CD28 bispecific antibodies for targeted T cell activation

Abstract

The present invention relates to antibody arms that bind to human CD28 agonists. The present invention relates to bispecific antibodies that bind to human carcino-embryonic antigen (CEA) and human CD28 using the agonist anti-CD28 arm described herein. The present invention relates to bispecific antibodies that bind to human mesothelin (MSLN) and human CD28 with the anti-CD28 arm using the agonist described herein.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 317,491, filed on March 7, 2022, and U.S. Provisional Application No. 63 / 323,893, filed on March 25, 2022, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The contents of the electronic sequence listing (NOVI_050_001WO_SeqList_ST26.xml; size: 129,730 bytes; creation date: March 6, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention generally relates to agonist fully human and κλ-body compatible CD28 binding domains with defined affinity that can be used to generate bispecific antibodies (bsAbs). Specifically, the CD28 binding domain is paired with an anti-tumor associated antigen (TAA) binding domain to generate TAAxCD28 bsAbs, particularly CEAxCD28 and MSLNxCD28 bispecific antibodies. Background Art

[0006] Over the past few years, new approaches have been developed to stimulate the body's own immune cells to better attack and kill cancer cells. Examples of successful cancer immunotherapies are monoclonal antibodies that can 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 name Keytruda and cemiplimab, sold under the trade name Libtayo), and PD-L1 (e.g., atezolizumab, sold under the trade name Tecentriq). Durable anti-tumor responses can be obtained in a range of cancer types using ICIs. Unfortunately, responses are limited to certain subsets of patients, and many cancer types are known to be intrinsically resistant to ICI monotherapy.

[0007] Other approved cancer immunotherapies include T cell bispecific antibodies - bridging T cells via the CD3 receptor on T cells and chimeric antigen receptor (CAR) T cells to target cells expressing tumor-associated antigens (TAAs). Although very good anti-tumor responses have been observed using treatment with T cell bispecific antibodies or CAR T cells in hematological malignancies, these approaches have not achieved significant breakthroughs in the case of solid cancers to date, leaving many cancer patients without treatment options.

[0008] Therefore, despite the success of these immunotherapies in some cancer types, a large proportion of cancer patients lack effective treatment options, highlighting the need for new treatments. The use of molecules capable of activating the immune system by targeting co-stimulatory signals on T cells is underexplored and may open up new treatment options for patients with solid cancers.

[0009] There is a need for compositions and methods for targeting T cell activation that can be used to treat solid cancers. Provided herein are methods and compositions that address this need. Summary of the invention

[0010] The present disclosure provides a bispecific antibody comprising: a. a first antigen-binding domain that binds to CD28; wherein the first antigen-binding domain comprises: i. a first heavy chain variable region comprising a complementary determining region 1 (CDRH1) comprising an amino acid sequence of (SEQ ID NO: 1), a complementary determining region 2 (CDRH2) comprising an amino acid sequence of (SEQ ID NO: 2), and a complementary determining region 3 (CDRH3) comprising an amino acid sequence of (SEQ ID NO: 3); and ii. a first light chain variable region comprising: 1. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 23; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 24; and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 25; 2. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 26; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 27; and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 28; 3. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 29; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 30 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; 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 CDRL3 comprising the amino acid sequence of SEQ ID NO:46. NO:45 amino acid sequence; 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.comprising the amino acid sequence of SEQ ID NO:50; CDRL1 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. 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. NO:64 amino acid sequence; 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. ID NO:79; and b. a second antigen binding domain that binds to a tumor-associated antigen (TAA), wherein the second antigen binding domain comprises: i. a second heavy chain variable region comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1; DRH2 comprising the amino acid sequence of SEQ ID NO:2; and CDRH3 comprising the amino acid sequence of SEQ ID NO:3.

[0011] In some embodiments, the TAA is CEA. In some aspects, the TAA is mesothelin (MSLN).

[0012] 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; e. the first light chain variable region of part a.ii.5. comprises the amino acid sequence of SEQ ID NO:84; 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; j. the first light chain variable region of part a.ii.10. comprises the amino acid sequence of SEQ ID NO:89; 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. the first light chain variable region of part a.ii.15. comprises the amino acid sequence of SEQ ID NO:94; p. 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.

[0013] 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:103; 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; j. the first light chain of part a.ii.10. comprises the amino acid sequence of SEQ ID NO:108; k. the first light chain of part a.ii.11. comprises the amino acid sequence of SEQ ID NO:109; l. the first light chain of part a.ii.12. comprises the amino acid sequence of SEQ ID NO:110; m. the first light chain of part a.ii.13. comprises the amino acid sequence of SEQ ID NO:111; n. the first light chain of part a.ii.14. comprises the amino acid sequence of SEQ ID NO:112; o. the first light chain of part a.ii.15. comprises the amino acid sequence of SEQ ID NO:113; p. the first light chain of part a.ii.16. comprises the amino acid sequence of SEQ ID NO:114; q. the first light chain of part a.ii.17. comprises the amino acid sequence of SEQ ID NO:115; r. the first light chain of part a.ii.18. comprises the amino acid sequence of SEQ ID NO:116; or s. the first light chain of part a.ii.19. comprises the amino acid sequence of SEQ ID NO:117.

[0014] In some embodiments, the second antigen binding domain comprises: ii. a second light chain variable region comprising: 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.

[0015] 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.

[0016] In some embodiments, a. the second light chain of part b.ii.1. comprises the amino acid sequence of SEQ ID NO:20; b. the second light chain of part b.ii.2. comprises the amino acid sequence of SEQ ID NO:21; or c. the second light chain of part b.ii.3. comprises the amino acid sequence of SEQ ID NO:22.

[0017] In some embodiments, the second antigen binding domain comprises: ii. a second light chain variable region comprising: 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;

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In some embodiments, the first light chain is κ and the second light chain is λ. In some embodiments, the first light chain is λ and the second light chain is κ.

[0023] In some embodiments, the bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce binding to activated Fc receptors and / or reduce effector functions. In some embodiments, the amino acid substitutions include L234A and L235A substitutions. In some embodiments, the amino acid substitutions include P329A, P329G or P329R substitutions. In some embodiments, the antibody has an IgG isotype. In some embodiments, the antibody is human.

[0024] 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.

[0025] The present disclosure provides a method of reducing the proliferation of tumor cells and / or killing tumor cells, comprising contacting the cells with any one of the compositions of the present disclosure.

[0026] 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.

[0027] The present disclosure provides an antibody comprising an antigen binding domain that binds to CD28; wherein the antigen binding domain comprises: 1. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 23; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 24; and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 25; 2. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 26; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 27; and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 28; 3. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 29; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 30; and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 31; 4. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 32; a CDRL2 comprising an amino acid sequence of SEQ ID NO: 33; and a CDRL3 comprising an amino acid sequence of SEQ ID NO: 34; 5. a CDRL1 comprising an amino acid sequence of SEQ ID NO: 35;

[0028] 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

[0029] 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 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;

[0030] 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; DRL2 comprising the amino acid sequence of SEQ ID NO: 54; and

[0031] comprising the amino acid sequence of SEQ ID NO:55; 12. 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; 13. 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; 14. 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; 15. 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; or 16. 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. 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.

[0032] In some embodiments, the antibody is a F(ab) fragment, a F(ab')2 fragment, and a Fv fragment or a single-chain Fv fragment. In some embodiments, the antibody is monospecific. In some embodiments, the antibody is monovalent.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Suitable methods and materials will be described below, but methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be limiting.

[0034] Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A-1C is a series of graphs showing the concentration-dependent binding of the anti-CD28 arm of the invention to Jurkat cells expressing CD28. The anti-CD28 arm was tested as a bivalent monoclonal antibody (hlgG1). hIgG1: isotype control; TGN1412 (hlgG4): positive control. Figure 1 A, 1B, and 1C show candidates identified during sequential phage display selection campaigns.

[0036] Figure 2 A-2D shows the interaction between the CEAxCD28 bispecific antibody of the present invention and Jurkat cells expressing CD28 ( Figure 2 A and Figure 2 B) LS174T cells expressing CEA ( Figure 2 C) and CD28 and CEA double negative TIB153 cells ( Figure 2 A series of graphs showing concentration-dependent binding of CEAxCD28 bsAbs (D). A panel of CEAxCD28 bsAbs was generated using the high affinity anti-CEA arm AC84, an IgG1 Fc with three mutations LALAPA, and various anti-CD28 arms of varying affinities. hIgG1: isotype control; TGN1412: positive control; 1a28 / AC84 / N, CEA_CD28_V8, and CEA_CD28_V15: CEAxCD28 reference bispecific antibodies.

[0037] Figure 3 A-3D shows the so-called wet coating of antibodies on the test plate ( Figure 3 A and 3C) or dry coating ( Figure 3 Proliferation of human CD4+ and CD8+ T cells from PBMCs of healthy donors mediated by the CEAxCD28 bispecific antibody of the present invention after 3D (respectively, Figure 3A and 3C and 3B and 3D). A panel of anti-CD28 arms were tested for superagonist activity in a bispecific format. All anti-CD28 arms of the invention, except "AI19", did not show superagonist activity in this 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.

[0038] Figure 4 A series of graphs showing concentration-dependent IL-2 reporter cell activation of the CEAxCD28 bispecific antibodies of the invention, as measured by luminescence readout in the presence of CEA-expressing MKN-45 cells and a fixed concentration (1 nM) of CEAxCD3. Different levels of CD28 co-stimulation induced by the CEAxCD28 bsAbs of the invention were observed, reflecting the different binding capacities of these anti-CD28 arms. DR = dose response; no target: CD3 and CD28 bsAbs were incubated in the presence of effector / reporter cells but not TAA-positive target cells.

[0039] Figure 5 A-5D are a series of graphs showing T-cell retargeted killing / lysis of LS174 T cells by the CEAxCD28 bispecific antibodies of the present invention when combined with CEAxCD3 bsAbs. Effector cells were PBMCs ( Figure 5 A and 5B). CEAxCD28 bsAb synergizes with CEAxCD3 to kill CEA-positive target cells. Killing is not induced in the absence of signal 1 ( Figure 5 C and 5D, the data are respectively used with Figure 5 5B). Y4L3-1 / N: non-targeting CD3 monovalent antibody; 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody. CD28bsAb concentration used: 1 μg / mL.

[0040] Figure 6 A-6B is a graph showing the effect of the CEAxCD28 bispecific antibody of the present invention on KATO-III ( Figure 6 A) and HT-29( Figure 6B) Series of graphs of T-cell retargeted killing / lysis of cells. Synergy between CEAxCD3 and CEAxCD28 bsAbs in killing CEA-positive target cells was also observed with these lower expressing cell lines. 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody. CD28 bsAb concentration used: 2.5 μg / mL.

[0041] Figure 7 A-7B shows human CD4+ and CD8+ T cells (respectively, Figure 7 A and 7B) A series of graphs showing upregulation of the T cell activation marker CD25 on PBMCs. PBMCs were co-cultured with CEA-positive LS174 T cells in the presence of CEAxCD3 alone (to trigger T cell activation signal 1) or in combination with the CEAxCD28 bispecific antibody of the invention (providing T cell activation signal 2). The combination of CEAxCD3 and CEAxCD28 bsAbs induces much stronger activation of both CD4+ and CD8+ T cells than CEAxCD3 monotherapy. 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody.

[0042] Figure 8 A-8B shows human CD4+ and CD8+ T cells (respectively, Figure 8 A and 8B). PBMCs were co-cultured with CEA-positive LS174 T cells in the presence of CEAxCD3 alone (to trigger T cell activation signal 1) or in combination with the CEAxCD28 bispecific antibody of the present invention (providing T cell activation signal 2). The combination of CEAxCD3 and CEAxCD28 bsAb greatly increased the percentage of proliferating CD4+ and CD8+ T cells compared to CEAxCD3 monotherapy. 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody.

[0043] Fig. 9 A-9F is a graph showing the expression of granzyme B (GMB) mediated by the CEAxCD28 bispecific antibody of the invention after co-culture of PBMCs with CEA-positive LS174T tumor cells when combined with the CEAxCD3 bsAb. Fig. 9 A) IFN-γ ( Fig. 9 B) TNF-α( Fig. 9 C) IL-2 ( Fig. 9 D) IL-6 ( Fig. 9 E), IL-10 ( Fig. 9F) A series of graphs showing the secretion of CEAxCD3. Cytokine induction by CEAxCD3 is increased when T cells are co-stimulated simultaneously with CEAxCD28 bsAb. 1a28 / AC84 / N: CEAxCD28 reference bispecific antibody.

[0044] Fig.10 for Fig.11 Schematic diagram of the experimental design of the in vivo efficacy study shown in , where the CEAxCD28 bispecific antibodies AI3AC84 / N and AI5AC84 / N were tested in combination with CEAxCD3 in the LS174T:PBMC co-transplant model of NOG mice.

[0045] Fig.11 A-11B shows Fig.10 A series of graphs depicting the in vivo anti-tumor efficacy of the CEAxCD28 bispecific antibodies AI3AC84 / N and AI5AC84 / N in the LS174T:PBMC co-transplant model. Mean tumor volumes ( Fig.11 A) and tumor growth in individual mice ( Fig.11 B). Both CEAxCD28 bsAbs enhanced the in vivo antitumor activity of CEAxCD3.

[0046] Fig.12 for Fig.13 Schematic representation of the experimental design of the in vivo efficacy study is shown, in which the CEAxCD28 bispecific antibodies AI3AC84 / N (over a dose range) and AI10AC84 / N were tested in combination with CEAxCD3 in the LS174T:PBMC co-transplant model of NOG mice.

