 containing the amino acid sequence of SEQ ID NO: 74, CDRL2 containing the amino acid sequence of SEQ ID NO: 75, and CDRL3 containing the amino acid sequence of SEQ ID NO: 76, or 19. CDRL1 containing the amino acid sequence of SEQ ID NO: 77, CDRL2 containing the amino acid sequence of SEQ ID NO: 78, and CDRL3 containing the amino acid sequence of SEQ ID NO: 79 The first light chain variable region having The first antigen-binding domain, including, b. A second antigen-binding domain that binds to tumor-associated antigens (TAAs), i. A second heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO:

3. The second antigen-binding domain, including the above.

2. a. The first light chain variable region of part a. ii.

1. contains the amino acid sequence of SEQ ID NO: 80, b. The first light chain variable region of part a. ii.

2. contains the amino acid sequence of SEQ ID NO: 81, c. The first light chain variable region of part a. ii.

3. contains the amino acid sequence of SEQ ID NO: 82, d. The first light chain variable region of part a. ii.

4. contains the amino acid sequence of sequence number 83, e. Part a. ii.

5. The first light chain variable region contains the amino acid sequence of Sequence ID No. 84, f. The first light chain variable region of part a. ii.

6. contains the amino acid sequence of SEQ ID NO: 85, g. The first light chain variable region of part a. ii.

7. contains the amino acid sequence of SEQ ID NO: 86, h. Part a. ii.

8. The first light chain variable region contains the amino acid sequence of Sequence ID No. 87, i. Part a. ii.

9. The first light chain variable region contains the amino acid sequence of Sequence ID No. 88, j. Part a. ii.

10. The first light chain variable region contains the amino acid sequence of Sequence ID No. 89, k. Part a. ii.

11. The first light chain variable region contains the amino acid sequence of Sequence ID No. 90, l. Part a. ii.

12. The first light chain variable region contains the amino acid sequence of SEQ ID NO: 91, m. Part a. ii.

13. The first light chain variable region contains the amino acid sequence of Sequence ID No. 92, n. Part a. ii.

14. The first light chain variable region contains the amino acid sequence of Sequence ID No. 93, o. Part a. ii.

15. The first light chain variable region contains the amino acid sequence of Sequence ID No. 94, p. Part a. ii.

16. The first light chain variable region contains the amino acid sequence of Sequence ID No. 95, q. Part a. ii.

17. The first light chain variable region contains the amino acid sequence of SEQ ID NO: 96, r. Part a. ii.

18. The first light chain variable region contains the amino acid sequence of Sequence ID No. 97, or The first light chain variable region of part a. ii.

19. contains the amino acid sequence of sequence number 98. The bispecific antibody according to claim 1.

3. a. Part a. iii.

1. The first light chain contains the amino acid sequence of Sequence ID No. 99, b. The first light chain in part a. ii.

2. contains the amino acid sequence of SEQ ID NO: 100, c. The first light chain of part a. ii.

3. contains the amino acid sequence of SEQ ID NO: 101, d. The first light chain of part a. ii.

4. contains the amino acid sequence of SEQ ID NO: 102, e. Part a. ii.

5. The first light chain contains the amino acid sequence of Sequence ID No. 103, f. Part a. ii.

6. The first light chain contains the amino acid sequence of Sequence ID No. 104, g. The first light chain of part a. ii.

7. contains the amino acid sequence of SEQ ID NO: 105, h. Part a. ii.

8. The first light chain contains the amino acid sequence of Sequence ID No. 106, i. Part a. ii.

9. The first light chain contains the amino acid sequence of Sequence ID No. 107, j. Part a. ii.

10. The first light chain contains the amino acid sequence of Sequence ID No. 108, k. Part a. ii.

11. The first light chain contains the amino acid sequence of Sequence ID No. 109, l. Part a. ii.

12. The first light chain of part a. ii.

12. contains the amino acid sequence of SEQ ID NO: 110, m. Part a. ii.

13. The first light chain of part a. ii.

13. contains the amino acid sequence of sequence number 111, n. Part a. ii.

