Bispecific GPC3xCD28 and GPC3xCD3 antibodies and combinations thereof for targeted killing of GPC3-positive malignant cells

Abstract

The present invention relates to a GPC3xCD3 bispecific antibody, a GPC3xCD28 bispecific antibody, and a combination of two agonistic fully human κλ antibodies, one targeting GPC3xCD3 and the other targeting GPC3xCD28, both of which do not compete for binding to GPC3. The present invention also relates to the use of the bispecific antibodies for the treatment of GPC3-positive malignancies.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 319,709, filed March 14, 2022, and U.S. Provisional Patent Application No. 63 / 328,586, filed April 7, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Incorporation by reference of sequence listing The contents of the electronic sequence listing (NOVI_725_001WO_SeqList_ST26.xml, size: 52,994 bytes, and creation date: March 13, 2023) are incorporated herein by reference in their entirety.

[0003] Field The present invention relates to fully human bispecific antibodies (such as κλ bodies) targeting GPC3xCD28 (CD28 agonistic), bispecific antibodies targeting GPC3xCD3, and the combination of GPC3xCD3 and GPC3xCD28 of the present invention. Due to the involvement of GPC3 in GPC3-expressing tumor cells and the involvement of CD28 in T cells, the combination therapy of the present invention can boost T cell activation and tumoricidal activity compared to GPC3xCD3 monotherapy. The present invention further relates to the method of using such antibodies in the treatment of GPC3-positive malignancies. [Background technology]

[0004] 2. Background of the Invention In the past few years, new ways have been developed to stimulate the body's own immune cells to better attack and kill cancer cells. Examples of successful cancer immunotherapy are monoclonal antibodies that can block so-called immune checkpoints. Currently approved immune checkpoint inhibitors (ICIs) block CTLA-4 (e.g., ipilimumab, sold under the brand name Yervoy), block PD-1 (e.g., pembrolizumab, sold under the brand name Keytruda, and cemiplimab, sold under the brand name Libtayo), and block PD-L1 (e.g., atezolizumab, sold under the brand name Tecentriq). ICIs can induce durable antitumor responses in some (but not all) cancer types, with responses limited to a subpopulation of patients.

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

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

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

[0008] The present disclosure provides a method for the production of a medicament for ... and ii. a second heavy chain variable region having a CDRH1 comprising the amino acid sequence of SEQ ID NO: 16, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 18, or 2. a second light chain variable region having a CDRL1 comprising the amino acid sequence of SEQ ID NO: 21, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 23.

[0009] 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: 9. In some embodiments, the first heavy chain and the second heavy chain comprise the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the first light chain comprises the amino acid sequence of SEQ ID NO: 15.

[0010] In some embodiments, the a. portion b.ii.1. of the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 19, or the b. portion b.ii.2. comprises the amino acid sequence of SEQ ID NO: 24.

[0011] In some embodiments, the a. portion b.ii.1. of the second light chain comprises the amino acid sequence of SEQ ID NO:20, or the b. portion b.ii.2. comprises the amino acid sequence of SEQ ID NO:25.

[0012] The present disclosure provides a method for the production of a antibody that binds to CD28, the antibody comprising: a. a first antigen-binding domain that binds to CD28, the first antigen-binding domain comprising: i. a first heavy chain variable region having 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 ii. a first antigen-binding domain comprising: 1. a CDRL1 comprising the amino acid sequence of SEQ ID NO: 41, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 42, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 43, or 2. a CDRL1 comprising the amino acid sequence of SEQ ID NO: 46, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 48, or 3. a first light chain variable region having a CDRL1 comprising the amino acid sequence of SEQ ID NO: 51, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 53; and b. a second antigen-binding domain that binds to GPC3, comprising: i. a second heavy chain variable region having 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 ii. and a second antigen-binding domain comprising: 1. 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, or 2. a CDRL1 comprising the amino acid sequence of SEQ ID NO:31, a CDRL2 comprising the amino acid sequence of SEQ ID NO:32, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:33, or 3. a second light chain variable region having CDRL1 comprising the amino acid sequence of SEQ ID NO:36, CDRL2 comprising the amino acid sequence of SEQ ID NO:37, and CDRL3 comprising the amino acid sequence of SEQ ID NO:38.

[0013] 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: 5.

[0014] In some embodiments, a. portion a.ii.1. of the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 44, b. portion a.ii.2. comprises the amino acid sequence of SEQ ID NO: 49, or c. portion a.ii.3. comprises the amino acid sequence of SEQ ID NO: 54.

[0015] In some embodiments, a. portion a. ii. 1. of the first light chain comprises the amino acid sequence of SEQ ID NO: 45, b. portion a. ii. 2. comprises the amino acid sequence of SEQ ID NO: 50, or c. portion a. ii. 3. comprises the amino acid sequence of SEQ ID NO: 55.

[0016] In some embodiments, a. portion b.ii.1. of the second light chain variable region comprises the amino acid sequence of SEQ ID NO:29, b. portion b.ii.2. comprises the amino acid sequence of SEQ ID NO:34, or c. portion b.ii.3. comprises the amino acid sequence of SEQ ID NO:39.

[0017] In some embodiments, a. portion b.ii.1. of the second light chain comprises the amino acid sequence of SEQ ID NO: 30, b. portion b.ii.2. comprises the amino acid sequence of SEQ ID NO: 35, or c. portion b.ii.3. comprises the amino acid sequence of SEQ ID NO: 40.

[0018] In some embodiments, the first light chain is kappa and the second light chain is lambda, hi some embodiments, the first light chain is lambda and the second light chain is kappa.

[0019] In some embodiments, the bispecific antibody comprises an Fc domain comprising one or more amino acid substitutions that reduce binding to an activating Fc receptor and / or reduce effector function. In some embodiments, the amino acid substitutions comprise an L234A and an L235A substitution. In some embodiments, the amino acid substitutions comprise (i) an L234A substitution, (ii) an L235A substitution, and (iii) a P329A, P329G, or P329R substitution.

[0020] In some embodiments, the antibody has an IgG isotype. In some embodiments, the antibody is a human antibody.

[0021] The present disclosure provides a composition comprising any one of the bispecific antibodies of the present disclosure.

[0022] The present disclosure provides a first antigen-binding domain that binds to CD3, comprising: i. a first heavy chain variable region having a complementarity determining region 1 (CDRH1) comprising the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDRH2) comprising the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDRH3) comprising the amino acid sequence of SEQ ID NO:8; and ii. a first light chain variable region having a CDRL1 comprising the amino acid sequence of SEQ ID NO:11, a CDRL2 comprising the amino acid sequence of SEQ ID NO:12, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:13. a second antigen-binding domain that binds to GPC3, comprising: i. a CDRH1 comprising the amino acid sequence of SEQ ID NO:6, a CDRH2 comprising the amino acid sequence of SEQ ID NO:7, and a CDRH3 comprising the amino acid sequence of SEQ ID NO:8; and ii. a second heavy chain variable region having: 1. a CDRL1 comprising the amino acid sequence of SEQ ID NO:16, a CDRL2 comprising the amino acid sequence of SEQ ID NO:17, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:18; or 2. a CDRL1 comprising the amino acid sequence of SEQ ID NO:21, and a CDRL2 comprising the amino acid sequence of SEQ ID NO:22. and a second light chain variable region having 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 ii. a first antigen-binding domain that binds to CD28, comprising: i. a first heavy chain variable region having 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 ii.a second heavy chain variable region having 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 second antigen-binding domain comprising: ii. a second light chain variable region having: 1. 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, or 2. a CDRL1 comprising the amino acid sequence of SEQ ID NO:31, a CDRL2 comprising the amino acid sequence of SEQ ID NO:32, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:33, or 3. a CDRL1 comprising the amino acid sequence of SEQ ID NO:36, a CDRL2 comprising the amino acid sequence of SEQ ID NO:37, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:38.

[0023] In some embodiments, the composition enables tumor-specific T cell activation.

[0024] The present disclosure provides a method of reducing the proliferation of and / or killing cancer cells comprising contacting the cancer cells with any one of the compositions of the present disclosure.

[0025] 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. In some embodiments, the cancer is GPC3 positive.