[0047] Fig.13 A-13B shows Fig.12 A series of graphs depicting the in vivo anti-tumor efficacy of the CEAxCD28 bispecific antibodies AI3AC84 / N (over a range of doses) and AI10AC84 / N in the LS174T:PBMC co-transplant model. Mean tumor volumes ( Fig.13 A) and tumor growth in individual mice ( Fig.13 B). Both CEAxCD28bsAbs enhanced the in vivo antitumor activity of CEAxCD3. However, at equivalent therapeutic doses, antibody AI3AC84 / N resulted in better tumor control than AI10AC84 / N.

[0048] Fig.14 A-14D is a graph showing the interaction between the MSLNxCD28 bispecific antibody of the present invention and H226 ( Fig.14 A) and OVCAR-3( Fig.14B) cells, Jurkat cells expressing CD28 ( Fig.14 C) and CD28 and MSLN double negative TIB153 cells ( Fig.14 D) A panel of huIgGl-LALAPA MSLNxCD28bsAbs was generated using high affinity anti-MSLN arms O30, O35 and O41 coupled to either "AI5" or "AI10" anti-CD28 arms. hIgGl: Isotype control.

[0049] Fig.15 A-15F shows the killing of target cells by the combination of MSLNxCD3 and MSLNxCD28 bispecific antibodies ( Fig.15 A and 15D), T cell activation ( Fig.15 B and 15E) and T cell proliferation ( Fig.15 Figure 15A shows a series of graphs showing the effects of the MSLNxCD28 bsAb on MSLN-positive target cells and induced T cell activation and proliferation. When the HPN536 analog was replaced with Y4L3-1 / N (a non-targeted CD3 monovalent antibody that cannot provide T cell activation signal 1), no killing, T cell activation or T cell proliferation was induced.

[0050] Fig.16 A-16F is a graph showing the expression of granzyme B (GNB) mediated by the MSLNxCD28 bispecific antibody of the present invention after co-culture of PBMCs with MSLN-positive H226 tumor cells when combined with the HPN536 analog. Fig.16 A) IFN-γ ( Fig.16 B) TNF-α( Fig.16 C) IL-2 ( Fig.16 D) IL-6 ( Fig.16 E), IL-10 ( Fig.16 F) A series of graphs showing the secretion of HPN536. When T cells were co-stimulated with MSLNxCD28 bsAb, the induction of cytokines by HPN536 analogs was greatly enhanced.

[0051] Fig.17 A-17D is a graph showing that an exemplary MSLNxCD28 bispecific antibody of the present invention binds to H226 ( Fig.17 A) and OVCAR-3( Fig.17 B) cells, Jurkat cells expressing CD28 ( Fig.17 C) and CD28 and MSLN double negative TIB153 cells ( Fig.17D) is a series of graphs showing concentration-dependent binding of MSLNxCD28. Exemplary MSLNxCD28 bsAbs generated using high affinity anti-MSLN arms O35 and O41 coupled to an AI3 anti-CD28 arm are shown. hIgG1: isotype control.

[0052] Fig.18 A-18D shows the presence of different E:T ratios (10:1 ( Fig.18 A) 3:1( Fig.18 B) 1:1( Fig.18 C) and 1:3( Fig.18 D)) A series of graphs of T cell retargeted killing / lysis of OVCAR3 cells by an exemplary MSLNxCD28 bispecific antibody of the invention when combined with the HPN536 analog MSLNxCD3 bsAb. The MSLNxCD28 bsAb synergizes with MSLNxCD3 to kill MSLN-positive target cells. The synergy is particularly evident in cases where the E:T ratio is unfavorable, when the combination compensates for the loss of efficacy of the MSLNxCD3 bsAb alone. Killing is not induced in the absence of signal 1 (CD28 bsAb alone). CD28 bsAb concentration used: 2.5 μg / mL.

[0053] Fig.19 A-19D shows the presence of different E:T ratios (10:1 ( Fig.19 A) 3:1( Fig.19 B) 1:1( Fig.19 C) and 1:3( Fig.19 D)) A series of graphs of T cell retargeted killing / lysis of CEA / MSLN double positive HPAC cells by an exemplary CD28 bispecific antibody of the invention when combined with CEAxCD3. Both CEAxCD28 and MSLNxCD28 bsAbs synergize with CEAxCD3 to kill target cells. The synergy is particularly evident in cases where the E:T ratio is unfavorable, when the combination compensates for the loss of efficacy of the CEAxCD3 bsAb alone. Killing is not induced in the absence of signal 1 (CD28 bsAb alone). CD28 bsAb concentration used: 2.5 μg / mL.

[0054] Fig. 20 A-20D shows human CD4+ and CD8+ T cells (respectively, Fig. 20 A and 20B) and upregulation of the T cell activation marker CD25 and human CD4+ and CD8+ T cells (respectively, Fig. 20C and 20D) a series of graphs of proliferation of PBMC. PBMC and CEA / MSLN double positive HPAC cells are co-cultured in the presence of CEAxCD3 alone (to trigger T cell activation signal 1) or in the presence of CEAxCD28 or MSLNxCD28 bispecific antibodies of the present invention in combination (providing T cell activation signal 2). Under the different E:T ratios tested, the combination of CEAxCD3 and CD28bsAb induced much stronger activation of both CD4+ and CD8+T cells than CEAxCD3 monotherapy. Similarly, under all tested E:T ratios, the combination of CEAxCD3 and CD28 bsAb also resulted in a higher percentage of proliferative CD4+ and CD8+T cells compared to CEAxCD3 monotherapy.

[0055] Fig.21 for Fig. 22 Schematic representation of the experimental design of the in vivo efficacy study is shown, in which the CEAxCD28 bispecific antibody AI3AC84 / N was tested in combination with CEAxCD3 in 67 mice implanted subcutaneously with HPAF-II cells.

[0056] Fig. 22 A-22B is shown Fig.21 A series of graphs showing the in vivo anti-tumor efficacy of the CEAxCD28 bispecific antibody AI3AC84 / N in the mouse model described above. The survival rate at day 56 after implantation is shown for all treatment groups ( Fig. 22 A) and tumor growth in individual mice ( Fig. 22 B). AI3AC84 / N enhanced the antitumor activity of CEAxCD3 in vivo. DETAILED DESCRIPTION

[0057] The present invention is based in part on bispecific antibodies (bsAbs) that can achieve tumor-dependent T cell activation and tumor cell killing. Specifically, the present invention is based on the co-engagement of bsAbs of tumor-associated antigens (TAAs) expressed on the surface of target cells to mediate CD28 clustering and thus mediate tumor-specific T cell activation. The present invention also provides CD28 antigen binding domains, antigen binding fragments and antibodies.

[0058] The bsAb of the present invention is characterized by a single agonist CD28 antigen binding domain to achieve monovalent co-stimulation of CD28 and a second antigen binding domain that can specifically and monovalently bind to a tumor-associated antigen to target the bsAb to the tumor microenvironment. The bsAb of the present invention is referred to herein as CD28xTAAbsAb or TAAxCD28 bsAb.

[0059] CD28 is a key co-stimulatory receptor expressed on the surface of T lymphocytes. It belongs to a subfamily of co-stimulatory molecules characterized by an extracellular variable immunoglobulin-like domain. Other members of this family of molecules include CTLA-4, ICOS, PD-1, and BTLA.

[0060] In humans, CD28 is expressed as a disulfide-linked homodimer at the cell surface of T lymphocytes and is found on approximately 80% of human CD4+ T cells and 50% of CD8+ T cells.

[0061] Despite the lack of intrinsic enzymatic activity, engagement of CD28 with its ligand results in specific phosphorylation and transcriptional signaling, which ultimately leads to metabolic changes and production of key cytokines, chemokines, and survival signals that are essential for long-term expansion and differentiation of T cells.

[0062] The main ligands of CD28 are CD80 (B7.1) and CD86 (B7.2), which are mainly expressed on the surface of professional antigen presenting cells (APCs). CD80 and CD86 differ in their expression patterns, polymer states, and functions. Because CD28 and CTLA-4 are highly homologous, they compete for the same ligands. However, since CTLA-4 binds to these ligands with a higher affinity than CD28, CTLA-4 competes with CD28 for ligands and ultimately inhibits T cell responses.

[0063] Several anti-CD28 monoclonal antibodies have been proposed for therapeutic targeting of CD28. It was found that a portion of the identified anti-CD28 antibodies, called superagonist (SA) antibodies, induced complete activation of primary resting T cells via clustering of CD28 on the surface of T cells even in the absence of TCR connection (signal 1). However, the first in vivo study of TGN1412, one of these SA anti-CD28 antibodies, led to severe inflammatory reactions and chronic organ failure in all healthy volunteers treated. Neither in vivo nor in vitro preclinical safety studies predicted that cytokine storms were the cause of these adverse events.

[0064] To avoid the safety issues associated with SA antibodies or with systemic CD28 co-stimulation, tumor-targeted CD28 bispecific antibodies can be designed to specifically co-stimulate T cells within the tumor microenvironment. By pairing an agonist anti-CD28 binding domain with an anti-TAA binding domain, a molecule capable of bridging T cells to malignant cells expressing the selected TAA will be generated. Since CD28 bsAbs can only bind monovalently to CD28, in the absence of TAA-positive target cells, CD28 will not be able to successfully cluster, thereby preventing systemic T cell activation.

[0065] Furthermore, even in the presence of TAA-positive cancer cells that allow CD28 clustering at the T cell surface, the full cytotoxic potential of T cells can be unleashed by TAAxCD28 bispecific antibodies only in the presence of primary T cell stimulation via the TCR. This is in contrast to the bivalent superagonist CD28 monoclonal antibodies described above. Preclinical studies have demonstrated the benefit of adding a co-stimulatory TAAxCD28 bsAb to treat solid tumors, which would enhance the efficacy of bispecific T cell engagers or PD-(L)1 checkpoint inhibitors. Examples of agonist 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).

[0066] 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, lewisY, sLe, LY6K, muthsp70-2, 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, asparagine endopeptidase, 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 β, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, CLDN18.2, TSHR, UPK2 and mesothelin (MSLN).

[0067] In specific embodiments, the TAA is CEA, PSMA, EpCAM, GPC3, MSLN, HER2, HER3, or BCMA. In some embodiments, the TAA is CEA. In some embodiments, the TAA is MSLN.

[0068] CEA

[0069] 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.

[0070] CEA was originally discovered as a specific carcinoembryonic antigen for the human gastrointestinal (GI) system. It was found to be highly expressed by GI tissues during fetal development, but its expression sharply decreases before birth, and in healthy people, CEA is usually found to be expressed at low levels only on the apical side of epithelial cells of the gastrointestinal epithelium and on other mucosal epithelia such as the nasopharynx, lungs, and urogenital tract. Such polarization and limited expression are lost during tumorigenesis, and CEA is overexpressed in solid tumors of multiple epithelial origins, including colorectal cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, and breast cancer.

[0071] CEA represents a promising TAA for the development of antibody-based therapies because different expression patterns and levels are observed between cancer cells and normal epithelial cells. Normal epithelial cells express CEA only at the apical surface of the epithelial cells, a region that excludes therapeutic antibodies due to tight junctions.

[0072] A variety of monoclonal antibodies targeting CEA have been developed for research purposes, as diagnostic tools and for therapeutic purposes (see, e.g., WO2012117002). In the past few years, several bispecific antibodies carrying an anti-CEA arm as a targeting arm have been described, including the CEAxCD3 bispecific antibody Cerbituximab ((Bacac et al., 2016) and US20140242079), MEDI-565 ((Oberst et al., 2014) and WO2016036678A1) or those described in WO2021053587, and more recently, CEAxCD28 bsAb (e.g., WO2020127628A1 and WO2020127618) and CEAxCD47 bsAb (e.g., WO2021110647).

[0073] Anti-CEA antibodies

[0074] Exemplary anti-CEA antibodies of the disclosure are described below (eg, "AC22," "AC61," "AC84"). Table 1 shows the amino acid sequences of the antibodies described herein.

[0075] In some embodiments, the AC22 antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:8, CDRL2 comprising the amino acid sequence of SEQ ID NO:9, and CDRL3 comprising the amino acid sequence of SEQ ID NO:10.

[0076] 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.

[0077] 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.

[0078] 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

[0079] A CDRL3 comprising the amino acid sequence of SEQ ID NO:13.

[0080] 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.

[0081] 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.

[0082] In some embodiments, the AC84 antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0083] 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.

[0084] 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.

[0085] Mesothelin

[0086] Mesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein 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 into a 40-kDa membrane-bound mature form, thereby releasing a 31-kDa fragment called megakaryocyte potentiating factor (MPF).

[0087] Mesothelin is 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 is believed to play a role in cancer cell proliferation, local invasion, and tumor metastasis, and its expression on tumor cells is associated with increased tumor aggressiveness and poor clinical outcomes.

[0088] Due to the differential expression observed between cancer cells and healthy tissues, mesothelin represents an interesting TAA for the development of therapeutics targeting mesothelin, some of which are currently undergoing clinical trials.

[0089] Anti-MSLN antibody

[0090] Exemplary anti-MSLN antibodies of the disclosure are described below (eg, "O30," "O25," "O35," "O38," "O41"). Table 1 shows the amino acid sequences of the antibodies described herein.

[0091] 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

[0092] CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0093] In some embodiments, the 030 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.

[0094] In some embodiments, the 030 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.