14. The first light chain contains the amino acid sequence of Sequence ID No. 112, o. Part a. ii.

15. The first light chain contains the amino acid sequence of Sequence ID No. 113, p. Part a. ii.

16. The first light chain in part a. ii.

16. contains the amino acid sequence of sequence number 114, q. Part a. ii.

17. Does the first light chain contain the amino acid sequence of Sequence ID No. 115? r. Part a. ii.

18. The first light chain contains the amino acid sequence of Sequence ID No. 116, or s. Part a. ii. 19 The first light chain of part a. ii. 19 contains the amino acid sequence of sequence number 117, The bispecific antibody according to claim 1.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the TAA is carcinoembryonic antigen (CEA).

5. The second antigen-binding domain is ii. The second light chain variable region, 1. CDR1 containing the amino acid sequence of SEQ ID NO: 8, CDR2 containing the amino acid sequence of SEQ ID NO: 9, and CDR3 containing the amino acid sequence of SEQ ID NO: 10, 2. CDR1 containing the amino acid sequence of SEQ ID NO: 11, CDR2 containing the amino acid sequence of SEQ ID NO: 12, and CDR3 containing the amino acid sequence of SEQ ID NO: 13, or 3. CDR1 containing the amino acid sequence of SEQ ID NO: 14, CDR2 containing the amino acid sequence of SEQ ID NO: 15 and CDR3 containing the amino acid sequence of SEQ ID NO: 16 The second light chain variable region having A bispecific antibody according to claim 4, comprising:

6. a. Part b. ii.

1. The second light chain variable region contains the amino acid sequence of SEQ ID NO: 17, b. The second light chain variable region of part b. ii.

2. contains the amino acid sequence of SEQ ID NO: 18, or c. Part b. ii.

3. The second light chain variable region includes the amino acid sequence of Sequence ID No.

19. The bispecific antibody according to claim 5.

7. a. Part b. ii.

1. The second light chain contains the amino acid sequence of Sequence ID No. 20, b. The second light chain of part b. ii.

2. contains the amino acid sequence of SEQ ID NO: 21, or c. Part b. ii.

3. The second light chain of part b. ii.

3. contains the amino acid sequence of SEQ ID NO:

22. The bispecific antibody according to claim 5.

8. The bispecific antibody according to any one of claims 1 to 3, wherein the TAA is mesothelin (MSLN).

9. The second antigen-binding domain is ii. The second light chain variable region, 1. CDR1 containing the amino acid sequence of SEQ ID NO: 128, CDR2 containing the amino acid sequence of SEQ ID NO: 129, and CDR3 containing the amino acid sequence of SEQ ID NO: 130, 2. CDR1 containing the amino acid sequence of SEQ ID NO: 131, CDR2 containing the amino acid sequence of SEQ ID NO: 132, and CDR3 containing the amino acid sequence of SEQ ID NO: 133, 3. CDR1 containing the amino acid sequence of SEQ ID NO: 134, CDR2 containing the amino acid sequence of SEQ ID NO: 135, and CDR3 containing the amino acid sequence of SEQ ID NO: 136, 4. CDR1 containing the amino acid sequence of SEQ ID NO: 137, CDR2 containing the amino acid sequence of SEQ ID NO: 138, and CDR3 containing the amino acid sequence of SEQ ID NO: 139, or 5. CDR1 containing the amino acid sequence of SEQ ID NO: 140, CDR2 containing the amino acid sequence of SEQ ID NO: 141, and CDR3 containing the amino acid sequence of SEQ ID NO: 142 The second light chain variable region having A bispecific antibody according to claim 8, comprising:

10. a. Part b. ii.

1. The second light chain variable region contains the amino acid sequence of Sequence ID No. 143, b. Part b. ii.

2. The second light chain variable region contains the amino acid sequence of SEQ ID NO: 144, c. Part b. ii.

3. The second light chain variable region contains the amino acid sequence of Sequence ID No. 145, d. Part b. ii.

4. The second light chain variable region contains the amino acid sequence of SEQ ID NO: 146, or e. Part b. ii.

5. The second light chain variable region includes the amino acid sequence of Sequence ID No.

147. The bispecific antibody according to claim 9.

11. a. Part b. ii.