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

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

[0028] [Figure 1] 1A-1C are a series of graphs showing concentration-dependent binding of the GPC3xCD3 bispecific antibody of the invention (AD84L3-1 / N) to Hep G2 cells expressing GPC3 (FIG. 1A), Jurkat cells expressing CD3 (FIG. 1B), and GPC3 and CD3 double negative SK-HEP-1 cells (FIG. 1C). FIG. 1A, FIG. 1B, and FIG. 1C show a comparison of binding between AD84L3-1 / N and ERY974, the latter used as a reference comparator. hIgG1 is used as an isotype control. [Diagram 2] Figures 2A-2C are a series of graphs showing concentration-dependent binding of GPC3xCD28 bispecific antibodies of the invention (AI3P44 / N and AI3P30 / N) to GPC3-expressing FU97 cells (Figure 2A), CD28-expressing Jurkat cells (Figure 2B), and GPC3 and CD28 double negative TIB-153 cells (Figure 2C). These two GPC3xCD28 biAbs contain the same anti-CD28 AI3 arm paired with two GPC3 arms (either P44 or P30), both of which bind to the distal part of the GPC3 receptor. hIgG1 is used as an isotype control. [Diagram 3]Figures 3A-3D are a series of graphs showing T cell retargeting killing / lysis of GPC3-positive Hep G2 cells (Figures 3A and 3B) and GPC3-negative SK-HEP-1 cells (Figures 3C and 3D) by one of the GPC3xCD28 bispecific antibodies of the invention (AI3P44 / N) in combination with the GPC3xCD3 biAb of the invention (AD84L3-1 / N). Concentration (dose) dependent lysis of tumor cells by AD84L3-1 / N alone and in combination with different fixed concentrations (0.5, 0.1, 0.05 or 0.025 μg / mL) of AI3P44 / N is shown. Effector cells are PBMCs derived from two different healthy donors (Figures 3A and 3C: donor 1 and Figures 3B and 3D: donor 2). CD28 biAb synergizes with GPC3xCD3 to kill GPC3-positive Hep G2 cells. Killing is dose-dependent with respect to the concentration of GPC3xCD28 biAb. No killing is induced using the GPC3-negative SK-HEP-1 cell line. Y4L3-1 / N is a non-targeting CD3 monovalent antibody and is used as a negative control, and hIgG1 is used as an isotype control. A single treatment of ERY974 is used as a reference control. [Figure 4]4A-4C are a series of graphs showing T cell retargeting killing / lysis of GPC3 positive Hep G2 cell lines (FIG. 4A, FIG. 4B, FIG. 4C). Dose response of GPC3xCD3 bispecific antibody AD84L3-1 / N when combined with fixed doses (0.5, 0.1, or 0.05 μg / mL) of a GPC3xCD28 biAb of the invention (either AI3P44 / N (FIG. 4A), AI3P30 / N (FIG. 4B) or AI3P111 (FIG. 4C)). GPC3 arms P44 and P30 target membrane distal regions within GPC3 and therefore do not compete with membrane proximal GPC3 arms of GPC3xCD3 biAbs (e.g., AD84). The P111 GPC3 arm binds membrane proximal but also does not compete with GPC3 arms of CD3 biAbs (e.g., AD84) for binding to GPC3. Synergy is observed for all combinations of GPC3xCD3 and GPC3xCD28 biAbs in killing GPC3-positive Hep G2 target cells. Killing is dose-dependent with respect to the concentration of GPC3xCD28 biAb. Comparative TDCC data show that pairing the P111 arm to AI3 is less efficient in boosting AD84L3-1 / N activity compared to the AI3P44 / N combination (Figure 4A and Figure 4C, respectively). Y4L3-1 / N is a CD3 targeting only monovalent antibody that does not crosslink tumor cells and T cells and is therefore used as a negative control, and hIgG1 is used as an isotype control. ERY974, which is being tested in a clinical trial (Clin. Trials Gov. ref NCT05022927), was included as a monotherapy and used as a reference comparator for activity. Data are presented using representative PBMC donors in each condition. See text for details. [Diagram 5]5A-5D are a series of graphs showing a comparison of two GPC3xCD28 biAbs generated using the same membrane-distal GPC3 binding arm P44 paired to anti-CD28 arms of different binding affinity to CD28, AI3 or AI10. Concentration-dependent binding of the GPC3xCD28 biAbs of the invention (AI3P44 / N and AI10P44 / N) to the GPC3-expressing cell line HuH-7 (FIG. 5A) and CD28-expressing Jurkat cells (FIG. 5B). The GPC3-targeting arms demonstrated similar binding signals to GPC3-positive cells for AI3P44 / N and AI10P44 / N (FIG. 5A). In contrast, the CD28-binding portion of the two GPC3xCD28 biAbs shows superior binding signals with AI3 compared to AI10 on CD28-positive cells (FIG. 5B). T cell retargeting killing / lysis of the GPC3-positive cell line Hep G2 is observed with a dose response of GPC3xCD3 bispecific antibody AD84L3-1 / N both as monotherapy and in combination with fixed doses (0.5, 0.1 or 0.05 μg / mL) of the GPC3xCD28 biAb of the invention, namely AI3P44 / N (FIG. 5C) and AI10P44 / N (FIG. 5D) (FIG. 5C and FIG. 5D). Synergy is observed between all combinations of GPC3xCD3 and GPC3xCD28 biAbs in killing GPC3-positive Hep G2 target cells. Killing is dose-dependent with respect to the concentration of GPC3xCD28 biAb. Based on TDCC data, the affinity of the CD28 arm influences activity. At intermediate concentrations of GPC3xCD28 κλ bodies, lower TDCC activity is observed in combination with AI10P44 / N compared to AI3P44 / N. Y4L3-1 / N is a CD3 targeting only monovalent antibody that does not crosslink tumor cells and T cells and is therefore used as a negative control. ERY974, which is being tested in a clinical trial (Clin. Trials Gov.ref NCT05022927), was included as a monotherapy and used as a reference comparator for activity. As for controls, in the absence of GPC3xCD3 biAb providing GPC3-driven signal 1, no killing is observed (Figure 5C and Figure 5D). Data are presented using a representative PBMC donor in each condition. [Figure 6-1] Figures 6A-6E show a series of graphs showing T cell retargeted killing / lysis of the GPC3 expressing cell line Hep 3B by the combination of GPC3xCD3 biAb (AD84L3-1 / N) and GPC3xCD28 bispecific antibodies of the invention (i.e. AI3P30 / N (one PBMC donor) (Figure 6A), AI3P44 / N (three different PBMC donors) (Figures 6B, 6C, 6D), AI10P44 / N (Figure 6E)). There is no or only minimal killing with GPC3xCD3(AD84L3-1 / N) alone in this cell line with lower GPC3 expression (i.e. Hep 3B, 60,000 GPC3 / cell). The combination of CD3 and CD28 biAb has a potent and synergistic killing effect in killing GPC3 positive target cells. Killing can be adjusted by varying the concentration of GPC3xCD28 biAb. As a negative control, Y4L3-1 / N (a monovalent antibody that targets only CD3) is used alone and in combination with the GPC3xCD28 biAb of the present invention, and hIgG1 is used as an isotype control. ERY974, currently being tested in a clinical trial (ClinicalTrials.gov identifier: NCT05022927), is included as a single agent treatment and used as a reference comparator for activity. [Figure 6-2] See description of Figure 6-1. [Figure 7]Figures 7A-7D are a series of graphs showing simultaneous measurement of T cell retargeting killing / lysis and secretion of pro-inflammatory cytokines. T cell retargeting killing / lysis of GPC3 expressing cell line Hep G2 (700,000 GPC3 / cell) by one of the GPC3xCD28 bispecific antibodies of the invention (AI3P44 / N) in combination with GPC3xCD3 biAb (AD84L3-1 / N) (Figure 7A). Secretion of IFN-γ, IL-6, TNF-α is shown in Figures 7B, 7C, and 7D, respectively. The combination of CD3 and CD28 biAbs results in a stronger induction of secretion of pro-inflammatory cytokines compared to GPC3xCD3 monotherapy with AD84L3-1 / N. Cytokine levels are dependent on the concentration of CD28 biAb (AI3P44 / N). ERY974, used as a clinical comparator, in combination with 0.05 or 0.1 μg / mL AI3P44 / N shows robust induction of cytokines at levels mostly higher than those observed with the highest concentration of AD84L3-1 / N. hIgG1 is used as an isotype control. Single treatment of ERY974 is used as the clinical reference control. [Figure 8]Figures 8A-8D are a series of graphs showing T cell retargeting killing / lysis and secretion of pro-inflammatory cytokines using AI3P30 / N instead of AI3P44 / N, the latter results are shown in Figure 7. Simultaneous measurement of T cell retargeting killing / lysis and secretion of pro-inflammatory cytokines. T cell retargeting killing / lysis of GPC3 expressing cell line Hep G2 by one of the GPC3xCD28 bispecific antibodies of the invention (AI3P30 / N) in combination with GPC3xCD3 biAb (AD84L3-1 / N) (Figure 8A). Secretion of IFN-γ, IL-6, TNF-α is shown in Figures 8B, 8C, and 8D, respectively. The combination of CD3 and CD28 biAbs results in a stronger induction of secretion of pro-inflammatory cytokines compared to GPC3xCD3 monotherapy with AD84L3-1 / N. Cytokine levels are dependent on the concentration of CD28 biAb (AI3P30 / N). ERY974, used as a clinical comparator, in combination with 0.05 or 0.1 μg / mL AI3P30 / N shows robust induction of cytokines at levels mostly higher than those observed with the highest concentration of AD84L3-1 / N. hIgG1 is used as an isotype control. Single treatment of ERY974 is used as the clinical reference control. [Figure 9-1]Figures 9A-9H are a series of graphs showing T cell retargeting killing / lysis and secretion of pro-inflammatory cytokines comparing AI3P44 / N (Figures 9A-9D) and AI10P44 / N (Figures 9E-9H). AI10 has a lower binding affinity for CD28 than AI3. The same PBMC donor is used as the source of effector cells for each experiment for comparability purposes. The GPC3 expressing cell line Hep G2 is used as the target. T cell retargeting killing activity is shown for the combination of AI3P44 / N (Figure 9A) and AI10P44 / N (Figure 9E). Secretion of IFN-γ (Figures 9B and 9F), IL-6 (Figures 9C and 9G), TNF-α (Figures 9D and 9H). Higher killing rates are achieved compared to ERY974 at 0.5 μg / mL with similar or lower cytokine release. Cytokine release can be reduced, especially in the case of AI10P44 / N, by lowering the concentration of GPC3xCD28. At 0.1 μg / mL, killing by AI3P44 / N is similar to ERY974 (Figure 9A), with correspondingly lower cytokine release (Figures 9B-9D). AI10P44 / N also promotes killing at 0.1 μg / mL, but the overall killing level is below that achieved by ERY974 (Figure 9E). The resulting cytokine release is low (FH, IFN-γ<5000 pg / mL, IL-6<50 pg / mL, TNF-α<100 pg / mL) despite the maintenance of some killing activity (E, up to 20-25% of maximum killing activity when AI10P44 / N is added at 0.1 μg / mL). hIgG1 is used as an isotype control. A single treatment of ERY974 will be used as the reference control. [Figure 9-2] See description of Figure 9-1. [Figure 10-1]Figures 10A-10F are a series of graphs showing T cell retargeting killing / lysis using CellTiter-Glo readout. As target cells, three GPC3 expressing cell lines, Hep G2 (Figures 10A-10B, showing two PBMC donors), Hep 3B (Figures 10C-10D, showing two PBMC donors) and HuH-7 (Figures 10E-10F, showing two PBMC donors) are used by GPC3xCD3 biAb (AD84L3-1 / N) in combination with GPC3xCD28 bispecific antibody of the invention (AI3P44 / N). GPC3xCD3 (AD84L3-1 / N) alone in Hep 3B (60,000 GPC3 / cell) or HuH-7 (18,000 GPC3 / cell) caused no or only minimal killing. The combination of CD3 and CD28 biAbs has a synergistic killing effect in killing all GPC3 positive target cells, regardless of their target expression level. As a negative control, Y4L3-1 / N (a monovalent antibody that targets only CD3) is used alone and in combination with the GPC3xCD28 biAb of the present invention, and hIgG1 is used as an isotype control. Single treatment of ERY974 is used as a reference control drug. [Figure 10-2] See description of Figure 10-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The present invention includes compositions comprising GPC3xCD3 bispecific antibody, GPC3xCD28 bispecific antibody, and combination of GPC3xCD3 bispecific antibody and GPC3xCD28 bispecific antibody to induce T cell activation and killing of glypican-3 (GPC3) positive tumor cells. Specifically, the present invention is based in part on the combination of two bispecific antibodies, one for co-engagement of GPC3 expressed on the surface of target (e.g., tumor cell) cells, and the other for co-engagement of GPC3 (expressed on tumor cells) and CD28 (expressed on T cells) to trigger CD3 positive T cell activation. This results in cluster formation and thus boosts tumor specific T cell activation.

[0030] The present invention provides a fully human GPC3xCD3 bispecific antibody designed to be administered in parallel with the GPC3xCD28 bispecific antibody of the present invention. Importantly, there is no competition between the GPC3xCD3 bispecific antibody and the GPC3xCD28 bispecific antibody for binding to the target GPC3. The present invention provides an agonist CD28 antigen binding molecule that allows co-engagement of T cells with the GPC3 receptor on tumor cells. The present invention also provides a GPC3xCD3 bispecific antibody that mediates tumor-specific T cell activation (i.e., signal 1) and mediates an increase in tumor-specific T cell activation (signal 2) when administered in combination with the GPC3xCD28 bispecific antibody of the present invention. The GPC3xCD28 bispecific antibody of the present invention is the first GPC3xCD28 antibody described in the literature, providing a new treatment option for patients with GPC3+ solid tumors. Furthermore, the combination of the GPC3xCD3 bispecific antibody and the GPC3xCD28 bispecific antibody of the present invention is a novel mechanism for activating the immune system by targeting costimulatory signals on T cells.

[0031] Glypican-3 (GPC3) Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG). There are six glypican subtypes, namely GPC1-6, with a similar structure consisting of a 60-70 kDa protein connected to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, 14 conserved cysteine ​​residues, and the last 50 residues of the carboxyl terminus modified by heparan sulfate (HS) side chains. GPC3 has been implicated in cell proliferation, differentiation, and migration (Glypicans as Cancer Therapeutic Targets. Li N. at al Trends Cancer. 2019 Nov 1, The Role of Glypicans in Cancer Progression and Therapy. Li N et al. J Histochem Cytochem. 2020 Dec; 68(12): 841-862). GPC3 is a highly tumor-specific antigen that is expressed during fetal development but physiologically constitutively suppressed in adult tissues. Abnormal expression of GPC3 has been reported in hepatocellular carcinoma (HCC), lung squamous cell carcinoma (LSCC), testicular tumors, ovarian follicular cyst tumors, melanoma, ovarian clear cell carcinoma, and other cancers (Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells Fedra Castillo L. et al, Oncotarget. 2016 Sep 13; 7(37): 60133-60154, GPC-3 in hepatocellular carcinoma: current perspectives. Wu Y et al. J Hepatocell Carcinoma. 2016; 3: 63-67).GPC3 expression correlates with poor prognosis in HCC patients (Prognostic and clinicopathological significance of glypican-3 overexpression in hepatocellular carcinoma: A meta-analysis. Li J. et al World J Gastroenterol. 2014 May 28; 20(20): 6336-6344). Therefore, GPC3 is a biomarker and prognostic factor for HCC and an attractive immunotherapy target (Cancer immunotherapy‐targeted glypican‐3 or neoantigens. Shimizu Y. et al. Cancer Sci. 2018 Mar; 109(3): 531-541, Next-Generation Cancer Immunotherapy Targeting Glypican-3. Shimizu Y et al. Front Oncol.2019;9:248).

[0032] CAR T cell therapy has shown clinical benefit in hematological malignancies but limited efficacy in solid tumors. Monoclonal antibodies targeting GPC3 have entered clinical trials exemplified by codrituzumab (GC33, RO5137382, ClinicalTrials.gov NCT04928677), but response rates have been low. A bispecific antibody with potentially higher activity is exemplified by "ERY974" as described in WO 2017 / 159287 A1 (incorporated by reference in its entirety), which comprises as CDRs the CDRs shown in SEQ ID NOs: 42-45 of WO 2017 / 159287 A1.

[0033] ERY974 is a humanized IgG antibody with a common light chain that can bind both GPC3 and CD3, promoting cytotoxicity through T cell effector actions. The GPC3 binder used in ERY974 is a humanized, affinity matured, and stability engineered version of the hGC33 antibody (An anti-glypican 3 / CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Ishiguro T. et al., Sci Transl Med. 2017 Oct 04; 9(410); Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974. Shiraiwa et al., Methods, 2018). The CD3 binder (rCE115) used in ERY974 was derived from rat immunization and humanized by CDR grafting method (Results of a phase 1 dose escalation study of ERY974, an anti-glypican 3(GPC3) / CD3 bispecific antibody, in patients with advanced solid tumors. Safran et al. Cancer Res(2021)81(13_Supplement):CT111). The binding arm is specific for CD3ε. Lead antibodies against GPC3 and CD3ε were multidimensionally optimized to generate ERY974 using a hIgG4 scaffold with reduced Fc effector function. ERY22 is a lead bispecific antibody consisting of two types of H chains and two types of L chains. ERY22 was humanized and a common L chain was identified to generate humanized ERY (hERY) with a common L chain.hERY was further engineered to improve its physicochemical properties, including its binding affinity to the antigen and enhanced stability, resulting in ERY974 (Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974. Shiraiwa et al., Methods, 2018).

[0034] ERY974 showed significant antitumor effects in preclinical tumor models that did not respond to treatment with immune checkpoint inhibitors (such as PD-1 and CTLA-4) (An anti-glypican 3 / CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Ishiguro T. et al., Sci Transl Med. 2017 Oct 04; 9(410)). Further studies showed that ERY974 induced high inflammation in the tumor microenvironment, and toxicity studies in cynomolgus monkeys showed short-term elevated levels of cytokines (An anti-glypican 3 / CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Ishiguro T. et al., Sci Transl Med. 2017 Oct 04; 9(410)). Significant improvement in antitumor activity in xenografts is achieved using a combination of ERY974 and chemotherapy (An anti-glypican 3 / CD3 bispecific T cell-redirecting antibody for treatment of solid tumors. Ishiguro T. et al., Sci Transl Med. 2017 Oct 04; 9(410)). Results of the first phase I clinical trial of ERY974 (ClinicalTrials.gov NCT05022927) in solid tumors were presented at the 2021 American Association for Cancer Research (AACR) Annual Meeting (Results of a phase 1 dose escalation study of ERY974, an anti-glypican 3(GPC3) / CD3 bispecific antibody, in patients with advanced solid tumors. Safran et al. Cancer Res(2021)81(13_Supplement): CT111). Steroid prophylaxis and two-step intrapatient titration were implemented to reduce toxicity of cytokine release syndrome (CRS).Twenty-nine patients were enrolled at very low dose levels ranging from 0.003 μg / kg to 0.81 μg / kg. Dose-limiting toxicity in terms of CRS (grade 2 and grade 3) was found at the 0.12 / 0.81 μg / kg dosing schedule (Results of a phase 1 dose escalation study of ERY974, an anti-glypican 3(GPC3) / CD3 bispecific antibody, in patients with advanced solid tumors. Safran et al. Cancer Res(2021)81(13_Supplement):CT111). Increases in IL-6, TNF-a, and IL-8 were observed in patients, explaining the occurrence of CRS. ERY974 was poorly tolerated. With a steep dose-response curve determined preclinically, clinical dosing is likely to be complicated due to a small therapeutic window with a simultaneous low probability of eliciting a response.