[0095] 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

[0096] CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0097] In some embodiments, the 025 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.

[0098] 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.

[0099] In some embodiments, the O35 antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:134, CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0100] 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.

[0101] 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.

[0102] In some embodiments, the O38 antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:137, CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0103] 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.

[0104] 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.

[0105] In some embodiments, the O41 antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:140, CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0106] 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.

[0107] 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.

[0108] Bispecific Antibodies

[0109] The bsAb antibodies according to the present invention can be generated de novo or engineered from existing monospecific CD28 antibodies and TAA antibodies. The bispecific antibodies of the present invention have an antigen binding region specific for CD28 and a second antigen binding region specific for TAA. In some embodiments, TAA is CEA. In some embodiments, TAA is MSLN. In some aspects, the bispecific antibodies are monovalent for CD28, CEA, and MSLN.

[0110] The bsAb of the present invention can be based on any different antibody formats previously described. In general, IgG-like formats are preferred because they provide favorable properties such as long half-life and potential reduced immunogenicity, but any other molecular bispecific formats can also be used in the present invention. In some embodiments, bispecific antibodies share a common heavy chain. In some embodiments, the heavy chain is a native heavy chain (i.e., without any mutation ("wild type")). In some embodiments, the heavy chain comprises at least one mutation (i.e., with "LALA" mutation or "LALAPA" mutation). Optionally, bispecific antibodies have different types of light chains. For example, a light chain is a κ light chain, and another light chain is a λ light chain (i.e., kl-body). Using κ and λ selection resins, different light chains allow easy purification of bispecific antibodies.

[0111] The heavy and light chain amino acid sequences of the antibodies are identified by their United States Adopted Names (USANs available, for example, through the American Medical Association https: / / www.ama-assn.org / or through CAS accession numbers).

[0112] Monospecific CD28 and TAA binding variable domains can be selected de novo, for example, from a phage display library, where the phage is engineered to express a human immunoglobulin or a portion thereof, such as a Fab, a single chain variable fragment (scFv), or an unpaired or paired antibody variable region, and subsequently engineered into a bispecific format. CD28 and TAA variable domains can be isolated, for example, from a phage display library expressing antibody heavy and light chain variable regions as a fusion protein using a phage g3 protein (scFv-g3 fusion protein).

[0113] The antibody library is screened to bind CD28 antibody and TAA, and the obtained positive clones are further characterized. Such phage display methods for isolating human antibodies are mature in the art. See, for example, U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915 and 6,593,081. The obtained de novo variable region is combined and engineered into a bispecific format using methods known in the art and described herein.

[0114] In addition, the bispecific antibodies of the present invention can be prepared using the following techniques, including those disclosed in WO 2012 / 023053 filed on August 16, 2011, the contents of which are hereby incorporated by reference in their entirety. The method described in WO 2012 / 023053 generates bispecific antibodies that are structurally identical to human immunoglobulins. This type of molecule consists 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 binding site exhibits a different antigenic specificity, to which both the heavy chain and the light chain contribute. The light chain variable region can 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 connections.

[0115] However, the bispecific antibodies of the present invention can also be obtained by fusing a κ light chain variable domain to a constant λ domain to obtain a first specificity and fusing a λ light chain variable domain to a constant κ domain to obtain a second specificity. The bispecific antibodies described in WO2012 / 023053 are referred to as IgGκλ antibodies or "κλ bodies", which are a new fully human bispecific IgG format. This κλ-body format allows affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules with properties that are indistinguishable from standard monoclonal antibodies, and is therefore advantageous compared to previous formats.

[0116] In addition to the above methods, the bispecific antibodies of the present invention can also be generated in a cell-free environment in vitro according to the method described in International Patent Publication No. WO2011 / 131746: by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parental monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization. In the method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that will promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow disulfide bond isomerization of cysteine ​​in the hinge region; thereby generating a bispecific antibody by Fab arm exchange.

[0117] 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.

[0118] As used herein, "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) is composed of the well-known heavy chain variable and constant domains VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) is composed of the well-known light chain variable and constant domains VL and CL. A full-length antibody may lack a C-terminal lysine (K) in one or both heavy chains.

[0119] The term "Fab-arm" or "half molecule" refers to one heavy chain-light chain pair that specifically binds to an antigen.

[0120] The full-length bispecific antibodies of the present invention can be generated, for example, as follows: using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, by introducing substitutions at the heavy chain CH3 interface in each half molecule to promote the formation of heterodimers of two antibody half molecules with different specificities in a cell-free environment in vitro or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody 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 a second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and re-formed by dissociation-association. The CH3 domain of the Fab arm can be engineered to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each of which binds to a different epitope.

[0121] As used herein, "homodimerization" refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, "homodimer" refers to an antibody having two heavy chains having the same CH3 amino acid sequence.

[0122] As used herein, "heterodimerization" refers to the interaction of two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody with two heavy chains with different CH3 amino acid sequences.

[0123] The "knob-in-hole" strategy (see, e.g., PCT International Publication No. WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In brief, selected amino acids that form the interface of the CH3 domains in human IgG can be mutated at positions that affect the interaction of the CH3 domains to promote heterodimer formation. Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form knob- and hole-like structures are (expressed 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.

[0124] Other strategies can be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, as described in US Patent Publication No. US2010 / 0015133, US Patent Publication No. US2009 / 0182127, US Patent Publication No. US2010 / 028637, or US Patent Publication No. US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following replacements (represented as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_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, as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849.

[0125] 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, necituzumab, panitumumab, and ervantumab (EGFR); labetuzumab and cerbituximab (CEA); amelezumab (mesothelin); cotrastuzumab (glypican 3); atezolizumab, avelumab, and durvalumab (PD-L1); blinatumomab (CD19); brentuximab (CD30); daratumumab (CD38); gemtuzumab (CD33); tositumomab (9CD22) or obinutuzumab, ocrelizumab, ofatumumab, rituximab, and ibritumomab tiuxetan (CD20).

[0126] Exemplary CD28, CEA and MSLN antibodies that can be used for engineering bispecific molecules include antibodies disclosed herein. Exemplary anti-CD28 antibodies from which the CD28 antigen binding region can be derived include "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 can be derived include "AC22", "AC61" or "AC84" antibodies. Exemplary anti-MSLN antibodies from which the MSLN antigen binding region can be derived include "O30", "O25", "O35", "O38" or "O41" antibodies. Table 1 shows the amino acid sequences of the regions of the antibodies disclosed herein.

[0127] Table 1. Exemplary amino acid sequences of the present disclosure

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] Table 2 shows exemplary bispecific antibodies of the present disclosure. The nomenclature of each antibody is shown, and the individual κ light chains, λ light chains, and heavy chain regions of the κλ-bodies of the present disclosure are described. "AI" represents the anti-human CD28 antigen binding region, "AC" represents the anti-human CEA antigen binding region, and "O" represents the anti-human MSLN antigen binding region. / N represents the heavy chain with the LALAPA mutation. Alternative nomenclature is shown in parentheses.

[0149] Table 2: Exemplary bispecific antibodies of the present disclosure

[0150]

[0151]

[0152]

[0153] 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

[0154] 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.

[0155] 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.

[0156] In some embodiments, the AI3AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0157] 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, 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

[0158] 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.

[0159] 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.

[0160] In some embodiments, the AI5AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0161] 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, 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

[0162] 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.

[0163] 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.

[0164] In some embodiments, the AI8AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0165] In some embodiments, the AI9AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0166] 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.

[0167] In some embodiments, the AI9AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0168] In some embodiments, the AI10AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0169] 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.

[0170] In some embodiments, the AI10AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0171] In some embodiments, the AI11AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0172] 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.

[0173] In some embodiments, the AI11AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0174] In some embodiments, the AI12AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0175] 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.

[0176] In some embodiments, the AI12AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0177] In some embodiments, the AI13AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0178] 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.

[0179] In some embodiments, the AI13AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0180] In some embodiments, the AI14AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0181] 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.

[0182] In some embodiments, the AI14AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0183] In some embodiments, the AI15AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0184] 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.

[0185] In some embodiments, the AI15AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0186] In some embodiments, the AI16AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0187] 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.

[0188] In some embodiments, the AI16AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0189] In some embodiments, the AI17AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0190] 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.

[0191] In some embodiments, the AI17AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0192] In some embodiments, the AI18AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0193] 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 κ light chain variable region comprising the amino acid sequence of SEQ ID NO:93, and a λ light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0194] In some embodiments, the AI18AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0195] In some embodiments, the AI19AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0196] 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.

[0197] In some embodiments, the AI19AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0198] In some embodiments, the AI20AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0199] 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.

[0200] In some embodiments, the AI20AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0201] In some embodiments, the AI21AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0202] 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.

[0203] In some embodiments, the AI21AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0204] In some embodiments, the AI22AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0205] 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.

[0206] In some embodiments, the AI22AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0207] In some embodiments, the AI23AC84 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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 λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:14, CDRL2 comprising the amino acid sequence of SEQ ID NO:15, and CDRL3 comprising the amino acid sequence of SEQ ID NO:16.

[0208] 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.

[0209] In some embodiments, the AI23AC84 / N bispecific antibody has heavy chain variable and constant regions 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.

[0210] In some embodiments, the AC61AI7 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:11, CDRL2 comprising the amino acid sequence of SEQ ID NO:12, and CDRL3 comprising the amino acid sequence of SEQ ID NO:13; and a λ light chain comprising: 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.

[0211] 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.

[0212] In some embodiments, the AC61AI7 / N bispecific antibody has heavy chain variable and constant regions 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.

[0213] In some embodiments, the AI3030 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:128, CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0214] In some embodiments, the AI3030 / 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.

[0215] In some embodiments, the AI3030 / N bispecific antibody has heavy chain variable and constant regions 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.

[0216] In some embodiments, the AI3025 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:131, CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0217] In some embodiments, the AI3025 / 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.

[0218] In some embodiments, the AI3025 / N bispecific antibody has heavy chain variable and constant regions 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.

[0219] In some embodiments, the AI3035 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:134, CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0220] In some embodiments, the AI3035 / 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.

[0221] In some embodiments, the AI3035 / N bispecific antibody has heavy chain variable and constant regions 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.

[0222] In some embodiments, the AI3038 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:137, CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0223] In some embodiments, the AI3038 / 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.

[0224] In some embodiments, the AI3038 / N bispecific antibody has heavy chain variable and constant regions 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.

[0225] In some embodiments, the AI3041 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:140, CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0226] In some embodiments, the AI3041 / 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.

[0227] In some embodiments, the AI3041 / N bispecific antibody has heavy chain variable and constant regions 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.

[0228] In some embodiments, the AI5030 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:128, CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0229] In some embodiments, the AI5030 / 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.

[0230] In some embodiments, the AI5030 / N bispecific antibody has heavy chain variable and constant regions 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.

[0231] In some embodiments, the AI5025 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:131, CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0232] In some embodiments, the AI5025 / 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.

[0233] In some embodiments, the AI5025 / N bispecific antibody has heavy chain variable and constant regions 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.

[0234] In some embodiments, the AI5035 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:134, CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0235] In some embodiments, the AI5035 / 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.

[0236] In some embodiments, the AI5035 / N bispecific antibody has heavy chain variable and constant regions 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.

[0237] In some embodiments, the AI5038 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:137, CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0238] In some embodiments, the AI5038 / 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.

[0239] In some embodiments, the AI5038 / N bispecific antibody has heavy chain variable and constant regions 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.

[0240] In some embodiments, the AI5041 / N bispecific antibody has a heavy chain comprising: 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; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:140, CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0241] In some embodiments, the AI5041 / 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.

[0242] In some embodiments, the AI5041 / N bispecific antibody has heavy chain variable and constant regions 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.

[0243] In some embodiments, the AI10030 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:140, CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0244] In some embodiments, the AI10030 / 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.

[0245] In some embodiments, the AI10030 / N bispecific antibody has heavy chain variable and constant regions 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.

[0246] In some embodiments, the AI10025 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:131, CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0247] In some embodiments, the AI10025 / 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.

[0248] In some embodiments, the AI10025 / N bispecific antibody has heavy chain variable and constant regions 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.

[0249] In some embodiments, the AI10035 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:134, CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0250] In some embodiments, the AI10035 / 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.

[0251] In some embodiments, the AI10035 / N bispecific antibody has heavy chain variable and constant regions 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.

[0252] In some embodiments, the AI10038 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:137, CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0253] In some embodiments, the AI10038 / 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.

[0254] In some embodiments, the AI10038 / N bispecific antibody has heavy chain variable and constant regions 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.

[0255] In some embodiments, the AI10041 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:140, CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0256] In some embodiments, the AI10041 / 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.

[0257] In some embodiments, the AI10041 / N bispecific antibody has heavy chain variable and constant regions 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.

[0258] In some embodiments, the AI13030 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:128, CDRL2 comprising the amino acid sequence of SEQ ID NO:129, and CDRL3 comprising the amino acid sequence of SEQ ID NO:130.

[0259] In some embodiments, the AI13030 / 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.

[0260] In some embodiments, the AI13030 / N bispecific antibody has heavy chain variable and constant regions 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.

[0261] In some embodiments, the AI13025 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:131, CDRL2 comprising the amino acid sequence of SEQ ID NO:132, and CDRL3 comprising the amino acid sequence of SEQ ID NO:133.

[0262] In some embodiments, the AI13025 / 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.

[0263] In some embodiments, the AI13025 / N bispecific antibody has heavy chain variable and constant regions 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.