1. The second light chain contains the amino acid sequence of Sequence ID No. 148, b. Part b. ii.

2. The second light chain contains the amino acid sequence of Sequence ID No. 149, c. Part b. ii.

3. The second light chain contains the amino acid sequence of Sequence ID No. 150, d. Part b. ii.

4. The second light chain contains the amino acid sequence of Sequence ID No. 151, or e. Part b. ii.

5. The second light chain of part b. ii.

5. contains the amino acid sequence of SEQ ID NO:

152. The bispecific antibody according to claim 9.

12. The bispecific antibody according to any one of claims 1 to 3, wherein the first heavy chain variable region and the second heavy chain variable region include the amino acid sequence of SEQ ID NO:

4.

13. The bispecific antibody according to any one of claims 1 to 3, wherein the first heavy chain and the second heavy chain include the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:

7.

14. A bispecific antibody according to any one of claims 1 to 3, wherein (i) the first light chain is copper and the second light chain is lambda, or (ii) the first light chain is lambda and the second light chain is copper.

15. A bispecific antibody according to any one of claims 1 to 3, comprising an Fc domain having one or more amino acid substitutions that reduce binding to an activated Fc receptor and / or reduce effector function.

16. The bispecific antibody according to claim 15, wherein the amino acid substitution comprises (i) L234A and L235A substitutions, or (ii) P329A, P329G, or P329R substitutions.

17. The bispecific antibody according to any one of claims 1 to 3, wherein the antibody has an IgG isotype.

18. The bispecific antibody according to any one of claims 1 to 3, wherein the antibody is a human antibody.

19. The bispecific antibody according to any one of claims 1 to 3, wherein the antibody enables tumor-specific T cell activation.

20. A composition comprising a bispecific antibody according to any one of claims 1 to 3.

21. The composition according to claim 20 for use in a method for reducing the proliferation of tumor cells and / or killing tumor cells.