[0035] Different GPC3xCD3 biAb formats are in preclinical development (A novel targeted GPC3 / CD3 bispecific antibody for the treatment hepatocellular carcinoma. Yu L. et al. Cancer Biol Ther. 2020; 21(7): 597-603, Development of a Tetravalent T-Cell Engaging Bispecific Antibody Against Glypican-3 for Hepatocellular Carcinoma. Yu L. et al. , J Immunother. 2021 Apr 1;44(3):106-113, Combination Therapy of Hepatocellular Carcinoma by GPC3-Targeted Bispecific Antibody and Irinotecan is Potent in Suppressing Tumor Growth in Mice. Chen X. et al. Mol Cancer Ther. 2022 Jan;21(1):149-158). One of them is the ScFv of anti-human CD3 antibody and the VH domain of anti-GPC3 antibody derived from monoclonal antibodies L2K and HN3, respectively. The ScFv fragments linked by a 15 amino acid long polyglycine / serine linker consisting of (G4 S)3 were fused to the Fc of IgG1 through the hinge region. Similarly, the C-terminus of the VH domain was fused to the Fc domain. Each Fc region was introduced with P329G / L234A / L235A mutations (to suppress Fc-mediated activity) and knob-in-hole mutations for production issues. Preclinical data show in vitro and in vivo tumoricidal activity (A novel targeted GPC3 / CD3 bispecific antibody for the treatment hepatocellular carcinoma. Yu L. et al. Cancer Biol Ther. 2020; 21(7): 597-603).Highly engineered biAb formats should generate anti-drug antibodies (ADA) with the risk of loss of exposure. No clinical trials have been reported to date.

[0036] CD28 is an important costimulatory receptor expressed on the surface of T cells. It belongs to a subfamily of costimulatory molecules characterized by an extracellular variable immunoglobulin-like domain, which also includes CTLA-4, ICOS, PD-1, and BTLA. CD28 is expressed on the cell surface of T cells as a disulfide-linked homodimer and is found on approximately 80% of human CD4+ T cells and 50% of CD8+ T cells (Mir, MA (2015). Introduction of costimulatory molecules and costimulatory molecules. Developing Costimulatory Molecules for Immunotherapy of Diseases, (Elsevier), pp. 1-43).

[0037] Despite lacking intrinsic enzymatic activity, engagement of CD28 by its ligands results in specific phosphorylation and transcriptional signaling, leading to metabolic changes and the production of important cytokines, chemokines, and survival signals that are essential for the long-term expansion and differentiation of T cells.

[0038] The primary ligands for CD28 are CD80 (B7.1) and CD86 (B7.2), which are primarily expressed on the surface of professional antigen-presenting cells (APCs). CD80 and CD86 are distinct in their expression patterns, multimer status, and functionality. CD28 and CTLA-4 are highly homologous and therefore compete for the same ligands. However, CTLA-4 binds to these ligands with higher affinity than CD28, so CTLA-4 competes with CD28 for the ligands and ultimately suppresses T cell responses.

[0039] Several anti-CD28 monoclonal antibodies have been developed. Some of these, called superagonist (SA) antibodies, were found to induce full activation of primary resting T cells even in the absence of TCR ligation (so-called "signal 1"). The first administration test in humans of one of these SA anti-CD28 antibodies, TGN1412, resulted in a severe inflammatory response including a cytokine storm, which was unexpected in previous in vitro and in vivo tests, and led to chronic organ failure in all healthy volunteers receiving TGN1412.

[0040] Targeted costimulation of CD28 using bispecific antibodies To avoid safety issues associated with superagonist antibodies or systemic CD28 costimulation, tumor-targeted CD28 bispecific antibodies can be designed to limit costimulation of T cells in the vicinity of tumor cells. By pairing an agonist anti-CD28 arm to an anti-tumor associated antigen (TAA) arm, a molecule is generated that can bridge T cells to malignant cells expressing the selected TAA. Because CD28 bispecific antibodies can only bind CD28 monovalently, they cannot accidentally cluster CD28 in the absence of TAA-positive target cells, thus preventing systemic T cell activation. Even in the presence of TAA-positive cancer cells that allow clustering of CD28 on the surface of T cells, the full potential of T cell cytotoxicity can only be exerted in the presence of primary T cell stimulation via the TCR. This is in contrast to the bivalent superagonist CD28 monoclonal antibodies described above.

[0041] As mentioned above, the results of the first clinical trials with the T cell bispecific antibody TAAxCD3 in patients with advanced solid tumors were inconclusive. Preclinical trials for the treatment of solid tumors have explored the use of CD3 bispecific antibodies (which induce T cell activation, i.e., signal 1), costimulatory CD28 bispecific antibodies (which provide additional T cell activation, i.e., signal 2) (Skokos, D., Waite, JC, Haber, L., Crawford, A., Hermann, A., Ullman, E., Slim, R., Godin, S., Ajithdoss, D., Ye, X., et al.(2020). A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies. Sci. Transl. Med. 12, eaaw7888), or PD-(L)1 checkpoint inhibitors (Waite, JC, Wang, B., Haber, L., Hermann, A., Ullman, E., Ye, X., Dudgeon, D., Slim, R., Ajithdoss, DK, Godin, SJ, et al.(2020). Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy. Sci. Transl. Med. 12, eaba2325) have been shown to be beneficial. Examples of agonistic TAAxCD28 bispecific antibodies are described in WO 2019246514, WO 2020198009, WO 2020132066, WO 2020132024, WO 2020127618, WO 2021259890, and WO 2021155071, each of which is incorporated by reference in its entirety.Some corresponding molecules are currently in clinical trials (ClinicalTrials.gov identifiers: NCT04590326, NCT03972657, NCT04626635).So far, such conditional CD28 co-stimulation for T cell activation has not been applied to GPC3-positive malignancies, and has not been combined with GPC3xCD3 bispecific antibodies to mediate cell-mediated tumoricidal activity and fine-tune related cytokine release.Another method of providing T cell activation signal 2 in the case of GPC3-positive malignancies has been developed by Pieris pharmaceuticals, aiming to induce 4-1BB co-stimulatory effect in a tumor-localized manner. PRS-342 is a 4-1BB / GPC3 preclinical immuno-oncologically engineered anticalin antibody bispecific fusion protein, the results of which were published at AACR 2019 (Costimulatory T-cell engagement by PRS-342, a GPC3 / 4-1BB bispecific molecule, leads to activation of T-cells and tumor growth inhibition in a HCC humanized mouse model. Bossenmeier et al Cancer Res(2019)79(13_Supplement): 3268) and show potent T-cell activation strictly dependent on the presence of GPC3 positive tumor cells. No clinical trials have been reported to date.

[0042] The present invention provides a new fully human GPC3xCD3 bispecific antibody designed to be administered in parallel with the GPC3xCD28 bispecific antibody provided in the present invention.The GPC3xCD3 bispecific antibody and the GPC3xCD28 bispecific antibody of the present invention are not cross-reactive with GPC3 binding, i.e., do not compete for binding to GPC3.The present invention provides an agonist CD28 antigen-binding molecule that allows co-engagement with GPC3 on tumor cells.These GPC3xCD28 bispecific antibody and GPC3xCD3 bispecific antibody combinations mediate strong tumor-specific T cell activation.

[0043] The present invention describes a novel GPC3xCD3 bispecific antibody and a novel GPC3xCD28 bispecific antibody, as well as their combination. The comparative molecule "ERY974" clinically shows CRS already at low doses, which prevents potential therapeutic activity. The GPC3xCD3 bispecific antibody of the present invention can be administered at higher levels with lower overall cytokine release levels compared to ERY974, which is advantageous for therapeutic use. In particular, this combination allows for administration schedules, such as parallel or sequential treatment, which are likely to allow better control of dose-limiting CRS and concomitant increased activity. In general, the strongest cytokine release is usually seen with the first dose of TAAxCD3, with much lower release with subsequent doses. To avoid this problem, in the present invention, initiating therapy with GPC3xCD3 and then continuing treatment with a combination of GPC3xCD3 and GPC3xCD28 bispecific antibodies provides a way to reduce cytokine release and achieve improved efficacy compared to GPC3xCD3 monotherapy. The activity of GPC3xCD28 in combination can be further fine-tuned using the panel of different molecules presented in this invention, which is advantageous for therapeutic applications.

[0044] Exemplary GPC3xCD3 and GPC3xCD28 Bispecific Antibodies GPC3xCD3 bispecific antibodies (further referred to as "bispecific antibody GPC3xCD3" or "GPC3xCD3 bispecific antibody" or "GPC3xCD3 bispecific antibody") comprise a first binding moiety that specifically binds to human GPC3 and a second binding moiety that specifically binds to human CD3ε. For GPC3xCD3 bispecific antibodies, the letter-number combination "AD84" or "AD95" refers to the anti-GPC3 antibody arm, and "L3-1" (also referred to as "1A4") refers to the anti-CD3 antibody arm of the bispecific antibody of the invention.

[0045] The GPC3xCD28 bispecific antibody comprises a first binding portion (i.e., antigen-binding region) that specifically binds to human CD28 and a second binding portion (i.e., antigen-binding region) that specifically binds to human GPC3 (a non-competitive binding domain compared to the GPC3xCD3 bispecific antibody). For the GPC3xCD28 bispecific antibody, "P44", "P30", and "P111" represent the anti-GPC3 antibody arm, and "AI3" or "AI10" represent the anti-CD28 antibody arm of the bispecific antibody of the present invention.

[0046] The structure of the κλ bispecific antibody (κλ antibody) of the present invention is nearly indistinguishable from that of natural IgG. The present invention provides a combination of low immunogenicity and high efficacy. The GCP3xCD3 bispecific antibody comprises a common heavy chain, in one embodiment, a kappa light chain at the GPC3 binding portion, and a lambda light chain at the CD3 binding portion. The GPC3xCD28 bispecific antibody comprises a different common heavy chain, in one embodiment, a kappa light chain at the GPC3 binding portion, and a lambda light chain at the CD28 binding portion. The GPC3 binding arm of the GPC3xCD3 bispecific antibody of the present invention and the GPC3 binding arm of the GPC3xCD28 bispecific antibody target different domains within GPC3, avoiding binding competition on target cells.

[0047] The GPC3xCD3 and GPC3xCD28 bispecific antibodies of the present invention require Fc portions with dramatically reduced binding to FcγR to avoid Fc-mediated effector functions or Fc receptor-mediated cross-linking of the bispecific antibodies.

[0048] In some embodiments, the heavy chain is a naturally occurring heavy chain (i.e., does not contain any mutations ("wild type")). In some embodiments, the heavy chain comprises at least one mutation (i.e., has a "LALA" mutation or a "LALAPA" mutation). In some embodiments, the bispecific antibody comprises an amino acid substitution in each subunit of the Fc domain that reduces binding to an activating Fc receptor and / or reduces effector function, wherein said amino acid substitution is L234A and L235A, and / or a substitution of P329 selected from the group consisting of P329A, P329G and P329R (Kabat EU index numbering). In one embodiment, the bispecific antibody comprises amino acid substitutions L234A and L235A and P329A (Kabat EU index numbering) in each subunit of the Fc domain. L234A and L235A (LALA) indicates that the amino acid leucine at position 234 / 235 is replaced by alanine. P329A(PA) indicates that the amino acid proline at position 329 is replaced by alanine. " / N" in the bispecific antibody indicates that the Fc part carries the mutations L234A and L235A and P329A(LALAPA).

[0049] The bsAbs of the present invention can be based on any of the different antibody formats already described. In general, IgG-like formats are preferred as they offer favorable properties such as long half-life and potentially reduced immunogenicity, but any other molecular bispecific format can also be used in the present invention. In some embodiments, the bispecific antibodies share a common heavy chain. The concept of using a common heavy chain to obtain bispecific antibodies has been described previously (Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Fischer N. et al. Nat Commun. 2015 Feb 12;6:6113; Optimizing assembly and production of native bispecific antibodies by codon de-optimization. Magistrelli G, MAbs. 2017 Feb / Mar;9(2):231-239). Kappa-lambda bispecific antibodies are described, for example, in WO2014087248, which is incorporated herein by reference in its entirety.

[0050] Optionally, the bispecific antibody has different types of light chains. For example, one light chain is a kappa light chain and the other is a lambda light chain (i.e., a kl body). The different light chains allow for easy purification of the bispecifics using kappa and lambda selective resins.

[0051] Furthermore, the bispecific antibodies of the present invention can be made using techniques including those disclosed in WO 2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO 2012 / 023053 produces bispecific antibodies that are identical in structure to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each combining site exhibits a different antigen specificity to which both the heavy and light chains 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 junctions.