[0264] In some embodiments, the AI13035 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:134, CDRL2 comprising the amino acid sequence of SEQ ID NO:135, and CDRL3 comprising the amino acid sequence of SEQ ID NO:136.

[0265] In some embodiments, the AI13035 / 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.

[0266] In some embodiments, the AI13035 / N bispecific antibody has heavy chain variable and constant regions 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.

[0267] In some embodiments, the AI13038 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:137, CDRL2 comprising the amino acid sequence of SEQ ID NO:138, and CDRL3 comprising the amino acid sequence of SEQ ID NO:139.

[0268] In some embodiments, the AI13038 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a κ light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a λ light chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0269] In some embodiments, the AI13038 / N bispecific antibody has heavy chain variable and constant regions 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.

[0270] In some embodiments, the AI13041 / N bispecific antibody has a heavy chain comprising: CDRH1 comprising the amino acid sequence of SEQ ID NO:1, CDRH2 comprising the amino acid sequence of SEQ ID NO:2, CDRH3 comprising the amino acid sequence of SEQ ID NO:3; a κ light chain comprising: 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; and a λ light chain comprising: CDRL1 comprising the amino acid sequence of SEQ ID NO:140, CDRL2 comprising the amino acid sequence of SEQ ID NO:141, and CDRL3 comprising the amino acid sequence of SEQ ID NO:142.

[0271] In some embodiments, the AI13041 / N bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a κ light chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a λ light chain variable region comprising the amino acid sequence of SEQ ID NO:147.

[0272] In some embodiments, the AI13041 / N bispecific antibody has heavy chain variable and constant regions 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.

[0273] How to use

[0274] Therapeutic formulations of the invention, comprising bsAbs of the invention, are used to treat or alleviate symptoms associated with cancer, non-limiting examples of which include 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, urethral bladder cancer, kidney cancer and renal pelvic cancer, oral cancer and pharyngeal cancer, uterine body cancer and / or melanoma. The invention also provides methods for treating or alleviating symptoms associated with cancer. The treatment regimen is performed by identifying a subject (e.g., a human patient having cancer (or at risk of developing cancer)) using standard methods.

[0275] The effectiveness of the treatment is determined in conjunction 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 confers a clinical benefit.

[0276] Pharmaceutical composition

[0277] The antibodies of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise 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 agents and absorption delaying agents compatible with drug administration, etc. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the field and 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. Liposomes and non-aqueous vehicles such as non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is covered. Supplementary active compounds may also be incorporated into the composition.

[0278] 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, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous applications may include the following components: sterile diluents, such as water for injection, saline solutions, non-volatile oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates or phosphates; and agents for regulating tension, such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be encapsulated in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0279] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where 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 TM(BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that there is easy injection. It must be stable under the conditions of manufacture and storage, and must prevent the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant. 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 preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. The absorption of the injectable composition can be extended by including agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0280] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount in an appropriate solvent together with one or a combination of ingredients listed above, followed by filtration sterilization if necessary. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other desired ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0281] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in a gelatin capsule or compressed into a tablet. For the purpose of oral therapeutic administration, the active compound can be combined with an excipient and used in the form of a tablet, lozenge or capsule. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and gargled and spit out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose, tragacanth or gelatin; an excipient, such as starch or lactose; a disintegrant, such as alginic acid, Primogel or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate or orange flavor.

[0282] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0283] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are well known in the art, and for example, for transmucosal administration, include detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream as well known in the art.

[0284] The compounds may 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.

[0285] In one embodiment, the active compound is prepared with a carrier (such as a controlled release formulation, which includes implants and microencapsulated delivery systems) that protects the compound from rapid elimination from the body. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such preparations will be apparent to those skilled in the art. These materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (which include liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0286] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such an active compound for individual treatment.

[0287] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0288] definition

[0289] As used herein, including the following claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0290] As used herein, "mAb V8" refers to "Molecule 11U: CD28 (SA_Variant 8) (PG-LALA), CD28 (SA_Variant 8) antibody in huIgGl PG-LALA isotype" as described in WO2020127618 (incorporated by reference in its entirety), which comprises the amino acid sequences of SEQ ID NO:377 and SEQ ID NO:378 of WO2020127618.

[0291] As used herein, "mAb V15" refers to "Molecule 11W: CD28 (SA_Variant 15) (PG-LALA), CD28 (SA_Variant 15) antibody in huIgGl PG-LALA isotype" as described in WO2020127618 (incorporated by reference in its entirety), which comprises the amino acid sequences of SEQ ID NO:381 and SEQ ID NO:382 of WO2020127618.

[0292] As used herein, "CEA_CD28_V8" refers to "Molecule 11B: CEA(A5H1EL1D)-CD28(SA variant 8) 1+1 format, bispecific huIgGl PGLALA CrossFab molecule with charged modification 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). It comprises the amino acid sequences of SEQ ID NOs: 351, 352, 355 and 356 of WO2020127618.

[0293] As used herein, "CEA_CD28_V15" refers to "Molecule 11C: CEA(A5H1EL1D)-CD28(SA variant 15) 1+1 format, bispecific huIgGl PGLALA CrossFab molecule with charged modification 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). It comprises the amino acid sequences of SEQ ID NOs: 351, 352, 357 and 358 of WO2020127618.

[0294] As used herein, "mAb 14226P2" refers to "mAb14226P2 (CD28 parent "A")" as described in WO2019246514 (incorporated by reference in its entirety), used to generate the anti-CD28 arm of bs16429D (PSMA x CD28 "A"), which comprises the amino acid sequence of SEQ ID NOs: 81 and 83 of WO2019246514.

[0295] As used herein, "1a28 / AC84 / N" refers to a benchmark CEAxCD28 bsAb generated by pairing the anti-CD28 arm of mAb14226P2 with the anti-CEA arm AC84 using crossMAb / knobs-in-hole technology and comprising the amino acid sequence of SEQ ID NOs: 122, 123, 124, and 125.

[0296] As used herein, “TGN1412” refers to a superagonist (SA) anti-huCD28 antibody in the human IgG4 isotype as described in WO2006050949, comprising the amino acid sequence of SEQ ID NOs: 126 and 127.

[0297] As used herein, "HPN536 analog" refers to a trispecific MSLNxCD3xHSA molecule capable of redirecting T cells to MSLN-positive target cells as described in WO2018209304 (incorporated by reference in its entirety), which comprises the amino acid sequence of SEQ ID NO: 100 of WO2018209304.

[0298] "Affinity" refers to the sum total strength 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 can generally be expressed by the dissociation constant (K D Affinity can be measured by common methods known in the art, including KinExA and Biacore.

[0299] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), fully human antibodies, and chimeric antibodies.

[0300] 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 an antigen bound by a full-length antibody, such as 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.

[0301] "Fab fragment" consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. "Fab fragment" can be the product of papain cleavage of an antibody.

[0302] The "Fc" region contains two heavy chain fragments comprising 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 domains.

[0303] A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain, said portion or fragment containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0304] A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. The F(ab')2 fragment is thus composed of two Fab' fragments held together by a disulfide bond between the two heavy chains. A "F(ab')2 fragment" may be the product of pepsin cleavage of an antibody. An "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant region.

[0305] "Isolated antibody" refers to a purified state and in this context means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of such materials or to the absence of water, buffers, or salts unless they are present in amounts that substantially interfere with experimental or therapeutic uses of the binding compounds as described herein.

[0306] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous antibody population, that is, except for possible naturally occurring mutations that may be present in trace amounts, the antibody molecules constituting the population are identical in amino acid sequence. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any specific method. For example, the monoclonal antibody to be used according to the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495 or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described in, for example, Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0307] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain mouse carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0308] In general, the basic "antibody" structural unit comprises a tetramer. In a monospecific antibody, 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 includes a "variable region" or "variable domain" of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain may define a constant region that is primarily responsible for effector function.

[0309] Usually, human constant light chains are divided into kappa light chains and lambda light chains. In addition, human constant heavy chains are usually divided into μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. The subtypes of these IgGs include, for example, IgG1 and IgG4.

[0310] As used herein, "variable region", "variable domain", "V region" or "V chain" means the segment of the IgG chain whose sequence varies between different antibodies. The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. The variable region of the heavy chain may be referred to as the "V H The variable region of the light chain may be referred to as the "V L ". Typically, the variable regions of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions to enable binding to specific epitopes. In general, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically based on the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; 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.

[0311] "CDR" refers to the V H One of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the β-sheet framework, or the antibody V LOne of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the β-sheet framework. Therefore, CDR is a variable region sequence interspersed within the framework region sequence. The CDR region is well known to those skilled in the art and has been defined, for example, by Kabat as a region with the greatest hypervariability within the antibody variable domain. The CDR region sequence has also been structurally defined by Chothia as those residues that are not part of the conserved .β-sheet framework and are therefore able to adapt to different conformations. Both terms are recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. The position of CDR in a typical antibody variable region has been determined by comparison of many structures (Al-Lazikani et al., 1997, J.Mol.Biol.273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues in the hypervariable region is different in different antibodies, in the typical variable region numbering scheme (Al-Lazikani et al., supra), the additional residues relative to the typical position are usually numbered with a, b, c, etc. next to the residue number. Such nomenclature is also well known to those skilled in the art. The corresponding relationship between the numbering system (including, for example, Kabat numbering and IMGT unique numbering system) is well known to those skilled in the art. In some embodiments, CDR is as defined by the Kabat numbering system. In other embodiments, CDR is as defined by the IMGT numbering system. In other embodiments, CDR is as defined by the AbM numbering system. In still other embodiments, CDR is as defined by the Chothia numbering system. In still other embodiments, CDR is as defined by the Contact numbering system.

[0312] Sequence identity refers to the extent to which the amino acids in two polypeptides are identical at equivalent positions when the two sequences are optimally aligned.

[0313] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids that share similar properties and are interchangeable are discussed above.

[0314] "Conservatively modified variants" or "conservative substitutions" refer to the use of other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) to replace amino acids in a protein, so that such changes can generally be made without changing the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide will not greatly change the biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition)). In addition, substitutions of amino acids that are similar in structure or function are less likely to destroy biological activity.

[0315] As used herein, the term "epitope" refers to a region or area on an antigen to which an antibody or antigen-binding fragment binds. The 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.

[0316] By "isolated" nucleic acid molecule or polynucleotide is meant a DNA or RNA (e.g., genomic), mRNA, cDNA, or synthetic origin, or some combination thereof, which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it is understood that a "polynucleotide" that "comprising" (or similar terms) a particular nucleotide sequence does not encompass an entire chromosome. In addition to the specified sequence, an isolated polynucleotide that "comprising" a particular nucleic acid sequence may contain coding sequences for up to ten or even up to twenty or more other proteins, or portions or fragments thereof, or may contain operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may contain vector sequences.

[0317] The phrase "control sequence" refers to a polynucleotide sequence that is necessary for or contributes to the expression of an operably linked coding sequence in a specific 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 invention, the polynucleotide is operably linked to a promoter, such as a viral promoter, a CMV promoter, a SV40 promoter, or a non-viral promoter or an elongation factor (EF)-1 promoter; and / or an intron.

[0318] A nucleic acid is "operably linked" when it is in a functional relationship with another polynucleotide. For example, if it is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of a presequence or secretory leader is operably linked to the DNA of a polypeptide; if it affects the transcription of a sequence, a promoter or enhancer is operably linked to a coding sequence; or if it is positioned to facilitate translation, a ribosome binding site is operably linked to a coding sequence. Usually, but not always, "operably linked" means that the polynucleotide sequences connected are continuous, and in the case of a secretory leader, they are continuous and in the reading phase. However, enhancers do not have to be continuous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0319] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that not all progeny have exactly the same DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as the original transformed cell from which it was selected are included. If a different name is intended, it should be clear from the context.

[0320] Host cells include eukaryotic and prokaryotic host cells, including mammalian cells. Among others, 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. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamster cells. Other cell lines that can be used include insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells.Fungal cells include yeast and filamentous fungal cells, including, 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 pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha), Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.The present invention includes any host cell (eg, a CHO cell or a Pichia cell, such as Pichia pastoris) containing an anti-ILT4 antibody or antigen-binding fragment thereof, or containing a polynucleotide encoding such an antibody or fragment, or containing a vector comprising the polynucleotide.

[0321] "Treatment" means administering an antibody or antigen-binding fragment thereof of the 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 suffering from cancer or an infectious disease, or in treating a subject suspected of suffering from cancer or an infectious disease, for which the agent has therapeutic activity. Typically, an antibody or fragment is administered in an "effective amount" or "effective dose" that will alleviate one or more symptoms (e.g., symptoms of a cancer or infectious disease) in the treated subject or population, either by inducing regression or elimination of such symptoms, or by inhibiting the progression of such symptoms (e.g., inhibiting cancer symptoms, such as tumor growth or metastasis, to any clinically measurable degree). The effective amount of an antibody or fragment may vary with factors such as the stage of the disease, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject.

[0322] Example

[0323] Example 1: Phage display selection of CD28FV using a human scFV library containing fixed variable heavy domains

[0324] Vaughan et al. (Nat. Biotech. 1996, 14: 309-314) describes a general procedure for constructing and processing a human scFv library displayed on M13 phage, which is hereby incorporated by reference in its entirety. The library encoding scFvs for selection and screening 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 WO 2012 / 023053, each of which is hereby incorporated by reference in its entirety. The following describes a procedure for identifying scFvs that bind to human CD28 (huCD28). Selection is performed in a solution with biotinylated huCD28 protein and / or on cells expressing huCD28. The selection strategy included up to 4 rounds of selection: (i) on recombinant protein, (ii) alternating recombinant protein and cells, (iii) 2 rounds on recombinant protein followed by 2 rounds on cells.