22. The composition according to claim 20 for use in a method for treating cancer in a subject.

23. An antibody containing an antigen-binding domain that binds to CD28, The antigen-binding domain, i. A first heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3 ii. The first light chain variable region, 1. CDRL1 containing the amino acid sequence of SEQ ID NO: 23, CDRL2 containing the amino acid sequence of SEQ ID NO: 24, and CDRL3 containing the amino acid sequence of SEQ ID NO: 25, 2. CDRL1 containing the amino acid sequence of SEQ ID NO: 26, CDRL2 containing the amino acid sequence of SEQ ID NO: 27, and CDRL3 containing the amino acid sequence of SEQ ID NO: 28, 3. CDRL1 containing the amino acid sequence of SEQ ID NO: 29, CDRL2 containing the amino acid sequence of SEQ ID NO: 30, and CDRL3 containing the amino acid sequence of SEQ ID NO: 31, 4. CDRL1 containing the amino acid sequence of SEQ ID NO: 32, CDRL2 containing the amino acid sequence of SEQ ID NO: 33, and CDRL3 containing the amino acid sequence of SEQ ID NO: 34, 5. CDRL1 containing the amino acid sequence of SEQ ID NO: 35, CDRL2 containing the amino acid sequence of SEQ ID NO: 36, and CDRL3 containing the amino acid sequence of SEQ ID NO: 37, 6. CDRL1 containing the amino acid sequence of SEQ ID NO: 38, CDRL2 containing the amino acid sequence of SEQ ID NO: 39, and CDRL3 containing the amino acid sequence of SEQ ID NO: 40, 7. CDRL1 containing the amino acid sequence of SEQ ID NO: 41, CDRL2 containing the amino acid sequence of SEQ ID NO: 42, and CDRL3 containing the amino acid sequence of SEQ ID NO: 43, 8. CDRL1 containing the amino acid sequence of SEQ ID NO: 44, CDRL2 containing the amino acid sequence of SEQ ID NO: 45, and CDRL3 containing the amino acid sequence of SEQ ID NO: 46, 9. CDRL1 containing the amino acid sequence of SEQ ID NO: 47, CDRL2 containing the amino acid sequence of SEQ ID NO: 48, and CDRL3 containing the amino acid sequence of SEQ ID NO: 49, 10. CDRL1 containing the amino acid sequence of SEQ ID NO: 50, CDRL2 containing the amino acid sequence of SEQ ID NO: 51, and CDRL3 containing the amino acid sequence of SEQ ID NO: 52, 11. CDRL1 containing the amino acid sequence of SEQ ID NO: 53, CDRL2 containing the amino acid sequence of SEQ ID NO: 54, and CDRL3 containing the amino acid sequence of SEQ ID NO: 55, 12. CDRL1 containing the amino acid sequence of SEQ ID NO: 56, CDRL2 containing the amino acid sequence of SEQ ID NO: 57, and CDRL3 containing the amino acid sequence of SEQ ID NO: 58, 13. CDRL1 containing the amino acid sequence of SEQ ID NO: 59, CDRL2 containing the amino acid sequence of SEQ ID NO: 60, and CDRL3 containing the amino acid sequence of SEQ ID NO: 61, 14. CDRL1 containing the amino acid sequence of SEQ ID NO: 62, CDRL2 containing the amino acid sequence of SEQ ID NO: 63, and CDRL3 containing the amino acid sequence of SEQ ID NO: 64, 15. CDRL1 containing the amino acid sequence of SEQ ID NO: 65, CDRL2 containing the amino acid sequence of SEQ ID NO: 66, and CDRL3 containing the amino acid sequence of SEQ ID NO: 67, 16. CDRL1 containing the amino acid sequence of SEQ ID NO: 68, CDRL2 containing the amino acid sequence of SEQ ID NO: 69, and CDRL3 containing the amino acid sequence of SEQ ID NO: 70, 17. CDRL1 containing the amino acid sequence of SEQ ID NO: 71, CDRL2 containing the amino acid sequence of SEQ ID NO: 72, and CDRL3 containing the amino acid sequence of SEQ ID NO: 73, 18. CDRL1 containing the amino acid sequence of SEQ ID NO: 74, CDRL2 containing the amino acid sequence of SEQ ID NO: 75, and CDRL3 containing the amino acid sequence of SEQ ID NO: 76, or 19. CDRL1 containing the amino acid sequence of SEQ ID NO: 77, CDRL2 containing the amino acid sequence of SEQ ID NO: 78, and CDRL3 containing the amino acid sequence of SEQ ID NO: 79 The first light chain variable region having The antibody, which includes the antibody.

24. The antibody according to claim 23, wherein (i) it is an F(ab) fragment, an F(ab')2 fragment, or an Fv fragment or a single-stranded Fv fragment, (ii) it is monospecific, or (iii) it is monovalent.

25. A bispecific antibody comprising the following: a. The first antigen-binding domain that binds to CD28, i. A first heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3, ii. A first light chain variable region having CDRL1 containing the amino acid sequence of SEQ ID NO: 23, CDRL2 containing the amino acid sequence of SEQ ID NO: 24, and CDRL3 containing the amino acid sequence of SEQ ID NO:

25. The first antigen-binding domain, including, b. A second antigen-binding domain that binds to CEA, i. A second heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3, ii. A second light chain variable region having CDRL1 containing the amino acid sequence of SEQ ID NO: 14, CDRL2 containing the amino acid sequence of SEQ ID NO: 15, and CDRL3 containing the amino acid sequence of SEQ ID NO:

16. The second antigen-binding domain, including the above.

26. i. The first heavy chain variable region and the second heavy chain variable region include the amino acid sequence of SEQ ID NO: 4, ii. The first light chain variable region includes the amino acid sequence of SEQ ID NO: 80, and iii. The second light chain variable region includes the amino acid sequence of SEQ ID NO: 19, The bispecific antibody according to claim 25.

27. ​​i. The first heavy chain and the second heavy chain contain the amino acid sequence of SEQ ID NO: 6, ii. The first light chain contains the amino acid sequence of SEQ ID NO: 99, and iii. The second light chain contains the amino acid sequence of SEQ ID NO: 22, The bispecific antibody according to claim 25.

Images