[0052] However, it is also possible to obtain the bispecific antibodies of the invention by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are called IgG κλ antibodies or "κλ bodies" (a new fully human bispecific IgG format). This κλ body format allows the affinity purification of bispecific antibodies indistinguishable from standard IgG molecules, with characteristics indistinguishable from standard monoclonal antibodies and therefore favorable characteristics compared to previous formats.

[0053] In addition to the above methods, bispecific antibodies of the invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization according to the method described in WO 2011 / 131746. In this method, a first monospecific bivalent antibody and a second monospecific bivalent antibody are engineered to have specific substitutions in the CH3 domains that promote heterodimer stability, and the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge regions to undergo disulfide bond isomerization, thereby generating the bispecific antibody by Fab arm exchange.

[0054] 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.

[0055] As used herein, a "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The full-length antibody heavy chain (HC) consists of the well-known heavy chain variable domain and constant domains VH, CH1, CH2, and CH3. The full-length antibody light chain (LC) consists of the well-known light chain variable domain and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine (K) in either one or both heavy chains.

[0056] The term "Fab arm" or "half molecule" refers to one heavy-light chain pair that specifically binds to an antigen.

[0057] The full-length bispecific antibodies of the invention can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example by introducing substitutions in the heavy chain CH3 interface in each half molecule, or by using co-expression, to favor heterodimer formation of two antibody half molecules with distinct specificities in an in vitro cell-free environment. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm may be engineered to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a distinct epitope.

[0058] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0059] As used herein, "heterodimerization" refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains having non-identical CH3 amino acid sequences.

[0060] A "knob-in-hole" strategy (see, for example, PCT Publication WO 2006 / 028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain "hole" with the heavy chain "knob". Exemplary CH3 substitution pairs that form the knob and hole are (represented as modification position in the first CH3 domain of the first heavy chain / modification 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.

[0061] Other strategies may be used, such as promoting heavy chain heterodimerization using electrical interactions by replacing positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface as described in US Patent Application Publication No. 2010 / 0015133, US Patent Application Publication No. 2009 / 0182127, US Patent Application Publication No. 2010 / 028637, or US Patent Application Publication No. 2011 / 0123532. In other strategies, heterodimerization may be promoted by the following substitutions (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): L351Y_F405A_Y407V / T394W, T366, as described in US Patent Application Publication No. 2012 / 0149876 or US Patent Application Publication No. 2013 / 0195849. I_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.

[0062] Exemplary CD28, CEA, and MSLN antibodies that can be used to engineer bispecific molecules include the antibodies disclosed herein. Exemplary anti-GPC3 antibodies from which the GPC3 antigen-binding region can be derived include the "AD843" or "AD95" antibodies. Exemplary anti-GPC3 antibodies from which the GPC3 antigen-binding region can be derived include the "P44", "P30" or "P111" antibodies. Exemplary anti-CD28 antibodies from which the CD28 antigen-binding region can be derived include the "AI3", "AI10" or "AI13" antibodies. Exemplary anti-CD3 antibodies from which the CD3 antigen-binding region can be derived include the "L3-1" (also known as "1A4") antibody. Table 1 shows the amino acid sequences of the regions of the antibodies of the present disclosure.

[0063] Table 1: Exemplary amino acid sequences of the present disclosure TIFF2025509702000001.tif242160TIFF2025509702000002.tif245160TIFF2025509702000003.tif246160 TIFF2025509702000004.tif235160TIFF2025509702000005.tif238160TIFF2025509702000006.tif146160

[0064] Tables 2 and 3 show exemplary bispecific antibodies of the disclosure. The nomenclature of each antibody is provided, and the individual first light chain, second light chain, and heavy chain regions of the κλ antibodies of the disclosure are described.

[0065] Table 2: Exemplary GPC3xCD3 bispecific antibodies of the present disclosure TIFF2025509702000007.tif25160

[0066] Table 3. Exemplary GPC3xCD28 bispecific antibodies of the present disclosure. TIFF2025509702000008.tif52160

[0067] In some embodiments, the AD84L3-1 / N bispecific antibody has a first heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:6, CDRH2 comprising the amino acid sequence of SEQ ID NO:7, and CDRH3 comprising the amino acid sequence of SEQ ID NO:8; a first 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; a second heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:6, CDRH2 comprising the amino acid sequence of SEQ ID NO:7, and CDRH3 comprising the amino acid sequence of SEQ ID NO:8; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:16, CDRL2 comprising the amino acid sequence of SEQ ID NO:17, and CDRL3 comprising the amino acid sequence of SEQ ID NO:18.

[0068] In some embodiments, the AD84L3-1 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, a first light chain variable region comprising the amino acid sequence of SEQ ID NO:14, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0069] In some embodiments, the AD84L3-1 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO: 10, a first light chain comprising the amino acid sequence of SEQ ID NO: 15, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 10, and a second light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0070] In some embodiments, the AD95L3-1 / N bispecific antibody has a first heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:6, CDRH2 comprising the amino acid sequence of SEQ ID NO:7, and CDRH3 comprising the amino acid sequence of SEQ ID NO:8; a first 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; a second heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:6, CDRH2 comprising the amino acid sequence of SEQ ID NO:7, and CDRH3 comprising the amino acid sequence of SEQ ID NO:8; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:21, CDRL2 comprising the amino acid sequence of SEQ ID NO:22, and CDRL3 comprising the amino acid sequence of SEQ ID NO:23.

[0071] In some embodiments, the AD95L3-1 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, a first light chain variable region comprising the amino acid sequence of SEQ ID NO:14, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO:24.

[0072] In some embodiments, the AD95L3-1 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO: 10, a first light chain comprising the amino acid sequence of SEQ ID NO: 15, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 10, and a second light chain comprising the amino acid sequence of SEQ ID NO: 25.

[0073] In some embodiments, the AI10P44 / N bispecific antibody has a first 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 first light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:46, CDRL2 comprising the amino acid sequence of SEQ ID NO:47, and CDRL3 comprising the amino acid sequence of SEQ ID NO:48; a second heavy chain comprising CDRH2 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; and a second 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.

[0074] In some embodiments, the AI10P44 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 49, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 29.

[0075] In some embodiments, the AI10P44 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:50, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:30.

[0076] In some embodiments, the AI10P30 / N bispecific antibody has a first 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 first light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:46, CDRL2 comprising the amino acid sequence of SEQ ID NO:47, and CDRL3 comprising the amino acid sequence of SEQ ID NO:48; a second heavy chain comprising CDRH2 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; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:31, CDRL2 comprising the amino acid sequence of SEQ ID NO:32, and CDRH3 comprising the amino acid sequence of SEQ ID NO:33.

[0077] In some embodiments, the AI10P30 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO:49, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, and a second light chain variable region of SEQ ID NO:34.

[0078] In some embodiments, the AI10P30 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:50, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:35.

[0079] In some embodiments, the AI10P111 / N bispecific antibody has a first 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 first light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:46, CDRL2 comprising the amino acid sequence of SEQ ID NO:47, and CDRL3 comprising the amino acid sequence of SEQ ID NO:48; a second heavy chain comprising CDRH2 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; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:36, CDRL2 comprising the amino acid sequence of SEQ ID NO:37, and CDRL3 comprising the amino acid sequence of SEQ ID NO:38.

[0080] In some embodiments, the AI10P111 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 49, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 39.

[0081] In some embodiments, the AI10P111 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:50, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:40.

[0082] In some embodiments, the AI3P44 / N bispecific antibody has a first 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 first 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; a second heavy chain comprising CDRH2 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; and a second 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.

[0083] In some embodiments, the AI3P44 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 44, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 29.

[0084] In some embodiments, the AI3P44 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:45, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:30.

[0085] In some embodiments, the AI3P30 / N bispecific antibody has a first 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 first 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; a second heavy chain comprising CDRH2 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; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:31, CDRL2 comprising the amino acid sequence of SEQ ID NO:32, and CDRH3 comprising the amino acid sequence of SEQ ID NO:33.

[0086] In some embodiments, the AI3P30 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 44, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 34.

[0087] In some embodiments, the AI3P30 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:45, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:35.

[0088] In some embodiments, the AI3P111 / N bispecific antibody has a first 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 first 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; a second heavy chain comprising CDRH2 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; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:36, CDRL2 comprising the amino acid sequence of SEQ ID NO:37, and CDRL3 comprising the amino acid sequence of SEQ ID NO:38.

[0089] In some embodiments, the AI3P111 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 44, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 39.

[0090] In some embodiments, the AI3P111 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:45, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:40.

[0091] In some embodiments, the AI13P44 / N bispecific antibody has a first 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 first light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:51, CDRL2 comprising the amino acid sequence of SEQ ID NO:52, and CDRL3 comprising the amino acid sequence of SEQ ID NO:53; a second heavy chain comprising CDRH2 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; and a second 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.

[0092] In some embodiments, the AI13P44 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 54, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 29.

[0093] In some embodiments, the AI13P44 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:55, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:30.

[0094] In some embodiments, the AI13P30 / N bispecific antibody has a first 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 first light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:51, CDRL2 comprising the amino acid sequence of SEQ ID NO:52, and CDRL3 comprising the amino acid sequence of SEQ ID NO:53; a second heavy chain comprising CDRH2 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; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:31, CDRL2 comprising the amino acid sequence of SEQ ID NO:32, and CDRH3 comprising the amino acid sequence of SEQ ID NO:33.

[0095] In some embodiments, the AI13P30 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO:54, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO:34.

[0096] In some embodiments, the AI13P30 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:55, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:35.

[0097] In some embodiments, the AI13P111 / N bispecific antibody has a first 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 first light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:51, CDRL2 comprising the amino acid sequence of SEQ ID NO:52, and CDRL3 comprising the amino acid sequence of SEQ ID NO:53; a second heavy chain comprising CDRH2 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; and a second light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:36, CDRL2 comprising the amino acid sequence of SEQ ID NO:37, and CDRL3 comprising the amino acid sequence of SEQ ID NO:38.

[0098] In some embodiments, the AI13P111 / N bispecific antibody has a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 54, a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a second light chain variable region of SEQ ID NO: 39.

[0099] In some embodiments, the AI13P111 / N bispecific antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:5, a first light chain comprising the amino acid sequence of SEQ ID NO:55, a second heavy chain comprising the amino acid sequence of SEQ ID NO:5, and a second light chain comprising the amino acid sequence of SEQ ID NO:40.

[0100] The present disclosure provides a composition comprising a first bispecific antibody of this disclosure (GPC3xCD3) and a second bispecific antibody of this disclosure (GPC3xCD28).

[0101] Table 4: Exemplary compositions TIFF2025509702000009.tif101160

[0102] In some embodiments, the composition comprises a first bispecific antibody of AD84L3-1 / N and a second bispecific antibody of AI10P44 / N. In some embodiments, the composition comprises a first bispecific antibody of AD84L3-1 / N and a second bispecific antibody of AI10P30 / N. In some embodiments, the composition comprises a first bispecific antibody of AD84L3-1 / N and a second bispecific antibody of AI10P111 / N. In some embodiments, the composition comprises a first bispecific antibody of AD84L3-1 / N and a second bispecific antibody of AI3P44 / N. In some embodiments, the composition comprises a first bispecific antibody of AD84L3-1 / N and a second bispecific antibody of AI3P30 / N. In some embodiments, the composition comprises a first bispecific antibody of AD84L3-1 / N and a second bispecific antibody of AI3P111 / N. In some embodiments, the composition comprises a first bispecific antibody to AD84L3-1 / N and a second bispecific antibody to AI13P44 / N. In some embodiments, the composition comprises a first bispecific antibody to AD84L3-1 / N and a second bispecific antibody to AI13P30 / N. In some embodiments, the composition comprises a first bispecific antibody to AD84L3-1 / N and a second bispecific antibody to AI13P111 / N.

[0103] In some embodiments, the composition comprises a first bispecific antibody of AD95L3-1 / N and a second bispecific antibody of AI10P44 / N. In some embodiments, the composition comprises a first bispecific antibody of AD95L3-1 / N and a second bispecific antibody of AI10P30 / N. In some embodiments, the composition comprises a first bispecific antibody of AD95L3-1 / N and a second bispecific antibody of AI10P111 / N. In some embodiments, the composition comprises a first bispecific antibody of AD95L3-1 / N and a second bispecific antibody of AI3P44 / N. In some embodiments, the composition comprises a first bispecific antibody of AD95L3-1 / N and a second bispecific antibody of AI3P30 / N. In some embodiments, the composition comprises a first bispecific antibody of AD95L3-1 / N and a second bispecific antibody of AI3P111 / N. In some embodiments, the composition comprises a first bispecific antibody to AD95L3-1 / N and a second bispecific antibody to AI13P44 / N. In some embodiments, the composition comprises a first bispecific antibody to AD95L3-1 / N and a second bispecific antibody to AI13P30 / N. In some embodiments, the composition comprises a first bispecific antibody to AD95L3-1 / N and a second bispecific antibody to AI13P111 / N.

[0104] How to use The therapeutic formulation of the present invention, comprising the bispecific antibody of the present invention, is used to treat cancer or to alleviate symptoms associated with cancer, such as, for example, hepatocellular carcinoma HCC and other GPC3-expressing cancers, as non-limiting examples.The present invention also provides a method of treating cancer or a method of alleviating symptoms associated with cancer.The therapeutic regimen is made by identifying a subject, for example, a human patient suffering from (or at risk of developing) cancer, using standard methods.

[0105] Efficacious treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody provides clinical benefit.