[0325] Protein selection

[0326] An aliquot of the scFv phage library was blocked with PBS containing 2% (w / v) skim milk. TM MyOne TM Streptavidin T1 magnetic beads or Sera-Mag SpeedBeadsNeutravidin TM The blocked phages were deselected on magnetic beads (coated magnetic particles) and then pre-incubated with 200nM, 100nM, 50nM or 5nM of biotinylated recombinant human CD28 (CD8-H82E5, Acro Biosystems). The phage + antigen mixture was then captured with blocked magnetic beads (the same species used for deselection) and washed with PBS / 0.1% The phages were washed five times with 20% PBS and twice with PBS alone. The phages were eluted with 1 mg / mL trypsin and added directly to exponentially growing TG1 cells after adding AEBSF to block trypsin activity. The infected TG1 aliquots were serially diluted to titrate the selected output. The output was then recovered and used for the next round of selection.

[0327] Cell surface selection

[0328] The supernatant containing phage was sealed with PBS containing 10% FBS. First, the sealed phage was deselected on CD28NEG TIB-153 cells (ATCC TIB 153), and then selected on CD28POS Jurkat cells (Jurkat CloneE6-1, ATCC TIB 152). The cells were precipitated and washed five times with PBS containing 10% FBS, and then washed once with only PBS. The phage was eluted with 1mg / mL trypsin, and after adding AEBSF to seal the trypsin activity, it was directly added to the TG1 cells growing exponentially. The aliquots of infected TG1 were serially diluted to select the output with titration. The output was then recovered and used for the next round of selection.

[0329] Example 2: Screening for SCFV that binds / does not bind to human CD28

[0330] Screening of scFvs binding to CD28 was tested by ELISA using biotinylated huCD28-His (or biotinylated huCEA_ECD as negative control) or by flow cytometry using CD28POS (Jurkat) and CD28NEG (TIB-153) cells.

[0331] For binding ELISA, neutravidin-coated plates were blocked with 1% casein in PBS. Biotinylated huCD28-His and biotinylated huCEA_ECD were captured at 5 nM. Dilutions of freshly prepared periplasmic extracts containing the selected scFv were applied to the plates and detected using a combination of mouse anti-c-myc antibody and donkey anti-mouse IgG HRP antibody. A microplate spectrophotometer was used to measure the brightness of the plates after addition of TM The OD at 450 nm generated after B. Hits were classified as specific binders if they were unable to bind to the irrelevant huCEA protein and if the OD450 on huCD28 was at least 3-fold higher than the background OD450.

[0332] For flow cytometry binding assays, cells were harvested, washed and dispensed into V-bottom 96-well plates at 150'000 or 200'000 cells / well. Dilutions of freshly prepared periplasmic extracts containing the selected scFv were pre-incubated 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 in the iQUE3 screening device (Sartorious). Hits were classified as positive and specific if at least 5% of the cells showed a binding signal 3 times higher than the GeoMFI of the background on CD28POS Jurkat cells and if no such signal was observed on CD28NEG TIB-153 cells.

[0333] Positive and specific hits were sequenced after extraction of DNA from individual clones.

[0334] Example 3: Fixed VH candidates reformatted to IGG and transiently expressed in mammalian cells

[0335] After screening and sequencing, scFv candidates with the desired binding properties were reformatted as IgG and expressed by transient transfection into PEAK cells. The VH and VL sequences of the selected scFv were amplified with specific oligonucleotides and cloned into expression vectors containing the heavy and light chain constant regions. The expression vectors were verified by sequencing and transfected into mammalian cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 4x10 ... 6PEAK cells. The transfected cells were cultured at 37 ° C for 5-6 days, and the production of IgG was quantified using the Octet RED96 instrument. The supernatant was harvested and IgG purification was performed on FcXL affinity resin (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, 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 then IgG was eluted in 50mM glycine (pH 3.5). The eluted IgG fraction was then dialyzed against histidine NaCl pH 6.0 buffer with Amicon 50kDa and the IgG content was quantified by absorption at 280nm. According to the manufacturer's instructions (Agilent Technologies), purity and IgG integrity were verified by electrophoresis using Agilent Bioanalyzer 2100.

[0336] Example 4: Binding of anti-CD28 MAB to CD28-positive JURKAT cells

[0337] The binding capacity of the anti-CD28 antibody arms of the invention (tested in bivalent mAb format) was assessed by flow cytometry using CD28-positive Jurkat cells (Jurkat clone E6-1, ATCC TIB 152).

[0338] Cells were harvested, checked for viability and counted. 200'000 cells were incubated at 4°C for 15 minutes with increasing concentrations of antibodies diluted in FACS buffer (PBS 2% BSA, 0.1% NaN3). Cells were washed twice with cold FACS buffer and incubated again at 4°C for 15 minutes with appropriate anti-human IgG secondary antibodies. Cells were washed twice with cold FACS buffer and resuspended in 150 μl FACS buffer with compatible viability markers. Antibody binding to live cells was measured by flow cytometry using the Cytoflex platform (Beckman Coulter). FlowJo TM Data were analyzed using v10 software (BD LifeSciences), and dose-response binding curves were plotted using GraphPad Prism 9 software.

[0339] Figure 1A-1C shows the resulting binding curves for a series of κλ-body compatible anti-CD28 arms. Superagonist anti-CD28 mAb TGN1412 and an unrelated hIgG1 mAb were used as positive and isotype controls, respectively, and were used for comparison in all experiments shown. The broad spectrum of binding curves representing anti-CD28 candidates identified during the sequential phage display campaigns shown on the three graphs highlights the fact that a set of anti-CD28 antibody arms with different binding affinities for CD28 were identified by phage display.

[0340] Example 5: Expression and purification of bispecific antibodies carrying λ and κ light chains

[0341] The simultaneous expression of one heavy chain and two light chains in the same cell can result in the assembly of three different antibodies. Simultaneous expression can be achieved in different ways, such as transfection of multiple vectors expressing one of the chains to be co-expressed or by using vectors driving the expression of multiple genes.

[0342] Here, two light chains are cloned into the previously generated vector pNoviκHλ to allow co-expression of a heavy chain, a κ light chain and a λ light chain, as described in US20120184716 and WO2012023053, each of which is hereby incorporated by reference in its entirety. The expression of these three genes is driven by the human cytomegalovirus promoter (hCMV), and the vector also contains a glutamine synthetase gene (GS), which enables selection and establishment of stable cell lines. The common VH and VL genes of anti-CD28 IgG and anti-CEA IgG are cloned into the vector pNoviκHλ for transient expression in mammalian cells. Expi293 cells are suspended in appropriate conical flasks with a suitable number of cells and culture medium volume. Plasmid DNA is transfected into Expi293 cells using PEI. The antibody concentration in the supernatant of transfected cells is measured during production using Octet RED96. Depending on the antibody concentration, the supernatant was harvested 5 to 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 TMFcXL affinity matrix (Thermo Fisher Scientific) was then added to the clarified supernatant. After incubation overnight at +4°C and 20rpm, the supernatant was centrifuged at 2000g for 10 minutes, the flow through was stored, and the resin was washed twice with PBS. The resin was then transferred to an Amicon Pro column and eluted using a solution containing 50mM glycine at pH 3.5. Several elution fractions were generated, neutralized with Tris-HCl pH7.4 and combined. The pool containing total human IgG (bispecific antibody and two monospecific antibodies) was quantified using a Nanodrop spectrophotometer (NanoDrop Technologies). A small portion of the sample was stored for further analysis, and the remaining sample was mixed with an appropriate volume of CaptureSelect TM KappaXL affinity matrix (Thermo Fisher Scientific) was incubated for 30 minutes. Resin recovery and washing, elution, and neutralization steps were performed as described above. TM The last affinity purification step was performed using the same procedure as for the κ purification step using λFab affinity matrix (ThermoFisher Scientific). Alternatively, purification was based on a two-step purification process in which only CaptureSelect TM KappaXL Affinity Matrix and CaptureSelect TM Lambda Fab affinity matrix. All elution fractions were pooled and desalted using a 50 kDa Amicon Ultra centrifugal filter unit (Merck Millipore) with His-NaCl pH 6.0 formulation buffer. The final product was quantified using Nanodrop.

[0343] Purified bispecific antibodies were analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer and a Protein 80 kit as described by the manufacturer (Agilent Technologies). Aggregation levels were determined by SEC-UPLC. All samples were tested for endotoxin contamination using a Limulus amebocyte lysate assay (LAL; Charles River Laboratories). Table 2 summarizes the generated CEAxCD28 and MSLNxCD28κλ-bispecific antibodies.

[0344] Example 6: In vitro characterization of CEAXCD28 bispecific antibodies

[0345] The binding of CEAxCD28 bsAb to CD28-positive Jurkat cells, CEA-positive LS174T cells, or CEA and CD28 double-negative TIB153 cells was tested. To confirm the binding of CD28xCEAκλ-body to target cells, a series of flow cytometry-based experiments were performed.

[0346] Examples of cells that can be used include CEA-positive cell lines such as the colorectal adenocarcinoma cell line LS174T, CD28-positive cell lines such as leukemia Jurkat T cells, and CEA and CD28 double negative cell lines such as leukemia TIB153 cells. Cell staining and binding assessment were performed as described in Example 4.

[0347] The binding curves of the CEAxCD28 bsAb of the present invention obtained using Jurkat, LS174T and TIB153 cells are respectively Figure 2 A, 2B, 2C and 2D are shown. On top of the κλ-body, other antibodies were introduced as reference antibodies: hIgG1 represents an unrelated monoclonal antibody of hIgG1 Fc, which serves as an isotype control; 1a28 / AC84 / N represents LALAPA-mutated huIgG1, a 1+1 bispecific molecule based on the anti-CD28 antibody arm mAb14226P2 described in WO2019246514A1, which uses the anti-CEA arm AC84 described in WO2021110647 as a targeting arm and comprises the amino acid sequences of SEQ ID NOs: 122, 123, 124 and 125; and CEA_CD28_V8 and CEA_CD28_V15 correspond to the CEAxCD28 bispecific antibodies described in WO2020127618, referred to therein as molecule 11B and molecule 11C, respectively.

[0348] Binding on Jurkat cells highlights the range of binding affinities of the selected anti-CD28 arms of the present invention to huCD28, which is consistent with published anti-CD28 antibody arms or has lower binding affinities ( Figure 2 Binding on LS174T cells highlights how antibodies sharing the same anti-CEA arm bind similarly to the cells, with all antibodies containing AC84 (high affinity anti-CEA arm) showing equivalent dose-range profiles, followed by two CEA_CD28 (V8 and V15), both of which contain the same medium affinity anti-CEA arm, and finally the only molecule based on the lower affinity anti-CEA arm AC61, AC61AI7 / N ( Figure 2 C). The absence of binding signals on TIB-153 indicates that all binding arms of the present invention are specific for the designated targets ( Figure 2 D).

[0349] Example 7: Wet Plate and Dry Plate Coating T-Cell Proliferation Assay

[0350] 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 coated T-cell proliferation assays.

[0351] 96-well polypropylene plates were coated overnight with antibodies diluted to 10 μg / ml in PBS, either at 4°C with 100 μl of antibody solution (wet coating) or at room temperature, unsealed in a Class II laminar flow cabinet (to allow buffer evaporation = dry coating) with 50 μL of antibody solution. After either coating procedure, the plates were washed twice with PBS. Simultaneously, 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 a 96-well plate in a final volume of 200 uL / well and incubated for 6 days at 37°C + 5% CO2. Cells were then harvested as detailed in Examples 4 and 6b and stained with anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (BioLegend, 301032) for flow cytometry evaluation. The proliferation rate of live CD4+ and CD8+ T cells was calculated by flow cytometry using CytoFLEX (Beckman Coulter), and the results of the two coating procedures were evaluated by FlowJo software ( Figure 3 A-3D). CD28 SA antibody TGN1412 served as a positive control while background proliferation rate of T cells was determined in the absence of any antibody (PBS only). Three CEAxCD28 bsAb 1a28 / AC84 / N, CEA_CD28_V8 and CEA_CD28_V15 (as described above) and their respective parental anti-CD28 monoclonal antibodies mAb14226P2, mAb V8 and mAb V15 were included as reference antibodies.

[0352] 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 vehicle control (PBS) with both coating procedures, thus ruling out unwanted hyperagonistic properties of these anti-CD28 arms. Candidate AI19AC84 / N was cautiously excluded from further analysis.

[0353] In contrast to all anti-CD28 arms of the present invention (except the AI19 arm), the anti-CD28 arms V8 and V15 described in WO2020127618 exhibited some super-agonistic activity, either as monoclonal antibodies (mAb V8 and mAb V15) or as bispecific CEAxCD28 antibodies (CEAxCD28 V8 and CEAxCD28 V15), as determined by induction of proliferation of resting CD4+ and CD8+ T cells under two experimental conditions (wet and dry coating). No super-agonistic activity was observed for the anti-CD28 arm 14226P2, either as a monoclonal antibody (mAb 14226P2) or as part of bsAb 1a28 / AC84 / N.

[0354] Example 8: T cell activation bioassay (IL-2 promoter)

[0355] Once possible super-agonistic activity was ruled out, a bispecific format of the IL-2 reporter system, CEAxCD28bsAb, was used to assess the ability of a range of anti-CD28 arms to agonize the CD28 co-stimulatory receptor on the surface of T cells.

[0356] The T cell activation bioassay (IL-2) kit developed by Promega (J1651) contains engineered Jurkat T cells that express a luciferase reporter gene driven by an IL-2 promoter. In the presence of both CD3 and CD28 stimulators, receptor-mediated signaling induces activation of the IL-2 pathway, which results in the luminescence of engineered Jurkat T cells. Since CD28 bsAb requires both T cell signal 1 providers and TAA-positive target cell lines to costimulate T cells, the assay is performed in the presence of CEA-positive MKN-45 cells and CEAxCD3 (a CEAxCD3 bispecific κλ-body, comprising SEQ ID NO: 118 common heavy chain HC, SEQ ID NO: 119 κ light chain, SEQ ID NO: 120 λ light chain, originally described in WO2021053587).

[0357] Briefly: 37,500 tumor target cells (MKN-45) were incubated with 75,000 IL-2 reporter cells (E:T=2:1) ​​at 37°C for 6 hours in the presence of a fixed dose (1 nM) of CEAxCD3 alone or in combination with increasing concentrations of the CEAxCD28 bispecific antibody. Alternatively, CEAxCD3 was replaced with Y4L3-1 / N, a non-targeted anti-CD3 bsAb comprising 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. The plate was equilibrated at room temperature for 15 minutes before measurement. 75ul of substrate (Bio-Glo reagent, Promega) was added to the cells and luminescence (relative luminescence units) was measured using a SpectraMax i3X luminometer (Molecular Devices) after incubation at room temperature in the dark for 5-10 minutes.

[0358] In the presence of basal CD3 stimulation (1 nM CEAxCD3), a concentration-dependent increase in IL-2 promoter-mediated luminescence was observed with increasing concentrations of CEAxCD28 bsAb, indicating appropriate CD28 stimulation via the anti-CD28 arm of the bsAb, leading to enhanced T cell activation. No T cell activation was observed in the absence of target cells or when CEAxCD3 was replaced with Y4L3-1 / N, a non-targeting CD3 bsAb that cannot deliver T cell activation signal 1 ( Figure 4 ).

[0359] This reporter gene assay demonstrated that the CEAxCD28 bispecific antibodies of the present invention can enhance T cell responses only in the presence of (1) CEA-positive target cells and (2) primary T cell activation (signal 1).

[0360] Example 9: T-cell dependent cytotoxicity (TDCC) mediated by CEAXCD28 bispecific antibody

[0361] TDCC of CEA-positive and CEA-negative cell lines

[0362] The T-cell dependent cytotoxicity (TDCC) induced by the CEAxCD28 bispecific antibodies of the invention on different CEA-positive and CEA-negative tumor cell lines was evaluated in combination with CEAxCD3 bsAb using human PBMCs or purified primary T-cells as effector cells.

[0363] After washing twice with PBS, the target cells were detached with trypsin or cell dissociation solution. After a centrifugation step, the cells were resuspended in assay medium, adjusted to the desired concentration, and plated in 96-well plates.

[0364] Effector cells can be human peripheral blood mononuclear cells (PBMC) or purified T-cells. TM Tube (Stemcell Technologies) and Lymphoprep TM PBMCs were isolated from buffy coats derived from healthy human donors using ELISA Buffer (Stemcell Technologies). If 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).

[0365] For TDCC assays, when PBMCs were used as effector cells, they were added to target cells at a final E:T ratio of 10:1 (unless otherwise stated); when purified T-cells were used, a final E:T ratio of 5:1 was used. A dose range of CEAxCD3 and a fixed dose of the CEAxCD28 antibody of the invention (2.5, 1.0, 0.5 or 0.1 μg / mL) were added to pre-plated target and effector cells. Alternatively, a non-targeted CD3 bsAb (Y4L3-1 / N) can be used instead of CEAxCD3. Target cell killing was assessed by quantifying (unless otherwise stated) LDH released into the culture medium by apoptotic / necrotic cells (Cytotoxicity Detection Kit PLUS (LDH), Roche) after incubation at 37°C, 5% CO2 for 24 hours, 48 ​​hours, 72 hours or 6 days. Maximum LDH release (=100% lysis) was obtained by incubating target cells with the lysate provided by the kit. Spontaneous LDH release (= 0% lysis) refers to target cells co-incubated with effector cells without adding any antibody. TDCC curves were plotted using GraphPad Prism 9 ( Figure 5 Alternatively, instead of using LDH release, an assay based on ATP quantification (Promega, Viability assay #G7571). In this case, the plate was shaken on a rocking platform to resuspend the PBMCs in the supernatant. The plate was then washed twice with PBS to remove all PBMCs. At the same time, the reagents from the kit were reconstituted and stabilized at room temperature for 10 minutes. Subsequently, 100 μL of the reconstituted reagent was added to each well and incubated at room temperature for 10 minutes, avoiding direct light. The luminescent signal from the adenosine triphosphate (ATP) contained in the remaining live target cells in the well 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)] × 100, where effector + target corresponds to the baseline value in the absence of antibody. The specific lysis results were then analyzed using GraphPad Prism software.

[0366] CEAxCD28 bsAb synergizes with CEAxCD3 bsAb to kill CEA-positive LS174T target cells expressing 26'000 CEA / cell ( Figure 5 A-5D). Killing induced by two PBMC donors in the presence of a dose range of CEAxCD3 and a fixed dose of 1 μg / mL CEAxCD28 antibody is shown. Synergy can be observed with lower EC50 or higher overall killing at the maximum concentration tested, or both. Importantly, if CEAxCD3 is replaced with Y4L3-1 / N (a non-targeting CD3 bsAb that cannot deliver signal 1 to effector cells), the CEAxCD28 bsAb does not induce killing, highlighting the importance of primary T cell stimulation (signal 1) for the activity of CD28 bsAbs. BsAbs 1a28 / AC84 / N and CEA_CD28_V8 (described above) were included as reference CEAxCD28 bsAbs and showed killing comparable to the tested κλ-bodies, with the exception of candidate AI11AC84 / N, which consistently resulted in weaker in vitro activity.

[0367] The synergistic effect between CEAxCD3 bsAb and CEAxCD28 bsAb was not cell line specific, as KATO-III and HT-29 cells expressing 15'000 and ~1'000 CEA / cell, respectively, were killed more efficiently by the combination of CEAxCD3 (in a dose response) and CEAxCD28 (2.5 μg / mL) bsAbs than by CEAxCD3 bsAb alone ( Figure 6 A-5B). bsAb 1a28 / AC84 / N (described above) was included as a reference CEAxCD28 bsAb.

[0368] T-cell activation after CEAxCD3 and CEAxCD28 bsAb combinations induce killing of CEA-expressing tumor cells Upregulation of markers

[0369] CEAxCD3 bsAb-induced killing of CEA-positive tumor cells is based on T cell activation. CD28 co-stimulation can further enhance the activation state of T cells. Therefore, the ability of CEAxCD28κλ-bodies to enhance T cell activation in the presence of appropriate signals was quantified by flow cytometry using antibodies recognizing specific T cell activation markers such as CD69 (early activation marker) or CD25 (late activation marker).

[0370] To evaluate the activation state of T cells at the end of the killing assay (described in detail in Example 9a), the following steps were followed: Floating cells (including 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 (PBS2% BSA, 0.1% NaN3), and then incubated at 4°C for 15 minutes with Fc-blocking reagent (BD Biosciences). After washing twice with FACS buffer, the cells were incubated at 4°C for 15 minutes with the following antibodies: anti-CD69-FITC (BioLegend), anti-CD8-PerCP-Cy5.5 (BioLegend), anti-CD25-PE (BioLegend) and anti-CD4-APC (ThermoFisher). The cells were washed and analyzed by flow cytometry using the Cytoflex platform (Beckman Coulter). FlowJo TM Data were analyzed using v10 software (BD Life Sciences). Figure 7 Shown in A-7B are the results of an experiment in which LS174T cells were co-cultured with PBMCs at an E:T ratio of 10:1 for 72 hours in the presence of a dose range of CEAxCD3 and a fixed dose of CEAxCD28 bsAb at 1 ug / mL.

[0371] T cell activation was measured by quantifying the late activation marker CD25 at the surface of both CD4+ and CD8+ T cells (respectively, Figure 7 A and 7B). The combination of CEAxCD3 and CEAxCD28 bsAb activated both CD4+ and CD8+ T cells to a greater extent than single treatment with CEAxCD3, with CD25 staining much brighter for the combined treatment. bsAb1a28 / AC84 / N (described above) was included as a reference CEAxCD28 bsAb.

[0372] CD28 co-stimulation mediated by CEAxCD28 bsAb affects T cell proliferation in the presence of CEAxCD3 bsAb impact.

[0373] The ability of CEAxCD28 bsAb to enhance the effect of CEAxCD3 in inducing T cell proliferation in the presence of CEA-positive tumor target cells was analyzed. 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 five to six days in the presence of a dose range of CEAxCD3 and the indicated fixed dose of CEAxCD28 bsAb at a final E:T ratio of 10:1 (unless otherwise stated). After co-culture, effector cells were harvested, washed, stained with appropriate viability markers to exclude dead cells, and stained with anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (BioLegend, 301032) to identify the population of interest. 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 CytoFLEX (Beckman Coulter). Data were evaluated by FlowJo software and plotted using GraphPad Prism ( Figure 8 A-8B).

[0374] The percentages of proliferating CD4+ and CD8+ T cells were Figure 8 The ability of CEAxCD3 bsAb to induce T cell proliferation was greatly enhanced by the addition of 2.5 μg / mL CEAxCD28 bsAb, and both CD4+ and CD8+ T cells responded equally well to the combined treatment.

[0375] Enhanced killing of CEA-expressing tumor cells by co-stimulation of CD28 with CEAxCD28 bsAb Cytokines released

[0376] The ability of the CD28 bispecific antibodies of the present invention to enhance cytokine release by T cells after killing CEA-expressing tumor cells in the presence of CEAxCD3 was evaluated by quantifying selected cytokines in the supernatant at the end of the TDCC assay. After co-culturing CEA-positive target cells and PBMCs containing T cells as described in Example 9, the culture supernatant was harvested by centrifugation and 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 a multiplex kit. Fig. 9Results of an experiment in which LS174T cells were co-cultured with PBMCs at an E:T ratio of 5:1 for 72 hours in the presence of a dose range of CEAxCD3 and a fixed dose of 1 μg / mL CEAxCD28 are shown in A-9F. bsAb 1a28 / AC84 / N (described above) was included as a reference CEAxCD28 bsAb.

[0377] Single-agent treatment with CEAxCD3 resulted in a moderate release of cytokines from T cells, whereas combination treatment of CEAxCD3 with any of the CEAxCD28 bsAbs tested greatly increased all measured cytokines, which was reflected in the better T cell activation and proliferation observed previously.

[0378] Example 10: Evaluation of the effect of CD28 BSAB-mediated T cell co-stimulation on the anti-tumor activity of CEAXCD3 T-cell retargeting BSAB in a humanized mouse tumor model

[0379] Antitumor activity of AI3AC84 / N and AI5AC84 / N in PBMC-humanized mouse tumor models

[0380] This efficacy study was designed to evaluate the antitumor effects of two CEAxCD28κλ-bodies carrying intermediate affinity anti-CD28 (AI3 and AI5) when combined with CEAxCD3.

[0381] The experimental design is summarized in Fig.10 In the figure, the treatment groups and treatment regimens are also shown. 6 LS174T cells and 1x10 6 PBMC (E:T = 1:1) were subcutaneously co-implanted into 7-8 week old NOG mice (NOD / Shi-scid / OL-2Rγnull mice, Taconic Biosciences) and treatment was started 7 days after implantation after randomization. CEAxCD3 and CEAxCD28 bsAb were co-injected at 10 mg / kg twice a week for a total of 6 injections. Mice were monitored for tumor development 2 to 3 times a week and tumors were measured with a digital caliper until the experimental endpoint (tumor volume = 1500 mm 3 or GvHD symptom onset). Usage (length x width 2 ) x 0.5 to calculate the tumor volume. Fig.11 A-11B shows each group ( Fig.11 A) and mice ( Fig.11 B) Tumor growth kinetics (mean + SEM).

[0382] In mice treated with CEAxCD3, only slight tumor growth inhibition relative to the vehicle group was observed. However, both AI3AC84 / N and AI5AC84 / N in combination with CEAxCD3 induced complete tumor regression, with no tumors detectable by palpation at day 28 post-implantation (when treatment was completed). The experiment was stopped at day 41: mice in the CEAxCD3+AI5AC84 / N group remained tumor-free, while the CEAxCD3+AI3AC84 / N group was the opposite, with 2 mice relapsing with slowly growing tumors.

[0383] AI3AC84 / N (tested across a dose range) and AI10AC84 / N in a PBMC-humanized mouse tumor model Antitumor activity

[0384] This efficacy study was designed to evaluate the anti-tumor effects of a dose range of CEAxCD28κλ-bodies carrying a medium-affinity anti-CD28 arm (AI3) in combination with CEAxCD3 and compare them with the anti-tumor effects of CEAxCD28κλ-bodies carrying a lower-affinity anti-CD28 arm (AI10).