[0106] Pharmaceutical Compositions The antibody of the present invention (also referred to herein as "active compound"), as well as its derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include an antibody and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0107] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can contain the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, or phosphates, and agents for adjusting osmolality such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0108] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols (e.g., mannitol, sorbitol), and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0109] Sterile injection solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above-listed components as required, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the other components listed above.For the preparation of sterile powder for sterile injection solution, the preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0110] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0111] Systemic administration may be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0112] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0113] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and the like, can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes that target infected cells with monoclonal antibodies against viral antigens, can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0114] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the composition in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unit dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for individual treatment.

[0115] The pharmaceutical compositions can be included in a container, pack, dispenser, or other together with instructions for administration.

[0116] definition As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0117] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein 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 molecule X for partner Y is generally determined by the dissociation constant (K D Affinity can be measured by common methods known in the art, including KinExA, and Biacore, and Octet.

[0118] The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), fully human antibodies, and chimeric antibodies.

[0119] As used herein, unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, and individual antibody heavy or light chains, as well as individual heavy or light chain variable regions.

[0120] A "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0121] The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0122] A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain that also contains the VH and CH1 domains, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0123] An "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody. The "Fv region" contains the variable regions of both the heavy and light chains but lacks the constant region.

[0124] "Isolated antibody" refers to a purified state, and in such context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other substances (such as cellular debris and growth medium). The term "isolated" is generally not intended to refer to the complete absence of such substances, or the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with the experimental or therapeutic use of the binding compounds described herein.

[0125] The term "monoclonal antibody" as used herein refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies having different amino acid sequences within the variable domains, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques such as those described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597. See Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0126] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse carbohydrate chains if produced in a mouse, or in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat carbohydrate chains if produced in a rat, or in a rat cell, or in a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0127] Generally, the basic "antibody" structural unit comprises a tetramer. In monospecific antibodies, each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain contains a "variable region" or "variable domain" of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.

[0128] Typically, human constant light chains are classified as kappa and lambda light chains. Furthermore, human constant heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. These IgG subtypes include, for example, IgG1 and IgG4.

[0129] As used herein, "variable region," "variable domain," "V region," or "V chain" refers to a segment of an IgG chain that is variable in sequence among different antibodies. An antibody "variable region" refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable region of the heavy chain is referred to as the "V H The light chain variable region may be referred to as "V L". Typically, both the heavy and light chain variable regions contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions and enable binding to a specific epitope. Generally, from N-terminus to C-terminus, both the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain generally follows the definitions in Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5th ed., NIH Publ. No. 91-3242(1991), Kabat (1978) Adv. Prot. Chem. 32:1-75, Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616, Chothia, et al., (1987) J Mol. Biol. 196:901-917, or Chothia, et al., (1989) Nature 342:878-883.

[0130] "CDR" is the antibody V H One of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the beta-sheet framework, or the antibody V LIt refers to one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of the beta-sheet framework. Thus, CDR is a variable region sequence interspersed within the framework region sequence. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable regions in antibody variable domains. CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved beta-sheet framework and therefore can fit into different structures. Both terms are well known in the art. CDR region sequences are also defined by AbM, Contact, and IMGT. The location of CDRs in standard antibody variable regions has been determined by comparison of multiple structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Since the number of residues in the hypervariable region varies in different antibodies, additional residues for standard positions are conventionally numbered with a, b, c, etc. next to the residue number in the standard variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those of skill in the art. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT specific numbering systems, is well known to those of skill in the art. In some embodiments, the CDRs are as defined by the Kabat numbering system. In other embodiments, the CDRs are as defined by the IMGT numbering system. In yet other embodiments, the CDRs are as defined by the AbM numbering system. In yet other embodiments, the CDRs are as defined by the Chothia numbering system. In yet other embodiments, the CDRs are as defined by the Contact numbering system. In Table 2, the sequences are listed using the IMGT nomenclature.

[0131] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same in equivalent positions when the two sequences are optimally aligned.

[0132] Sequence similarity includes identical residues and non-identical biochemically related amino acids. Biochemically related amino acids which share similar properties and may be interchangeable are discussed above.

[0133] "Conservatively modified variant" or "conservative substitution" refers to the substitution of amino acids in a protein with other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure and rigidity, etc.), so that changes can be frequently made without changing the biological activity of the protein. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Moreover, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity.

[0134] The term "epitope" as used herein refers to an area or region on an antigen to which an antibody or antigen-binding fragment binds. Binding of an antibody or antigen-binding fragment thereof disclosed herein to an epitope means that the antibody or antigen-binding fragment thereof binds to one or more amino acid residues within the epitope.

[0135] An "isolated" nucleic acid molecule or polynucleotide refers to DNA or RNA, such as DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, in which the isolated polynucleotide is not associated with all or a portion of a polynucleotide found in nature, or associated with a polynucleotide with which it is not associated in nature. For purposes of this disclosure, it should be understood that a "polynucleotide comprising" a particular nucleotide sequence (or the like) does not encompass an intact chromosome. An isolated polynucleotide "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include coding sequences for up to 10, or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0136] The term "control sequence" refers to polynucleotide sequences necessary or useful for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, enhancers. In one embodiment of the invention, the polynucleotide is operably linked to a promoter, such as a viral promoter, a CMV promoter, an SV40 promoter, or a non-viral promoter or an elongation factor (EF)-1 promoter, and / or an intron.

[0137] A nucleic acid is "operably linked" when it is placed into a functional relationship with another polynucleotide. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, but not necessarily, "operably linked" means that the polynucleotide sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0138] The terms "cell", "cell line" and "cell culture" used herein are used interchangeably, and all such designations include their progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and the cultures derived therefrom, regardless of the number of transformations. It is also understood that not all progeny have exactly the same DNA content due to deliberate or accidental mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where separate designations are intended, it will be clear from the context.

[0139] Host cells include eukaryotic and prokaryotic host cells, including mammalian cells. Host cells include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells, among others. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells.Fungal cells include, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophia, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindnen), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia piperi, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucnowens lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.Pichia spp., any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucnowens, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa. The invention includes any host cell (e.g., a CHO cell or a Pichia cell, such as Pichia pastoris) that contains an anti-ILT4 antibody or antigen-binding fragment thereof, or that contains a polynucleotide encoding such an antibody or fragment, or that contains a vector containing a polynucleotide.

[0140] "Treat" or "treating" refers to administering an antibody or antigen-binding fragment thereof of the present invention to a subject having one or more symptoms of a disease for which the antibody and antigen-binding fragment of the present invention is effective, e.g., in treating a subject having or suspected of having cancer or an infectious disease for which the agent has therapeutic activity. Typically, the antibody or fragment is administered in an "effective amount" or "effective dose" that will alleviate one or more symptoms (e.g., symptoms of cancer or infectious disease) in the subject or population being treated, by inducing regression or elimination of such symptoms, or by inhibiting to any clinically measurable extent the progression of such symptoms, e.g., cancer symptoms such as tumor growth or metastasis. The effective amount of the antibody or fragment may vary depending on factors such as the stage of the disease, age, weight of the patient, and the ability of the drug to elicit a desired response in the subject. EXAMPLES

[0141] Example 1: Phage display selection of GPC3 Fv using a human scFv library containing fixed variable heavy chain domains The general procedure for constructing and manipulating human scFv libraries displayed on M13 bacteriophage is described in Vaughan et al. (Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library. Vaughan, T. et al. Nat Biotechnol 14, 309-314(1996)), which is incorporated herein by reference in its entirety. The libraries for selection and screening all encode scFvs sharing the same VH domain and diversifying only in the VL domain. Different VH domains were used for the library identifying GPC3-binders of GPC3xCD3 bispecific antibodies on the one hand and for the library identifying GPC3-binders of GPC3xCD28 on the other hand. Methods for generating fixed VH libraries and their use for identifying and assembling bispecific antibodies are described in US Patent Publication No. 2012 / 0184716 and WO 2012 / 023053, each of which is incorporated herein by reference in its entirety. The approach for identifying scFvs that bind to human GPC3 (huGPC3) is described below. Selections were performed in solution with biotinylated huGPC3 protein and / or on cells expressing huGPC3. The selection strategy included (i) up to four rounds of selection on recombinant protein, (ii) two rounds on recombinant protein followed by two rounds on cells.

[0142] Protein Selection Aliquots of scFv phage libraries were blocked with PBS containing 2% (w / v) skim milk. Blocked phages were first deselected on streptavidin / neutravidin magnetic beads (Dynabeads™ MyOne™ streptavidin T1 magnetic beads or Sera-Mag SpeedBeads Neutravidin™ coated magnetic particles) and then pre-incubated with 100 nM, 50 nM or 5 nM biotinylated recombinant human GPC3 (GP3-H82E5 AcroBiosystems, or in-house generated). The phage+antigen mixture was then captured by blocked magnetic beads (same type as used for deselection) and washed five times with PBS / 0.1% Tween® 20 and twice with PBS only. Phages were eluted with 1 mg / mL trypsin and added directly to exponentially growing TG1 cells after adding AEBSF to block trypsin activity. An aliquot of infected TG1 was serially diluted to titer the selection output, which was then removed and used for the next round of selection.

[0143] Example 2: Phage display selection of CD28 Fv using a human scFv library containing fixed variable heavy chain domains The general procedure for constructing and handling a human scFv library displayed on M13 bacteriophage is the same as described above. The approach to identify scFvs that bind to human CD28 (huCD28) is described below. Selections were performed in solution with biotinylated huCD28 protein and / or on cells expressing huCD28. The selection strategy included (i) up to four rounds of selection on recombinant protein, (ii) up to four rounds of selection alternating between recombinant protein and cells, and (iii) two rounds on recombinant protein followed by two rounds on cells.

[0144] Protein Selection Aliquots of scFv phage libraries were blocked with PBS containing 2% (w / v) skim milk. Blocked phages were first deselected on streptavidin / neutravidin magnetic beads (Dynabeads™ MyOne™ streptavidin T1 magnetic beads or Sera-Mag SpeedBeads Neutravidin™ coated magnetic particles) and then pre-incubated with 200 nM, 100 nM, 50 nM or 5 nM biotinylated recombinant human CD28 (CD8-H82E5, Acro Biosystems). Phage+antigen mixtures were then captured by blocked magnetic beads (same type as used for deselection) and washed 5 times with PBS / 0.1% Tween® 20 and twice with PBS only. Phages were eluted with 1 mg / mL trypsin and added directly to exponentially growing TG1 cells after adding AEBSF to block trypsin activity. An aliquot of infected TG1 was serially diluted to titer the selection output. The results were then removed and used for the next selection round.

[0145] Cell surface selection Phage-containing supernatants were blocked with PBS containing 10% FBS. Blocked phages were first deselected on CD28-negative TIB-153 cells (ATCC TIB 153) and then selected on CD28-positive Jurkat cells (Jurkat clone E6-1, ATCC TIB 152). Cells were pelleted and washed five times with PBS containing 10% FBS, followed by one wash with PBS only. Phages were eluted with 1 mg / mL trypsin and added directly to exponentially growing TG1 cells after adding AEBSF to block trypsin activity. Aliquots of infected TG1 were serially diluted to titer the selection output. The results were then removed and used for the next selection round.

[0146] Example 3: Screening of scFv binding / non-binding to human GPC3 Screening of scFvs for binding to GPC3 was tested by ELISA using biotinylated huGPC3-His (or biotinylated unrelated protein huMSLN as a negative control) or by flow cytometry using GPC3-positive (Hep G2) and GPC3-negative (SK-HEP-1) cells.

[0147] ELISA For the binding ELISA, neutravidin-coated plates were blocked with a 1% casein solution in PBS. Biotinylated huGPC3-His and biotinylated huMSLN (mesothelin) were captured at 5 nM. Dilutions of freshly prepared periplasmic extracts containing selected scFvs were applied to the plates and detected using a combination of mouse anti-c-myc and donkey anti-mouse IgG-HRP antibodies. The OD at 450 nm generated after addition of TMB was measured using a microplate spectrophotometer. Hits were classified as specific binders if they could not bind to an unrelated huMSLN protein and if the OD450 on huCD28 was at least 3-fold higher than the background OD450.

[0148] Flow cytometry For flow cytometry binding assays, cells were harvested, washed, and distributed into V-bottom 96-well plates at 150,000 or 200,000 cells / well. Dilutions of freshly prepared periplasmic extracts containing selected scFvs were preincubated with mouse anti-c-myc antibody and added to the cells. After incubation, cells were washed, incubated with goat anti-mouse IgG-APC detection antibody, and analyzed on an iQUE3 screening instrument (Sartorious). Hits were classified as positive and specific if at least 5% of the cells showed a binding signal on Hep G2 (ATCC HB-8065) ​​cells 3-fold higher than background GeoMFI, and if no such signal was observed on SK-HEP-1 (ATCC HTB-52) cells.

[0149] After DNA extraction from single clones, positive and specific hits were sequenced.

[0150] Example 4: Screening for scFv binding / non-binding to human CD28 Screening of scFvs for binding to CD28 was tested by ELISA using biotinylated huCD28-His (or biotinylated huCEA_ECD as a negative control) or by flow cytometry using CD28 positive (Jurkat) and CD28 negative (TIB-153) cells.

[0151] ELISA A similar method was used as described above for anti-GPC3 scFv screening. Instead of using recombinant huGPC3, biotinylated huCD28-His and biotinylated huCEA_ECD were captured at 5 nM on neutravidin coated plates.

[0152] Flow cytometry A similar method was used as described above for anti-GPC3 scFv screening. Jurkat and TIB-153 cells were used as CD28 positive and negative cells, respectively.