[0385] The experimental design is summarized in Fig.12 In the figure, the treatment groups and treatment regimens are also shown. 6 LS174T cells and 1x10 6 PBMC (E:T = 1:1) were subcutaneously co-implanted into 7-8 week old NOG mice (NOD / Shi-scid / OL-2Rγnull mice, Taconic Biosciences) and treatment was started 7 days after implantation after randomization. CEAxCD3 and CEAxCD28 bsAbs were co-injected twice a week at the indicated doses for a total of 6 injections. Mice were monitored for tumor development 2 to 3 times a week and tumors were measured with a digital caliper until the experimental endpoint (tumor volume = 1500 mm 3 or GvHD symptom onset). Usage (length x width 2 ) x 0.5 to calculate the tumor volume. Fig.13 A-13B shows each group ( Fig.13 A) and mice ( Fig.13 B) Tumor growth kinetics (mean + SEM).

[0386] Only slight tumor growth inhibition relative to the vehicle group was observed in mice treated with CEAxCD3, while all combinations of CD28 bsAb with CEAxCD3 induced significant but variable antitumor activity. More specifically, the highest dose of AI3AC84 / N (10 mg / kg) resulted in complete tumor regression in 6 / 8 mice, with palpable but unmeasurable tumors in the remaining 2 mice; the intermediate dose of AI3AC84 / N (2 mg / kg) resulted in robust tumor control and no recurrence of tumors, with all tumor volumes eventually decreasing to <50 mm 3 (Complete tumor eradication was observed in only 1 mouse); the lowest dose (0.4 mg / kg) resulted in variable tumor control, with tumors being eradicated in half of the mice, while treatment in the other half resulted in only tumor growth delay. Treatment with 10 mg / kg AI10AC84 / N (a CEAxCD28 bsAb containing a lower affinity anti-CD28 arm (AI10)) was generally less effective than treatment with the same dose of AI3AC84 / N, with only tumor growth delay observed in 2 mice and complete tumor eradication observed in only 3 mice.

[0387] Example 11: In vitro characterization of MSLNXCD28 bispecific antibody

[0388] Binding of MSLNxCD28 bsAb to CD28-positive Jurkat cells, MSLN-positive H226 and OVCAR-3 cells, or MSLN and CD28 double-negative TIB153 cells.

[0389] To confirm the binding of MSLNxCD28κλ-bodies to target cells, a series of flow cytometry-based experiments were performed.

[0390] 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. The mesothelin cell surface expression of H226 and OVCAR-3 cells was measured to be 180'000 and 68'000 MSLN / cell, respectively. Cell staining and binding assessment were performed as described in Example 4.

[0391] The binding curves of the MSLNxCD28 bsAb of the present invention obtained using H226, OVCAR-3, Jurkat and TIB153 cells are respectively Fig.14The binding curves of additional MSLNxCD28 bsAbs of the present invention to H226, OVCAR-3, Jurkat and TIB153 cells are shown in Figures A, 14B, 14C and 14D, respectively. Fig.17 Shown in A, 17B, 17C and 17D. hIgG1 represents an irrelevant monoclonal antibody to hIgG1 Fc, which served as an isotype control.

[0392] Binding to H226 and OVCAR-3 cells highlights how antibodies sharing the same anti-MSLN arm bind similarly and with high affinity to MSLN-positive cells ( Fig.14 A and 14B and Fig.17 A and 17B). Binding to Jurkat cells reflects the difference in binding affinity between the AI5 and AI10 arms ( Fig.14 C), but highlights the similarity in binding affinity between AI5 and AI3 arms ( Fig.17 C), which is consistent with the data generated with the CEAxCD28 bsAb of the present invention. The absence of binding signal on TIB-153 indicates that all binding arms of the present invention are specific for the designated target ( Fig.14 D and 17D).

[0393] Example 12: T-cell dependent cytotoxicity (TDCC) mediated by MSLNXCD28 bispecific antibody

[0394] TDCC of MSLN-positive cell lines

[0395] T-cell dependent cytotoxicity (TDCC) induced by the MSLNxCD28 bispecific antibodies of the invention on different MSLN-positive cell lines was evaluated as described in Example 9. The MSLNxCD28 bsAb was tested in combination with the HPN536 analog, a trispecific MSLNxCD3xHSA molecule capable of redirecting T cells to MSLN-positive target cells. Fig.15 A and 15D show TDCC curves obtained with a dose range of HPN536 analogs and a fixed dose of 1 or 0.1 μg / mL of MSLNxCD28 bsAb targeting H226 cells. Fig.18 A-18D shows TDCC curves obtained with a dose range of HPN536 analogs and a fixed dose of 2.5 μg / mL of MSLNxCD28 bsAb targeting OVCAR3 cells (at different E:T ratios).

[0396] MSLNxCD28 bsAbs synergized with HPN536 analogs to kill MSLN-positive H226 target cells. Synergy was observed for most MSLNxCD28 bsAbs at either concentration tested (1 and 0.1 μg / mL), but was most pronounced for bsAbs carrying O35 or O41 as the targeting anti-MSLN arm. AI5O35 and AI5O41 achieved nearly identical specific lysis maxima (between 50% and 60%) at 0.1 and 1 ug / ml, whereas maximal lysis obtained with the lower affinity AI10 arm ranged between 40% and 60%. Importantly, MSLNxCD28 bsAbs did not induce killing if the HPN536 analog was replaced with Y4L3-1 / N, a non-targeting CD3 bsAb that is unable to deliver signal 1 to effector cells, highlighting the importance of primary T cell stimulation (signal 1) for the activity of CD28 bsAbs.

[0397] It is worth noting that reducing the E:T ratio (from 10:1 to 3:1, then to 1:1, and finally to 1:3, Fig.18 A-18D) reduced the killing potential of the HPN536 analog. If at E:T ratios of 10:1 and 3:1, more than 90% of target cells were killed at the highest concentrations of the HPN536 analog dose range, while at E:T ratios equal to 1:1 and 1:3, only 70% and 26% of target cells were killed at the highest MSLNxCD3 concentration, respectively. Addition of the MSLNxCD28 bsAb of the invention compensated for this reduction in activity, fully restoring the activity of the HPN536 analog at E:T=1:1 and resulting in up to 60% target cell killing at E:T=1:3.

[0398] Combination of MSLNxCD3 and MSLNxCD28 bsAbs induces post-T cell killing of MSLN-expressing tumor cells Upregulation of activation markers

[0399] The ability of MSLNxCD28κλ-bodies to enhance T cell activation in the presence of appropriate signal 1 was quantified by flow cytometry as described in Example 9 using antibodies recognizing specific T cell activation markers. Fig.15 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 1 μg / mL or 0.1 μg / mL CD28 bsAb are shown in B and 15E.

[0400] The combination of MSLNxCD3 and MSLNxCD28 bsAb activated CD4+ T cells to a greater extent than single treatment with HPN536 analogs, with brighter CD25 staining for most combination treatments. Differences between T cell activation levels were observed depending on the dose of the CD28 bsAb tested, with 1 μg / mL leading to better T cell activation than 0.1 μg / mL. The importance of both anti-MSLN and anti-CD28 arms was also demonstrated: bsAbs based on O35 and O41 activated T cells better than bsAbs based on O30, and the medium affinity AI5 arm induced T cell activation better than the low affinity AI10 arm.

[0401] Effect of CD28 co-stimulation mediated by MSLNxCD28 bsAb on T cell proliferation in the presence of MSLNxCD3 ring.

[0402] The ability of MSLNxCD28 bsAb to enhance the effect of MSLNxCD3 in inducing T cell proliferation in the presence of MSLN-positive tumor target cells was analyzed as described in Example 9. The percentage of proliferating CD4+ T cells was Fig.15 C and 15F. The ability of HPN536 analogs to induce T cell proliferation was greatly enhanced by the addition of MSLNxCD28 bsAb, but the greatest effect was obtained using AI5-based bsAbs carrying O35 or O41 as targeting arms at a dose of 1 μg / mL.

[0403] Enhanced killing of MSLN-expressing tumor cells by co-stimulation of CD28 with MSLNxCD28 bsAb Cytokines released in

[0404] As described in Example 9, the ability of the MSLNxCD28 bispecific antibodies of the invention to enhance cytokine release by T-cells after killing of MSLN-expressing tumor cells in the presence of MSLNxCD3 (HPN536 analogs) was evaluated by quantifying selected cytokines in the supernatant at the end of the TDCC assay. Fig.16 Shown in A-16F are the results of an experiment 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 HPN536 analogs and a fixed dose of 1 or 0.1 μg / mL MSLNxCD28.

[0405] Single-agent treatment with HPN536 analogs resulted in moderate cytokine release from T cells, whereas combination treatment of HPN536 analogs with any of the tested MSLNxCD28 bsAbs greatly increased all measured cytokines, especially IL-2, which was reflected in the better T cell activation and proliferation observed previously.

[0406] Example 13: T-cell dependent cellular cytotoxicity (TDCC) mediated by a combination of TAAXCD3 and TAAXCD28 bispecific antibodies targeting different TAAs.

[0407] TDCC of CEA / MSLN double-positive cells

[0408] As described in Example 9, using Viability Assay As readout, T-cell dependent cytotoxicity (TDCC) of CEA / MSLN double positive cell lines (HPAC cells, 280'000 CEA / cell and 14'000 CEA / cell) induced by the MSLNxCD28 bispecific antibodies of the invention was evaluated.CD28 bsAbs were tested in combination with CEAxCD3. Fig.19 A-19D shows TDCC curves obtained with a dose range of CEAxCD3 and a fixed dose of 2.5 μg / mL of CEAxCD28 or MSLNxCD28 bsAbs targeting HPAC cells (at different E:T ratios).

[0409] Both CEAxCD28 and MSLNxCD28 bsAbs synergized equally well with CEAxCD3 to kill CEA / MSLN double-positive HPAC target cells, indicating that TAAxCD28 bsAbs can be successfully paired with TAAxCD3 bsAbs targeting different TAAs. Synergy was observed at all E:T ratios tested but was most prominent at lower E:T ratios, where the addition of CD28 bsAb compensated for the lower activity of CEAxCD3.

[0410] Importantly, when tested alone (ie, in the absence of signal 1), the CD28 bsAb did not induce killing, highlighting the importance of primary T cell stimulation (signal 1) for the activity of the CD28 bsAb.

[0411] T-cell activation and T-cell proliferation induced by combinations of CD3 and CD28 bsAbs targeting different TAAs

[0412] exist Fig.19 The TDCC assay shown quantifies the ability of CD28κλ-bodies to enhance T cell activation and proliferation in the presence of appropriate signaling 1. Fig. 20 T cell activation data for CD4+ and CD8+ T cells are shown in A and B, respectively. Fig. 20 C and 20D show T cell proliferation of CD4+ and CD8+ T cells, respectively. The different E:T ratios tested are shown and clearly labeled at the top of the graph.

[0413] Combination of CEAxCD3 with either CEAxCD28 or MSLNxCD28 bsAb activated CD4+ T cells to a greater extent than single treatment with CEAxCD3, with more CD25-positive T cells for both combination treatments and across different E:T ratios. The synergy between CD3 and CD28 bsAbs in activating CD8+ T cells was more evident at lower E:T ratios, as CEAxCD3 bsAb alone induced CD8+ T cell activation well at E:T>1. Similar levels of T cell activation were observed with both bsAbs, suggesting that TAAxCD28 bsAbs can be successfully paired with TAAxCD3 bsAbs targeting different TAAs.

[0414] The proliferative potential of T cells is reflected in the higher T cell activation observed when the combination is used and results in enhanced T cell proliferation induced by the combination across the 4 E:T ratios tested. Notably, CEAxCD3 alone only minimally induced T cell proliferation at E:T ratios equal to or below 1, highlighting the benefit of combining with CD28 bsAb to induce T cell proliferation even at unfavorable E:T ratios.

[0415] Example 13: Evaluation of the effect of CD28 BSAB-mediated T cell co-stimulation on the anti-tumor activity of CEAXCD3 T-cell retargeted BSAB in BRGSF-HIS mice

[0416] This efficacy study was designed to evaluate the antitumor effects of AI3AC84 / N in combination with CEAxCD3 in BRGSF-HIS mice bearing subcutaneous CEA+HPAF-II tumors. The experimental design is summarized in Fig. 22 In the figure, the treatment groups and treatment regimens are shown. 6 HPAF-II cells were subcutaneously implanted into humanized BRGSF-HIS mice (genOway) using 4 different CD34+ hematopoietic stem cell donors (9 mice / donor, 3 mice per donor per treatment group). 3 Treatment was started at 1:10 pm. CEAxCD3 and CEAxCD28 bsAb were co-injected at 10 mg / kg twice a week for a total of 6 injections. Mice were monitored for tumor progression and tumors were measured with a digital caliper 2 to 3 times a week until the experimental endpoint (tumor volume = 1500 mm 3 ). Usage type (length x width 2 ) x 0.5 to calculate tumor volume. FIG23A shows the survival rate at day 56 after implantation, while FIG23B shows the tumor growth kinetics of individual mice in each treatment group.

[0417] For mice treated with CEAxCD3, only a slight survival advantage over the vehicle group was measured. However, the combination of AI3AC84 / N and CEAxCD3 induced lower tumor growth kinetics and higher chances of survival at day 56 (when the experiment was terminated): 8 / 12 mice in the CEAxCD3+AI3AC84 / N group had not yet reached the tumor endpoint, whereas only 1 / 12 and 3 / 12 mice in the vehicle and CEAxCD3 groups, respectively, had not yet reached the tumor endpoint.