[0153] Example 5: Fixed VH candidates reformatted into IgG and transiently expressed in mammalian cells After screening and sequencing, scFv candidates with the desired binding properties were reformatted into IgG and expressed by transient transfection into PEAK cells. The VH and VL sequences of the selected scFvs were amplified with specific oligonucleotides and cloned into an expression vector containing the heavy and light chain constant regions. The expression vectors were verified by sequencing and transfected into mammalian cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 4 × 106 PEAK cells were cultured in 25 mL of culture medium containing fetal bovine serum in a T75 flask. The transfected cells were cultured at 37 °C for 5-6 days and IgG production was quantified using an Octet RED96 instrument. The supernatant was harvested for IgG purification on FcXL affinity resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, the supernatant from the transfected cells was incubated with an appropriate amount of FcXL resin overnight at 4 °C. After washing the resin with PBS, the sample was loaded onto an Amicon Pro column and the resulting IgG was eluted with 50 mM glycine pH 3.5. The eluted IgG fraction was then dialyzed by Amicon 50 kDa against histidine NaCl pH 6.0 buffer and the IgG content was quantified by absorbance at 280 nm. Purity and IgG integrity were verified by electrophoresis using an Agilent Bioanalyzer 2100 according to the manufacturer's instructions (Agilent Technologies).

[0154] Example 6: Binding of anti-GPC3 mAb to GPC3-positive cells The binding capacity of the anti-GPC3 antibody arms of the present invention tested as bivalent mAbs was evaluated by flow cytometry using, for example, Hep G2, Hep 3B or SK-HEP-1 cells.

[0155] Cells were harvested, checked for viability and counted. 200,000 cells were incubated for 15 min at 4° C. with increasing concentrations of antibodies diluted in FACS buffer (PBS 2% BSA). Cells were washed twice with cold FACS buffer and re-incubated with the appropriate anti-human IgG secondary antibody for another 15 min at 4° C. Cells were washed twice with cold FACS buffer and resuspended in 150 μl FACS buffer with the compatible viability marker. Antibody binding to live cells was measured by flow cytometry using a Cytoflex Platform (Beckman Coulter). Data was analyzed using FlowJo™ v10 software (BD Life Sciences) and dose-response binding curves were generated using GraphPad Prism 9 software. The AD84 GPC3 arm of the GPC3xCD3 bispecific antibody of the present invention and the P44, P30, P111 GPC3 arms of the GPC3xCD28 bispecific antibody were selected from this screening process based on their specificity and binding signal strength for GPC3-positive cells.

[0156] Example 7: Binding of anti-CD28 mAb to CD28-positive cells The binding ability of the anti-CD28 antibody arms of the present invention tested as bivalent mAbs was evaluated by flow cytometry using Jurkat cells (Jurkat clone E6-1, ATCC TIB 152). The flow cytometry method was the same as described above. The AI3 and AI10 CD28 binding arms were selected from this screening process based on their specificity and binding signal strength to CD28 positive cells.

[0157] Example 8: Expression and purification of a bispecific antibody carrying lambda and kappa light chains Co-expression of one heavy chain and two light chains in the same cell can result in the assembly of three different antibodies. Co-expression can be achieved in different ways, such as by transfection of multiple vectors expressing one of the co-expressed chains, or by using vectors driving the expression of multiple genes.

[0158] Here, the two light chains were cloned into the vector pNovi κHλ, which was previously generated to allow for the co-expression of one heavy chain, one kappa light chain, and one lambda light chain as described in US Patent Publication No. 20120184716 and WO2012023053 (each of which is incorporated herein by reference in its entirety). Expression of the three genes is driven by the human cytomegalovirus promoter (hCMV), and the vector also contains the glutamine synthetase gene (GS), which allows for the selection and establishment of stable cell lines. The common VH and VL genes of anti-CD3 IgG (L3-1 / N) and anti-GPC3 IgG (AD84), or anti-CD28 IgG (AI3 or AI10), and anti-GPC3 (P44, P30, or P111) IgG were cloned into the vector pNovi κHλ for transient expression in mammalian cells. Expi293 cells were cultured in suspension in appropriate Erlenmeyer flasks with the appropriate number of cells and culture medium volume. Plasmid DNA was transfected into Expi293 cells using PEI. Antibody concentrations in the supernatants of transfected cells were measured during production using an Octet RED96. According to the antibody concentration, the supernatants were harvested 5-7 days after transfection and clarified by filtration after the addition of diatomaceous earth (Sartorius). Purification was based on a three-step purification process. First, the CaptureSelect™ FcXL affinity substrate (Thermo Fisher Scientific) was washed with PBS and then added to the clarified supernatant. After overnight incubation at +4°C and 20 rpm, the supernatant was centrifuged at 2000g for 10 min, the flow-through was saved and the resin was washed twice with PBS. The resin was then transferred to an Amicon Pro column and a solution containing 50 mM glycine at pH 3.5 was used for elution. Several elution fractions were generated, neutralized with Tris-HCl pH 7.4 and pooled. The pool containing total human IgG (bispecific and two monospecific antibodies) was quantified using a Nanodrop spectrophotometer (NanoDrop Technologies).A small aliquot was saved for further analysis and the remaining sample was incubated with an appropriate volume of CaptureSelect™ Kappa XL Affinity Substrate (Thermo Fisher Scientific) for 30 min at room temperature at 20 rpm. Resin recovery and washing, elution and neutralization steps were performed as described above. The final affinity purification step was performed using CaptureSelect™ Lambda Fab Affinity Substrate (Thermo Fisher Scientific) applying the same process as the Kappa purification step. Alternatively, purification was based on a two-step purification process using only CaptureSelect™ Kappa XL Affinity Substrate and CaptureSelect™ Lambda Fab Affinity Substrate. All elution fractions were pooled and desalted against His-NaCl pH 6.0 formulation buffer using 50 kDa Amicon Ultra centrifugal filter units (Merck Millipore). The final product was quantified using Nanodrop.

[0159] Purified bispecific antibodies were analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer with the Protein 80 kit as described by the manufacturer (Agilent Technologies). Aggregate levels were determined by SEC-UPLC. All samples were tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL, Charles River Laboratories).

[0160] Example 9: In vitro characterization of bispecific antibodies Binding of GPC3xCD3 bispecific antibodies to GPC3-positive Hep G2 cells, CD3-positive Jurkat cells, and GPC3-negative / CD3-negative SK-HEP-1 cells A series of flow cytometry-based experiments were carried out to demonstrate the binding of GPC3xCD3κλ bodies (e.g., AD84L3-1 / N) to target cells. Cell staining and binding evaluation were carried out as described in Example 6. Binding curves were obtained using GPC3-positive Hep G2 (Figure 1A), CD3-positive Jurkat (Figure 1B), and GPC3-negative / CD3-negative SK-HEP-1 cells (Figure 1C). hIgG1 indicates an irrelevant monoclonal antibody of hIgG1 Fc that served as an isotype control. ERY974, described in WO 2017 / 159287 A1, is used as a reference control drug.

[0161] The bispecific binding ability of AD84L3-1 / N was confirmed by flow cytometry using cells expressing GPC3 or CD3 antigens (Figure 1A and Figure 1B). The results in Figure 1A and Figure 1B demonstrated that ERY974 exhibited higher maximum binding activity to both Hep G2 cells and Jurkat cells. For both AD84L3-1 / N and ERY974, no binding to non-expressing GPC3 cells and CD3 cells (SK-HEP-1) was observed (Figure 1C).

[0162] Binding of GPC3xCD28 bispecific antibody to GPC3-positive FU97 cells, CD28-positive Jurkat cells, and GPC3-negative / CD28-negative TIB-153 cells A series of flow cytometry-based experiments were performed to demonstrate the binding of the two GPC3xCD28 κλ bodies (AI3P44 / N and AI3P30 / N) to target cells. Examples of cells that can be used include GPC3-positive cell lines such as the gastric cancer cell line FU97 (JCRB1074), CD28-positive cell lines such as leukemic Jurkat T cells, and GPC3 / CD28 double-negative cell lines such as leukemic TIB-153 cells. Cell staining and binding assessment were performed as described in Example 6. The resulting binding profiles are shown in Figures 2A-2C. An irrelevant hIgG1 mAb served as a negative isotype control and was used for comparison in all experiments shown. The binding data on FU97 cells (Figure 2A) highlight the range of binding affinities of the selected anti-GPC3 arms (P44 and P30) of the present invention to huGPC3. Both the P44 and P30 arms bind to the membrane-distal domain within GPC3. Binding on CD3 positive Jurkat cells highlights how these two CD28xGPC3 bispecific antibodies sharing the same anti-CD28 arm (e.g., AI3) bind similarly to CD28 positive cells (Figure 2B). The absence of binding signal on GPC3 / CD28 negative TIB-153 suggests that all binding arms of the present invention are specific for their designated targets (Figure 2C).

[0163] Example 10: Bispecific antibody-mediated T-cell dependent cytotoxicity (TDCC) Once the co-engagement of each bispecific antibody was confirmed, the ability of the combination of GPC3xCD3 and GPC3xCD28 bispecific antibodies to kill tumor cells in the presence of PBMC effector cells was tested using a panel of GPC3-positive malignant cells.

[0164] The T-cell dependent cytotoxicity (TDCC) of GPC3 positive and negative tumor cell lines induced by the GPC3xCD3 bispecific antibody, tested alone or in combination with the GPC3xCD28 bispecific antibody of the invention, was evaluated using human peripheral blood mononuclear cells (PBMC) as effector cells. A minimum of three different donors were used in each experiment.

[0165] After two washes with PBS, the target cells are detached with cell dissociation solution. After a centrifugation step, the cells are resuspended in assay medium, adjusted to the required concentration, and seeded into 96-well plates.

[0166] PBMCs were isolated from buffy coats derived from healthy human donors using SepMate™ Tubes (Stemcell Technologies) containing Lymphoprep™ buffer (Stemcell Technologies).

[0167] For TDCC assays, PBMCs were added to target cells at a final E:T ratio of 20:1 (Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974. Shiraiwa et al., Methods, 2018). A dose range of GPC3xCD3 antibody and a fixed dose of GPC3xCD28 antibody (0.5, 0.1, 0.05, or 0.025 μg / mL unless otherwise stated) of the present invention were added to pre-seeded target and effector cells. Alternatively, a non-targeting CD3 bispecific antibody (Y4L3-1 / N) was used instead of GPC3xCD3 (AD84L3-1 / N). hIgG1 was used as an isotype control. ERY974 monotherapy was used as a reference comparator. Target cell killing was assessed after 48 h incubation at 37 °C with 5% CO2 by quantifying LDH released in the medium by apoptotic / necrotic cells (Cytotoxicity Detection KitPLUS (LDH), Roche). Maximal LDH release (=100% lysis) was obtained by incubating target cells with the lysis solution provided in the kit. Spontaneous LDH release (=0% lysis) refers to target cells co-incubated with effector cells without the addition of any antibody. TDCC curves were plotted using GraphPad Prism 9. TDCC data are shown in Figures 3-6. EC50 (half maximal concentration to mediate killing) and Emax (maximal percentage killing at the dose range tested) were determined using GraphPad Prism software. EC50 and Emax values ​​are summarized using Hep G2 (Table 5A) or Hep 3B (Table 5B) target cells as a source of effector cells and two PBMC donors.10A-10F show the assessment of target cell killing by quantifying the number of viable, adherent cells remaining in culture after 72 hours using Promega's CellTiter-Glo® (G7570) and comparing the values ​​obtained in each treated well to the reference (i.e., untreated wells (=0% killing)).

[0168] TDCC with Hep G2 or Hep 3B CD3 bispecific antibody and CD28 bispecific antibody combination therapy demonstrated increased potency (lower EC50) and maximal killing (Emax) compared to GPC3xCD3 bispecific antibody as monotherapy in relation to killing of GPC3-expressing cancer cell lines Hep G2 (Table 5A) and Hep 3B (Table 5B) in the dose range tested. EC50 and Emax range values ​​are derived from two representative PBMC donors used as a source of effector cells.

[0169] Table 5A: TDCC in Hep G2 cells TIFF2025509702000010.tif29169

[0170] Table 5B: TDCC in Hep 3B cells TIFF2025509702000011.tif29169*Within the dose range tested

[0171] Tested GPC3xCD28 bispecific antibodies (e.g., AI3P44 / N) synergized with GPC3xCD3 bispecific antibodies of the invention (e.g., AD84L3-1 / N) killed GPC3-positive Hep G2 target cells expressing 700,000 GPC3 / cell. Killing induced by two different PBMC donors is shown in Figures 3A-3B. Compared to AD84L3-1 / N monotherapy, the synergistic effect of the combination can be observed as a lower EC50 and a higher overall killing at the highest concentration tested. Importantly, the dose of GPC3xCD28 bispecific antibodies can be adapted to better control the synergistic effect (see also Table 5A). AD84L3-1 / N GPC3xCD3 in combination with 100ng / mL-500ng / mL AI3P44 / N is at least as good as ERY974 single agent treatment (Figures 3A-3B). A GPC3 negative cell line (SK-HEP-1) is not killed by any treatment demonstrating the importance of engaging GPC3 to mediate TDCC (Figures 3C-3D).

[0172] The ability of GPC3xCD28 bispecific antibodies (e.g., AI3P44 / N) to enhance killing of GPC3-expressing tumor cells in the presence of GPC3xCD3 bispecific antibodies was tested and compared with other GPC3xCD28 bispecific antibodies of the present invention. These CD28 bispecific antibodies share the same CD28 arm (e.g., AI3) but are paired with different GPC3 arms, P30 or P111. The GPC3 arms P44 and P30 target the membrane-distal region within GPC3. The P111 GPC3 arm is binding membrane-proximal like AD84L3-1 / N and does not compete with the GPC3 arm of the CD3 bispecific antibody for binding to GPC3. Also, the P44 arm and the P30 arm do not compete with the GPC3 arm of the CD3 bispecific antibody. The resulting CD28 bispecific antibodies were tested under the same conditions as in Figure 3. Combinations of AD84L3-1 / N with AI3P44 / N, AI3P30 / N, and AI3P111 / N are shown in Figures 4A-4C, respectively. Using GPC3-positive Hep G2 target cells, the data show that synergistic TDCC is observed regardless of the GPC3xCD28 bispecific antibody used in the combination. The killing activity can be fine-tuned by lowering the concentration of each GPC3xCD28 bispecific antibody (Figures 4A-4C). ERY974, included as a reference, showed maximal killing comparable to the CD28 bispecific antibody κλ-body when tested at the highest concentration (0.5 or 0.1 m / mL), except for the candidate AI3P111 / N that was tested in combination (Figure 4C). This consistently resulted in weaker activity in vitro compared to the AI3P44 / N (Figure 4A) or AI3P30 / N (Figure 4B) combinations. Overall, the data presented herein demonstrate that GPC3xCD28κλ-bodies can boost GPC3xCD3κλ-bodies (AD84L3-1 / N) TDCC to levels similar to or greater than ERY974 monotherapy. The resulting TDCC activity can be attenuated by lowering the CD28-κλ-body concentration or by using another GPC3 arm (e.g., P111, GPC3 membrane proximal).Importantly, when GPC3xCD3 was replaced with Y4L3-1 / N, a non-targeting CD3 bispecific antibody unable to deliver signal 1 to T cells, no killing was elicited by any of the CD28 bispecific antibodies included in the combination, highlighting the importance of the primary T cell stimulus (signal 1) for the activity of CD28 bispecific antibodies (Figures 4A-4C).

[0173] Another set of TDCC experiments was performed to evaluate the effect of a lower anti-CD28 arm (e.g., AI10) on TDCC activity when paired with one of the GPC3 binding arms (e.g., P44) of the present invention. The data show that both CD28 bispecific antibodies (AI3P44 / N and AI10P44 / N) bind similarly to GPC3-positive cells, and all P44-containing antibodies show comparable dose range profiles (Figure 5A). The data showing the binding range with the AI3-containing bispecific antibody demonstrates a higher binding signal on CD28-positive cells (Jurkat) compared to the AI10-containing bispecific antibody (Figure 5B). The data demonstrates the lower binding affinity of the CD28 AI10-binding arm compared to the AI3-binding arm. The antibody combination was then tested using the previously fixed optimized dose range. The TDCC data using the GPC3-expressing cell line Hep G2 suggests that the activity reflects the affinity of the CD28 arm. At intermediate concentrations of GPC3xCD28 κλ bodies, higher TDCC activity was observed for AI3P44 / N compared to the AI10P44 / N combination (Figure 5C and Figure 5D, respectively). Nevertheless, synergistic killing activity could be further boosted above the level of ERY974 monotherapy by increasing the concentration of lower affinity CD28 bispecific antibodies (e.g., AI10P44 / N) (Figure 5D).

[0174] To further evaluate killing synergy, GPC3 cell lines with lower expression of the target were included. The synergy between CD3 and CD28 bispecific antibodies is not cell line specific, since the Hep 3B cell line (ATCC Hep 3B2.1-7, expressing 60,000 GPC3 / cell) was killed more efficiently by the combination of CD3 and CD28 bispecific antibodies than by GPC3xCD3 bispecific antibody alone. Increased killing is found for all CD28 bispecific antibodies claimed in this application. The combination of AD84L3-1 / N and AI3P30 / N at 2.5 μg / mL demonstrates killing similar to ERY974 single agent treatment, or slightly lower (Figure 6A). As shown in Figure 6B, Figure 6C, and Figure 6D, effector cells from three independent PBMC donors all induced superior killing with AI3P44 / N at 0.5 μg / mL compared to ERY974 single agent. As observed in HepG2 cells, in Hep 3B cells too, the synergistic killing activity can be fine-tuned by lowering the concentration of GPC3xCD28 bispecific antibody (Figure 6B-6D, Table 5B). Combining a CD3 bispecific antibody to a CD28 bispecific antibody, pairing the lower affinity CD28 arm (AI10) to the GPC3 arm (P44), also enhances killing of Hep 3B cells compared to the CD3 bispecific antibody alone. The efficacy in this case is comparable to ERY974 single agent treatment (Figure 6E).

[0175] Example 11: Cytokines released into the supernatant when CD28 costimulation with GPC3xCD28 bispecific antibody enhanced killing of GPC3-expressing tumor cells ERY974 monotherapy has been demonstrated to induce severe cytokine release syndrome (CRS) in clinical trials. Such CRS can be caused by T cell overactivation. Indeed, T cell activation leads to the release of effector cytokines, which may impair the therapeutic window in patients.

[0176] The ability of the CD28 bispecific antibodies of the invention to enhance cytokine release by T cells upon killing of GPC3-expressing tumor cells in the presence of GPC3xCD3 was assessed by quantifying selected cytokines in the supernatants at the end of the TDCC assay. Cytokine levels were compared to those induced by ERY974 monotherapy.

[0177] After co-culture of GPC3-positive target cells with T cell-containing PBMCs as described in Example 10, the culture supernatants were collected by centrifugation and stored frozen at -80°C until further analysis. Cytokines (IL-2, IL-6, TNF-α, and IFN-γ) were quantified using the Mesoscale Discovery Platform by using multiplex kits.

[0178] The results of an experiment in which Hep G2 cells were co-cultured for 48 h with PBMCs at a dose range of GPC3xCD3 and different fixed doses of GPC3xCD28 bispecific antibody (AI3P44 / N) at an E:T ratio of 20:1 are shown in Figures 7A-7D. The resulting TDCC is shown in Figure 7A, and IFN-g, IL-6, and TNF-a levels are shown in Figures 7B, 7C, and 7D, respectively. While monotherapy with GPC3xCD3 (AD84L3-1 / N) resulted in very low levels of cytokines released by T cells, combination treatment with the GPC3xCD28 bispecific antibody tested increased virtually all measured cytokines, reflecting a better activation of T cells as suggested by the increased TDCC activity mentioned above. By lowering the concentration of the CD28 bispecific antibody, the resulting cytokine levels can be reduced below those obtained with ERY974 monotherapy and, importantly, without a statistically significant impairment of the maximum killing activity provided by the combination (Figures 7A-7D). As illustrated in Figures 7A-7D, at 0.1 mg / mL, the maximum AI3P44 / N killing / TDCC exceeds the maximum achieved with ERY974. However, the release of IFN-g, IL-6, and TNF-a is below that observed with ERY974. Similar data were generated by using another CD28 bispecific antibody pairing the same CD28 arm (e.g., AI3) to another GPC3 arm (e.g., P30).

[0179] Both killing activity and cytokine release can be fine-tuned by decreasing the concentration of the CD28 module (Figures 8A-8D). The decrease in maximum killing is smaller than the decrease in cytokine release when the concentration of AI3P30 is decreased from 500 ng / mL to 100 or 50 ng / mL (Figures 8A-8D). The maximum killing achieved with 0.5 mg / mL of AI3P30 / N is close to that achieved with ERY974, but the cytokine release is lower, especially in the case of IL-6 and TNF-a. Furthermore, the data in Figures 9E-9H show that compared to ERY974 single agent application, the cytokine release with the combination of AD84L3-1 / N and 100 ng / mL of AI10P44 (lower affinity CD28 arm) is much lower, but the maximum efficacy / kill of GPC3 expressing cancer cells is about 30% with ERY974 and still about 20% with the combination (Figures 9E-9F). The GPC3xCD3 and GPC3xCD28 bispecific antibodies of the present invention allow the ability to modify the ratio of tumor cell killing and cytokine release, which allows the ability to reduce CRS, which is advantageous over current therapies (such as "ERY974") that failed to achieve clinical efficacy at low doses and induced high CRS (Results of a phase 1 dose escalation study of ERY974, an anti-glypican 3(GPC3) / CD3 bispecific antibody, in patients with advanced solid tumors. Safran et al. Cancer Res(2021)81(13_Supplement):CT111).

[0180] Finally, we demonstrate the TDCC activity of the combination using CellTiter-Glo readout. In contrast to LDH release assays that rely on dead cells to quantify TDCC, the CellTiter-Glo assay format quantifies ATP levels in remaining live cells, which better reflects TDCC induced by bispecific antibodies in the presence of effector cells and is recognized as more sensitive (Choosing the right cell-based assay for your research. Riss, T. et al. Promega Cell Note, Issue 6(2003)). We demonstrate concentration-dependent killing of three different GPC3-expressing cancer cell lines with high amounts of GPC3 expression, including Hep G2 (700,000 GPC3 / cell), Hep 3B (60,000 GPC3 / cell), and HuH-7 (18,000 GPC3 / cell). As observed in Hep G2 cells, the combination of AD84L3-1 / N and AI3P44 / N (0.5 or 0.1 mg / mL) enhances cancer cell killing by up to 80% with efficacy comparable to ERY974 monotherapy (Figures 10A-10B). The potent and synergistic killing effect of the combination is also observed using Hep 3B cells (up to 80% of maximum killing). Figures 10A-10F show that the efficacy of the combination can be driven to higher tumor cell killing potential than achieved with ERY974 (Figures 10C-10D). Using HuH-7 cells, AI3P44 / N (tested at 0.5 mg / mL) can boost AD84L3-1 / N-mediated killing / TDCC to levels approaching ERY974 monotherapy with maximum killing killing 60-80% of malignant cells (Figures 10E-10F).

[0181] Overall, the data of the present invention demonstrate that GPC3xCD28 κλ bodies can boost GPC3xCD3 κλ bodies (AD84L3-1 / N)-mediated killing / TDCC to levels close to or even higher than ERY974 monotherapy, especially in tumor cells with low expression of GPC3. TDCC and associated cytokine production can be attenuated while maintaining efficacy by lowering the concentration of CD28-κλ bodies, CD28 affinity, or by using alternative GPC3 arms (e.g., P30) or CD28 arms (e.g., AI10). Furthermore, with similar or equal efficacy / TDCC, cytokine release induced by the bispecific antibody combination of the present invention is significantly lower compared to ERY974 (Figures 9A-9H).

[0182] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0183] Additional embodiments of the present disclosure include the following. Embodiment 1. 1. A bispecific antibody comprising: a. a first antigen-binding domain that binds to CD3, i. a first heavy chain variable region having a complementarity determining region 1 (CDR1) comprising the amino acid sequence of (SEQ ID NO:6), a complementarity determining region 2 (CDR2) comprising the amino acid sequence of (SEQ ID NO:7), and a complementarity determining region 3 (CDR3) comprising the amino acid sequence of (SEQ ID NO:8); ii. a first light chain variable region, 1. A first light chain variable region having 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; A first antigen-binding domain comprising: b. a second antigen-binding domain that binds to GPC3, i. a second heavy chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO:6, a CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a CDR3 comprising the amino acid sequence of SEQ ID NO:8; ii. a second light chain variable region, 1. CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18, or 2. CDR1 comprising the amino acid sequence of SEQ ID NO:21, CDR2 comprising the amino acid sequence of SEQ ID NO:22, and CDR3 comprising the amino acid sequence of SEQ ID NO:23 and a second light chain variable region having and a second antigen-binding domain comprising A bispecific antibody comprising:

[0184] Embodiment 2. 1. A bispecific antibody comprising: c. a first antigen-binding domain that binds to CD28, i. a first heavy chain variable region having a complementarity determining region 1 (CDR1) comprising the amino acid sequence of (SEQ ID NO:1), a complementarity determining region 2 (CDR2) comprising the amino acid sequence of (SEQ ID NO:2), and a complementarity determining region 3 (CDR3) comprising the amino acid sequence of (SEQ ID NO:3); ii. a first light chain variable region, 1. CDR1 comprising the amino acid sequence of SEQ ID NO: 41, CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and CDR3 comprising the amino acid sequence of SEQ ID NO: 43, or 2. CDR1 comprising the amino acid sequence of SEQ ID NO: 46, CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR3 comprising the amino acid sequence of SEQ ID NO: 48, or 3. CDR1 comprising the amino acid sequence of SEQ ID NO:51, CDR2 comprising the amino acid sequence of SEQ ID NO:52, and CDR3 comprising the amino acid sequence of SEQ ID NO:53 and a first light chain variable region having A first antigen-binding domain comprising: d. a second antigen-binding domain that binds to GPC3, i. a second heavy chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; ii. a second light chain variable region, 1. CDR1 comprising the amino acid sequence of SEQ ID NO: 26, CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR3 comprising the amino acid sequence of SEQ ID NO: 28, or 2. CDR1 comprising the amino acid sequence of SEQ ID NO: 31, CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and CDR3 comprising the amino acid sequence of SEQ ID NO: 33, or 3. CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 38 and a second light chain variable region having and a second antigen-binding domain comprising A bispecific antibody comprising:

[0185] Embodiment 3. 3. The bispecific antibody of claim 2, wherein the first and second heavy chain variable regions comprise the amino acid sequence of SEQ ID NO:4.

[0186] Embodiment 4. 2. The bispecific antibody of claim 1 , wherein the first and second heavy chains comprise the amino acid sequence of SEQ ID NO: 9.