[0418] Other Implementations

[0419] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0420] Additional embodiments of the present disclosure include the following:

[0421] Embodiment 1. A bispecific antibody, comprising:

[0422] a. a first antigen binding domain that binds to CD28; wherein the first antigen binding domain comprises:

[0423] i. a first heavy chain variable region comprising 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

[0424] ii. a first light chain variable region having:

[0425] 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

[0426] 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

[0427] 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; or

[0428] 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

[0429] 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

[0430] 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

[0431] 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

[0432] 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

[0433] 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

[0434] 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

[0435] 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

[0436] 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

[0437] 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

[0438] 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

[0439] 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

[0440] 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; or

[0441] 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; or

[0442] 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

[0443] 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

[0444] b. a second antigen binding domain that binds a tumor associated antigen (TAA), wherein the second antigen binding domain comprises:

[0445] i. a second heavy chain variable region comprising: 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.

[0446] Embodiment 2. The bispecific antibody of claim 1, wherein the TAA is CEA.

[0447] Embodiment 3. The bispecific antibody of claim 2, wherein the second antigen binding domain comprises:

[0448] c. having the following second light chain variable region:

[0449] 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;

[0450] 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;

[0451] 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.

[0452] Embodiment 4. The bispecific antibody according to any of the preceding claims, wherein the first heavy chain variable region and the second heavy chain variable region comprise the amino acid sequence of SEQ ID NO:4.

[0453] Embodiment 5. The bispecific antibody according to any of the preceding claims, 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.

[0454] Embodiment 6. The bispecific antibody according to claim 1, wherein

[0455] d. the first light chain variable region of part ii(1) comprises the amino acid sequence of SEQ ID NO:80;

[0456] e. the first light chain variable region of part ii(2) comprises the amino acid sequence of SEQ ID NO:81;

[0457] f. the first light chain variable region of part ii(3) comprises the amino acid sequence of SEQ ID NO:82;

[0458] g. the first light chain variable region of part ii(4) comprises the amino acid sequence of SEQ ID NO:83;

[0459] h. The first light chain variable region of part ii(5) comprises the amino acid sequence of SEQ ID NO:84

[0460] i. the first light chain variable region of part ii(6) comprises the amino acid sequence of SEQ ID NO:85;

[0461] j. the first light chain variable region of part ii(7) comprises the amino acid sequence of SEQ ID NO:86;

[0462] k. the first light chain variable region of part ii(8) comprises the amino acid sequence of SEQ ID NO:87;

[0463] l. the first light chain variable region of part ii(9) comprises the amino acid sequence of SEQ ID NO:88;

[0464] m. the first light chain variable region of part ii (10) comprises the amino acid sequence of SEQ ID NO: 89;

[0465] n. The first light chain variable region of part ii (11) comprises the amino acid sequence of SEQ ID NO: 90;

[0466] o. the first light chain variable region of part ii(12) comprises the amino acid sequence of SEQ ID NO:91;

[0467] p. the first light chain variable region of part ii (13) comprises the amino acid sequence of SEQ ID NO: 92;

[0468] q. the first light chain variable region of part ii(14) comprises the amino acid sequence of SEQ ID NO:93;

[0469] r. the first light chain variable region of part ii (15) comprises the amino acid sequence of SEQ ID NO: 94;

[0470] s. The first light chain variable region of part ii (16) comprises the amino acid sequence of SEQ ID NO: 95;

[0471] t. the first light chain variable region of part ii (17) comprises the amino acid sequence of SEQ ID NO: 96;

[0472] u. the first light chain variable region of part ii (18) comprises the amino acid sequence of SEQ ID NO: 97; and

[0473] v. The first light chain variable region of part ii(19) comprises the amino acid sequence of SEQ ID NO:98.

[0474] Embodiment 7. The bispecific antibody according to claim 1, wherein

[0475] a. The first light chain of part ii(1) comprises the amino acid sequence of SEQ ID NO:99;

[0476] b. the first light chain of part ii(2) comprises the amino acid sequence of SEQ ID NO: 100;

[0477] c. the first light chain of part ii(3) comprises the amino acid sequence of SEQ ID NO: 101;

[0478] d. the first light chain of part ii(4) comprises the amino acid sequence of SEQ ID NO:102;

[0479] e. the first light chain of part ii(5) comprises the amino acid sequence of SEQ ID NO: 103;

[0480] f. The first light chain of part ii(6) comprises the amino acid sequence of SEQ ID NO:104;

[0481] g. The first light chain of part ii(7) comprises the amino acid sequence of SEQ ID NO: 105

[0482] h. the first light chain of part ii(8) comprises the amino acid sequence of SEQ ID NO:106;

[0483] i. the first light chain of part ii(9) comprises the amino acid sequence of SEQ ID NO:107;

[0484] j. the first light chain of part ii(10) comprises the amino acid sequence of SEQ ID NO:108;

[0485] k. the first light chain of part ii (11) comprises the amino acid sequence of SEQ ID NO: 109;

[0486] l. The first light chain of part ii (12) comprises the amino acid sequence of SEQ ID NO: 110;

[0487] m. The first light chain of part ii (13) comprises the amino acid sequence of SEQ ID NO: 111;

[0488] n. The first light chain of part ii (14) comprises the amino acid sequence of SEQ ID NO: 112;

[0489] o. The first light chain of part ii (15) comprises the amino acid sequence of SEQ ID NO: 113;

[0490] p. The first light chain of part ii (16) comprises the amino acid sequence of SEQ ID NO: 114;

[0491] q. the first light chain of part ii (17) comprises the amino acid sequence of SEQ ID NO: 115;

[0492] r. The first light chain of part ii (18) comprises the amino acid sequence of SEQ ID NO: 116; and

[0493] s. The first light chain of part ii(19) comprises the amino acid sequence of SEQ ID NO:117.

[0494] Embodiment 8. The bispecific antibody of claim 3, wherein:

[0495] a. The second light chain variable region of part a(i) comprises the amino acid sequence of SEQ ID NO: 17;

[0496] b. the second light chain variable region of part a(ii) comprises the amino acid sequence of SEQ ID NO: 18; and

[0497] c. The second light chain variable region of part a(iii) comprises the amino acid sequence of SEQ ID NO:19.

[0498] Embodiment 9. The bispecific antibody of claim 3, wherein:

[0499] a. The second light chain of part a(i) comprises the amino acid sequence of SEQ ID NO:20;

[0500] b. the second light chain of part a(ii) comprises the amino acid sequence of SEQ ID NO:21; and

[0501] c. The second light chain of part a(iii) comprises the amino acid sequence of SEQ ID NO:22.

[0502] Embodiment 10. The bispecific antibody according to any of the preceding claims, wherein the first light chain is κ and the second light chain is λ.

[0503] Embodiment 11. The bispecific antibody according to any of the preceding claims, wherein the first light chain is λ and the second light chain is κ.

[0504] Embodiment 12. The bispecific antibody of claim 1, wherein 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.

[0505] Embodiment 13. The bispecific antibody of claim 12, wherein the amino acid substitutions comprise L234A and L235A substitutions.

[0506] Embodiment 14. The bispecific antibody of claim 12, wherein the amino acid substitution comprises a P329A, P329G or P329R substitution.

[0507] Embodiment 15. The bispecific antibody according to any of the preceding claims, wherein said antibody is of the IgG isotype.

[0508] Embodiment 16. The bispecific antibody according to any of the preceding claims, wherein the antibody is human.

[0509] Embodiment 17. The bispecific antibody according to any of the preceding claims, wherein the composition enables tumor-specific T cell activation.

[0510] Embodiment 18. A composition comprising the bispecific antibody according to any of the preceding claims.

[0511] Embodiment 19. The composition of claim 18, further comprising a CD3xCEA bispecific antibody.

[0512] Embodiment 20. The composition of claim 19, wherein the CD3 x CEA 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.

[0513] Embodiment 21. A method of reducing the proliferation of tumor cells and / or killing tumor cells, the method comprising contacting the cells with a composition according to any one of claims 18-20.

[0514] Embodiment 22. A method of treating cancer in a subject, the method comprising administering to the subject a composition according to any one of claims 18-20.

[0515] Embodiment 23. An antibody comprising an antigen binding domain that binds to CD28; wherein the antigen binding domain comprises:

[0516] i. A heavy chain variable region comprising a complementary determining region 1 (CDRH1) comprising the amino acid sequence of (SEQ ID NO: 1), a complementary determining region 2 (CDRH2) comprising the amino acid sequence of (SEQ ID NO: 2), and a complementary determining region 3 (CDRH3) comprising the amino acid sequence of (SEQ ID NO: 3); and

[0517] ii. A light chain variable region having:

[0518] 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

[0519] 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

[0520] 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; or

[0521] 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

[0522] 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;

[0523] 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

[0524] 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

[0525] 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

[0526] 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

[0527] 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

[0528] 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

[0529] 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

[0530] 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

[0531] 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

[0532] 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

[0533] 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; or

[0534] 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; or

[0535] 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

[0536] 19. 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.

[0537] Embodiment 24. The antibody of claim 23, wherein the antibody is a F(ab) fragment, a F(ab')2 fragment, and a Fv fragment or a single-chain Fv fragment.

[0538] Embodiment 25. The antibody of claim 23, wherein said antibody is monospecific.

[0539] Embodiment 26. The antibody of claim 23, wherein said antibody is monovalent.

Claims

1. A bispecific antibody, comprising: a. a first antigen binding domain that binds to CD28; wherein the first antigen binding domain comprises: i. a first heavy chain variable region comprising 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 light chain variable region having:

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; 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 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. 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; and b. a second antigen binding domain that binds a tumor associated antigen (TAA), wherein the second antigen binding domain comprises: i. a second heavy chain variable region 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.

2. The bispecific antibody according to claim 1, wherein: 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; e. the first light chain variable region of part a.ii.

5. comprises the amino acid sequence of SEQ ID NO: 84; 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; j. the first light chain variable region of part a.ii.

10. comprises the amino acid sequence of SEQ ID NO: 89; 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. the first light chain variable region of part a.ii.

15. comprises the amino acid sequence of SEQ ID NO:94; p. 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.

3. The bispecific antibody according to any one of claims 1 to 2, wherein: 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: 103; 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; j. the first light chain of part a.ii.

10. comprises the amino acid sequence of SEQ ID NO: 108; k. the first light chain of part a.ii.

11. comprises the amino acid sequence of SEQ ID NO: 109; l. The first light chain of part a.ii.

12. comprises the amino acid sequence of SEQ ID NO:110; m. The first light chain of part a.ii.

13. comprises the amino acid sequence of SEQ ID NO:111; n. the first light chain of part a.ii.

14. comprises the amino acid sequence of SEQ ID NO:112; o. the first light chain of part a.ii.

15. comprises the amino acid sequence of SEQ ID NO: 113; p. The first light chain of part a.ii.

16. comprises the amino acid sequence of SEQ ID NO: 114; q. the first light chain of part a.ii.

17. comprises the amino acid sequence of SEQ ID NO: 115; r. the first light chain of part a.ii.

18. comprises the amino acid sequence of SEQ ID NO: 116; or s. The first light chain of part a.ii.

19. comprises the amino acid sequence of SEQ ID NO:

117.

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

5. The bispecific antibody of claim 4, wherein the second antigen binding domain comprises: ii. a second light chain variable region having:

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.

6. The bispecific antibody according to any one of claims 4-5, wherein: 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.

7. The bispecific antibody according to any one of claims 4 to 6, wherein: a. The second light chain of part b.ii.

1. comprises the amino acid sequence of SEQ ID NO: 20; b. the second light chain of part b.ii.

2. comprises the amino acid sequence of SEQ ID NO: 21; or c. The second light chain of part b.ii.

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

22.

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

9. The bispecific antibody of claim 8, wherein the second antigen binding domain comprises: ii. a second light chain variable region having:

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.

10. The bispecific antibody according to any one of claims 7-8, wherein: 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.

11. The bispecific antibody according to any one of claims 7 to 9, wherein: 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. 12 . The bispecific antibody according to claim 1 , wherein the first heavy chain variable region and the second heavy chain variable region comprise the amino acid sequence of SEQ ID NO:

4.

13. The bispecific antibody according to any one of claims 1 to 12, 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.

14. The bispecific antibody according to any one of claims 1-13, wherein the first light chain is κ and the second light chain is λ.

15. The bispecific antibody according to any one of claims 1-14, wherein the first light chain is λ and the second light chain is κ.

16. The bispecific antibody of any one of claims 1-15, wherein 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.

17. The bispecific antibody of claim 16, wherein the amino acid substitutions comprise L234A and L235A substitutions.

18. The bispecific antibody of claim 16, wherein the amino acid substitution comprises a P329A, P329G or P329R substitution.

19. The bispecific antibody according to any one of claims 1-18, wherein the antibody has an IgG isotype.

20. The bispecific antibody of any one of claims 1-19, wherein the antibody is human.

21. The bispecific antibody according to any one of claims 1-20, wherein the composition enables tumor-specific T cell activation.

22. A composition comprising the bispecific antibody according to any one of claims 1-21.

23. A method of reducing the proliferation of tumor cells and / or killing tumor cells, the method comprising contacting the cells with the composition according to claim 22.

24. A method of treating cancer in a subject, the method comprising administering to the subject the composition of claim 22.

25. An antibody comprising an antigen binding domain that binds to CD28; wherein the antigen binding domain comprises: i. a first heavy chain variable region comprising 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 light chain variable region having:

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; 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 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. 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. 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.

26. The antibody according to claim 25, wherein the antibody is a F(ab) fragment, a F(ab')2 fragment, a Fv fragment or a single-chain Fv fragment.

27. The antibody of claim 25, wherein the antibody is monospecific.

28. The antibody of claim 25, wherein the antibody is monovalent.

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