[0187] Embodiment 5. a. the second light chain variable region of portion ii(1) comprises the amino acid sequence of SEQ ID NO:29; b. the second light chain variable region of portion ii(2) comprises the amino acid sequence of SEQ ID NO: 34; or c. the second light chain variable region of portion ii(3) comprises the amino acid sequence of SEQ ID NO: 39; 3. The bispecific antibody of claim 2.

[0188] Embodiment 6. a. the second light chain variable region of portion ii(1) comprises the amino acid sequence of SEQ ID NO: 19; or b. the second light chain variable region of portion ii(2) comprises the amino acid sequence of SEQ ID NO:24; 2. The bispecific antibody of claim 1.

[0189] Embodiment 7. 2. The bispecific antibody of claim 1 , wherein the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 14.

[0190] Embodiment 8. 2. The bispecific antibody of claim 1 , wherein the first light chain comprises the amino acid sequence of SEQ ID NO: 15.

[0191] EMBODIMENT 9. a. the first light chain variable region of portion a(ii)1 comprises the amino acid sequence of SEQ ID NO: 44; b. the first light chain variable region of portion a(ii)2 comprises the amino acid sequence of SEQ ID NO: 49; or c. the first light chain variable region of portion a(ii)2 comprises the amino acid sequence of SEQ ID NO:54; 3. The bispecific antibody of claim 2.

[0192] Embodiment 10. a. the first light chain of portion a(ii)1 comprises the amino acid sequence of SEQ ID NO: 45; b. the first light chain of portion a(ii)2 comprises the amino acid sequence of SEQ ID NO: 50; or c. the first light chain of portion a(ii)2 comprises the amino acid sequence of SEQ ID NO:55; 3. The bispecific antibody of claim 2.

[0193] Embodiment 11. 11. The bispecific antibody according to claim 1, wherein the first light chain is kappa and the second light chain is lambda.

[0194] Embodiment 12. 2. The bispecific antibody of claim 1 , wherein the first light chain is lambda and the second light chain is kappa.

[0195] Embodiment 13. An Fc domain comprising one or more amino acid substitutions that reduce binding to an activating Fc receptor and / or reduce effector function. 5. The bispecific antibody of claim 1 , comprising:

[0196] Embodiment 14. 14. The bispecific antibody of claim 13, wherein the amino acid substitutions include L234A and L235A substitutions.

[0197] EMBODIMENT 15. 15. The bispecific antibody of claim 13 or claim 14, wherein the amino acid substitutions comprise L234A, L235A and P329A, or P329G or P329R substitutions.

[0198] EMBODIMENT 16. 10. The bispecific antibody of any one of the preceding claims, having an IgG isotype.

[0199] EMBODIMENT 17. 10. The bispecific antibody of claim 9, wherein the antibody is a human antibody.

[0200] EMBODIMENT 18. 4. The bispecific antibody of any one of the preceding claims, wherein the composition enables tumor-specific T cell activation.

[0201] EMBODIMENT 19. A composition comprising a bispecific antibody according to any one of the preceding claims.

[0202] EMBODIMENT 20. A composition comprising the bispecific antibody of claim 1 and claim 2.

[0203] EMBODIMENT 21. 21. A method for reducing the proliferation of and / or killing cancer cells, comprising contacting said cells with a composition according to any one of claims 19 to 20.

[0204] EMBODIMENT 22. A method for treating cancer in a subject, comprising administering to the subject a composition according to any one of claims 19 to 20.

[0205] Embodiment 23. The method of claim 21 or claim 22, wherein the cancer is GPC3 positive.

Claims

1. It is a bispecific antibody, a. The first antigen-binding domain that binds to CD3, i. A first heavy chain variable region having a heavy chain complementarity determination region 1 (CDRH1) containing the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determination region 2 (CDRH2) containing the amino acid sequence of SEQ ID NO: 7, and a heavy chain complementarity determination region 3 (CDRH3) containing the amino acid sequence of SEQ ID NO: 8, ii. A first light chain variable region having a light chain complementarity determination region 1 (CDRL1) containing the amino acid sequence of SEQ ID NO: 11, a light chain complementarity determination region 2 (CDRL2) containing the amino acid sequence of SEQ ID NO: 12, and a light chain complementarity determination region 3 (CDRL3) containing the amino acid sequence of SEQ ID NO:

13. The first antigen-binding domain, b. A second antigen-binding domain that binds to GPC3, i. A second heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 6, CDRH2 containing the amino acid sequence of SEQ ID NO: 7, and CDRH3 containing the amino acid sequence of SEQ ID NO: 8, ii. The second light chain variable region, 1. CDRL1 containing the amino acid sequence of SEQ ID NO: 16, CDRL2 containing the amino acid sequence of SEQ ID NO: 17, and CDRL3 containing the amino acid sequence of SEQ ID NO: 18, or 2. CDRL1 containing the amino acid sequence of SEQ ID NO: 21, CDRL2 containing the amino acid sequence of SEQ ID NO: 22, and CDRL3 containing the amino acid sequence of SEQ ID NO: 23 A second light chain variable region having The second antigen-binding domain includes A bispecific antibody containing [specific antibody].

2. The bispecific antibody according to claim 1, wherein the first heavy chain variable region and the second heavy chain variable region include the amino acid sequence of SEQ ID NO:

9.

3. The bispecific antibody according to claim 1, wherein the first heavy chain and the second heavy chain comprise the amino acid sequence of SEQ ID NO:

10.

4. The bispecific antibody according to claim 1, wherein the first light chain variable region comprises the amino acid sequence of SEQ ID NO:

14.

5. The bispecific antibody according to claim 1, wherein the first light chain comprises the amino acid sequence of SEQ ID NO:

15.

6. a. The second light chain variable region of part b.ii.

1. contains the amino acid sequence of SEQ ID NO: 19, or b. The second light chain variable region of part b.ii.

2. includes the amino acid sequence of SEQ ID NO:

24. The bispecific antibody according to claim 1.

7. a. The second light chain of part b.ii.

1. contains the amino acid sequence of SEQ ID NO: 20, or b. The second light chain of part b.ii.

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

25. The bispecific antibody according to claim 1.

8. It is a bispecific antibody, a. The first antigen-binding domain that binds to CD28, i. A first heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3, ii. The first light chain variable region, 1. CDRL1 containing the amino acid sequence of SEQ ID NO: 41, CDRL2 containing the amino acid sequence of SEQ ID NO: 42, and CDRL3 containing the amino acid sequence of SEQ ID NO: 43, or 2. CDRL1 containing the amino acid sequence of SEQ ID NO: 46, CDRL2 containing the amino acid sequence of SEQ ID NO: 47, and CDRL3 containing the amino acid sequence of SEQ ID NO: 48, or 3. CDRL1 containing the amino acid sequence of SEQ ID NO: 51, CDRL2 containing the amino acid sequence of SEQ ID NO: 52, and CDRL3 containing the amino acid sequence of SEQ ID NO: 53 Having a first light chain variable region and The first antigen-binding domain, b. A second antigen-binding domain that binds to GPC3, i. A second heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3, ii. The second light chain variable region, 1. CDRL1 containing the amino acid sequence of SEQ ID NO: 26, CDRL2 containing the amino acid sequence of SEQ ID NO: 27, and CDRL3 containing the amino acid sequence of SEQ ID NO: 28, or 2. CDRL1 containing the amino acid sequence of SEQ ID NO: 31, CDRL2 containing the amino acid sequence of SEQ ID NO: 32, and CDRL3 containing the amino acid sequence of SEQ ID NO: 33, or 3. CDRL1 containing the amino acid sequence of SEQ ID NO: 36, CDRL2 containing the amino acid sequence of SEQ ID NO: 37, and CDRL3 containing the amino acid sequence of SEQ ID NO: 38 A second light chain variable region having The second antigen-binding domain, which includes A bispecific antibody containing [specific antibody].

9. The bispecific antibody according to claim 8, wherein the first heavy chain variable region and the second heavy chain variable region include the amino acid sequence of SEQ ID NO:

4.

10. The bispecific antibody according to claim 8, wherein the first heavy chain and the second heavy chain contain the amino acid sequence of SEQ ID NO:

5.

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

1. includes the amino acid sequence of SEQ ID NO:

44. b. The first light chain variable region of part a.ii.

2. contains the amino acid sequence of SEQ ID NO: 49, or c. The first light chain variable region of part a.ii.

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

54. The bispecific antibody according to claim 8.

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

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

45. b. The first light chain of part a.ii.

2. contains the amino acid sequence of SEQ ID NO: 50, or c. The first light chain of part a.ii.

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

55. The bispecific antibody according to claim 8.

13. a. The second light chain variable region of part b.ii.

1. includes the amino acid sequence of SEQ ID NO:

29. b. The second light chain variable region of part b.ii.

2. contains the amino acid sequence of SEQ ID NO: 34, or c. The second light chain variable region of part b.ii.

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

39. The bispecific antibody according to claim 8.

14. a. The second light chain of part b.ii.

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

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

2. contains the amino acid sequence of SEQ ID NO: 35, or c. The second light chain of part b.ii.

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

40. The bispecific antibody according to claim 8.

15. i) the first light chain is kappa and the second light chain is lambda; or ii) the first light chain is lambda and the second light chain is kappa, according to any one of claims 1 to 14.

16. Fc domain comprising one or more amino acid substitutions that reduce binding to the activated Fc receptor and / or reduce effector function. A bispecific antibody according to any one of claims 1 to 14, comprising:

17. The bispecific antibody according to claim 16, wherein the amino acid substitution includes L234A and L235A substitutions.

18. The aforementioned amino acid substitution is (i) L234A substitution, (ii) Replacement of L235A, and (iii) P329A, P329G, or P329R substitution A bispecific antibody according to claim 16, comprising:

19. The bispecific antibody according to any one of claims 1 to 14, wherein the antibody has an IgG isotype.

20. The bispecific antibody according to any one of claims 1 to 14, wherein the antibody is a human antibody.

21. A composition comprising a bispecific antibody according to any one of claims 1 to 14.

22. A composition comprising a first bispecific antibody and a second bispecific antibody, a. The first bispecific antibody comprises a first antigen-binding domain that binds to CD3 and a second antigen-binding domain that binds to GPC3. i. The first antigen-binding domain is 1. A first heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 6, CDRH2 containing the amino acid sequence of SEQ ID NO: 7, and CDRH3 containing the amino acid sequence of SEQ ID NO: 8, 2. A first light chain variable region having CDRL1 containing the amino acid sequence of SEQ ID NO: 11, CDRL2 containing the amino acid sequence of SEQ ID NO: 12, and CDRL3 containing the amino acid sequence of SEQ ID NO:

13. Includes, ii. The second antigen-binding domain is 1. A second heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 6, CDRH2 containing the amino acid sequence of SEQ ID NO: 7, and CDRH3 containing the amino acid sequence of SEQ ID NO: 8, 2. A second light chain variable region having CDRL1 containing the amino acid sequence of SEQ ID NO: 16, CDRL2 containing the amino acid sequence of SEQ ID NO: 17, and CDRL3 containing the amino acid sequence of SEQ ID NO: 18; or CDRL1 containing the amino acid sequence of SEQ ID NO: 21, CDRL2 containing the amino acid sequence of SEQ ID NO: 22, and CDRL3 containing the amino acid sequence of SEQ ID NO:

23. Including; and b. The second bispecific antibody comprises a first antigen-binding domain that binds to CD28 and a second antigen-binding domain that binds to GPC3, i. The first antigen-binding domain is 1. A first heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3, 2. A first light chain variable region having CDRL1 containing the amino acid sequence of SEQ ID NO: 41, CDRL2 containing the amino acid sequence of SEQ ID NO: 42, and CDRL3 containing the amino acid sequence of SEQ ID NO: 43; or CDRL1 containing the amino acid sequence of SEQ ID NO: 46, CDRL2 containing the amino acid sequence of SEQ ID NO: 47, and CDRL3 containing the amino acid sequence of SEQ ID NO: 48; or CDRL1 containing the amino acid sequence of SEQ ID NO: 51, CDRL2 containing the amino acid sequence of SEQ ID NO: 52, and CDRL3 containing the amino acid sequence of SEQ ID NO:

53. Including; and ii. The second antigen-binding domain is 1. A second heavy chain variable region having CDRH1 containing the amino acid sequence of SEQ ID NO: 1, CDRH2 containing the amino acid sequence of SEQ ID NO: 2, and CDRH3 containing the amino acid sequence of SEQ ID NO: 3, 2. A second light chain variable region having CDRL1 containing the amino acid sequence of SEQ ID NO: 26, CDRL2 containing the amino acid sequence of SEQ ID NO: 27, and CDRL3 containing the amino acid sequence of SEQ ID NO: 28; or CDRL1 containing the amino acid sequence of SEQ ID NO: 31, CDRL2 containing the amino acid sequence of SEQ ID NO: 32, and CDRL3 containing the amino acid sequence of SEQ ID NO: 33; or CDRL1 containing the amino acid sequence of SEQ ID NO: 36, CDRL2 containing the amino acid sequence of SEQ ID NO: 37, and CDRL3 containing the amino acid sequence of SEQ ID NO:

38. including, The aforementioned composition.

23. A composition comprising a bispecific antibody according to claim 1 or 8, or the composition according to claim 22, which enables tumor-specific T cell activation.

24. A pharmaceutical composition comprising a bispecific antibody according to claim 1 or 8, or a composition according to claim 22, for reducing the proliferation of cancer cells and / or killing cancer cells.

25. A pharmaceutical composition comprising a bispecific antibody according to claim 1 or 8 or a composition according to claim 22 for treating cancer in a target.

26. The pharmaceutical composition according to claim 24, wherein the cancer cells are GPC3 positive.

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