PD-L1×CD28 bispecific antibody for immune checkpoint-dependent T cell activation

Abstract

The present invention relates to a PD-L1xCD28 bispecific antibody that acts as an immune checkpoint inhibitor by binding to and blocking PD-L1 (thus preventing its binding to PD-1 expressed on T cells), and can further deliver a costimulatory signal to T cells by clustering and agonistically binding CD28. TIFF2025535352000019.tif45159

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 418,264, filed October 21, 2022, and U.S. Provisional Application No. 63 / 446,987, filed February 20, 2023, each of which is incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to a fully human PD-L1xCD28 bispecific antibody (bsAb) in a kappa lambda body format that is capable of blocking PD-1 / PD-L1 interaction while providing a costimulatory T cell signal.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (NOVI_052_001WO_SeqList_ST26.xml, size: 148,790 bytes, and creation date: October 12, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] Background of the Invention In the past few years, novel approaches have been developed to stimulate the body's own immune cells to better attack and kill cancer cells. Examples of successful cancer immunotherapy include monoclonal antibodies that can block so-called immune checkpoints. Currently approved immune checkpoint inhibitors (ICIs) block CTLA-4 (e.g., ipilimumab, marketed under the trade name Yervoy), PD-1 (e.g., pembrolizumab, marketed under the trade name Keytruda, and cemiplimab, marketed under the trade name Libtayo), and PD-L1 (e.g., atezolizumab, marketed under the trade name Tecentriq). ICIs can be used to obtain durable antitumor responses in various cancer types. Unfortunately, responses are limited to a subset of patients, and many cancer types are known to be inherently resistant to ICI monotherapy.

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

[0006] T cell costimulatory bispecific antibodies (bsAbs) are a novel class of therapeutic agents that can elicit antitumor responses, particularly in combination with T cell bispecific antibodies or immune checkpoint inhibitors (ICIs). Preclinical studies have demonstrated the benefit of adding bsAbs to T cell engagers (Correnti et al. 2018; Skokos et al. 2020) or PD-(L)1 checkpoint inhibitors (Waite et al. 2020) for the treatment of solid tumors, enhancing the efficacy of these agents. They act by providing costimulatory signals 2 to T cells within the tumor microenvironment. The specificity of CD28 costimulatory bsAbs is conferred by their so-called effector arms, specifically the targeting anti-tumor-associated antigen (TAA) arm paired with the agonistic anti-CD28 arm. To date, several TAAxCD28 bsAbs have been described (Correnti et al. 2018, also WO 2019246514, WO 2020132066, WO 2020198009, WO 2020127618, WO 2020132024, WO 2021259890, and WO 2022040482), some of which are actively tested in early-stage clinical trials (ClinicalTrials.gov Identifiers NCT03972657, NCT04590326, NCT04626635, NCT05219513, or NCT05585034).

[0007] To further enhance the antitumor activity of CD28 bsAbs, the anti-TAA targeting arm can be replaced with an antibody arm with intrinsic therapeutic activity. As shown in Figure 1, the use of an anti-PD-L1 arm offers multiple advantages: (1) monovalent blockade of PD-L1 still prevents the binding of PD-1 on T cells; (2) PD-L1 expressed on tumor, immune, or stromal cells inhibits T cells through PD-1 binding, which negatively regulates CD28 costimulatory signals; and (3) PD-L1 expressed on APCs sequester CD80, preventing it from costimulating CD28 on T cells.

[0008] Such PD-L1×CD28 bsAbs are: (1) PD-L1 + It can prevent PD-1 / PD-L1 interaction while providing costimulatory signal 2 when bridging (tumor) cells and T cells, (2) it prevents PD-L1 from APCs from interacting with PD-1 on T cells in the tumor microenvironment, but also in the draining lymph nodes, (3) it prevents PD-L1 from DCs from sequestering CD80, thus supporting CD80 / CD28 interaction, and (4) it prevents PD-L1 on stromal cells and immune cells from interacting with PD-1 on T cells.

[0009] Therefore, there is a need for novel antibodies and therapeutic agents that allow dual targeting of CD28 and PD-L1. Summary of the Invention

[0010] The present invention provides immune checkpoint-driven costimulatory bispecific antibodies. In some embodiments, the antibodies are bispecific antibodies having a first antigen-binding domain that binds PD-L1 and a second binding domain that binds CD28, referred to herein as PD-L1xCD28 bsAb.

[0011] The PD-L1xCD28 bsAb shares a common heavy chain with complementarity determining region 1 (CDR1) comprising the amino acid sequence of (SEQ ID NO: 6), complementarity determining region 2 (CDR2) comprising the amino acid sequence of (SEQ ID NO: 7), and complementarity determining region 3 (CDR3) comprising the amino acid sequence of (SEQ ID NO: 8).

[0012] In some embodiments, the PD-L1xCD28 bsAb comprises a first light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15 [S79].

[0013] In some embodiments, the PD-L1xCD28 bsAb has a second light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20 [AI3]; or a second light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25 [AI13].

[0014] In some aspects, the first and second heavy chain variable regions comprise the amino acid sequence of SEQ ID NO: 10. In some aspects, the first and second heavy chains comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0015] In some embodiments, the first light chain variable region of SEQ ID NO: 16.

[0016] In other embodiments, the first light chain has the amino acid sequence of SEQ ID NO:17.

[0017] In some embodiments, the bispecific antibody has a second light chain variable region of SEQ ID NO:21, SEQ ID NO:26.

[0018] In another embodiment, the bispecific antibody has a second light chain of SEQ ID NO:22, SEQ ID NO:27.

[0019] Also included in the present invention are compositions of any of the bispecific antibodies disclosed herein. Optionally, the composition further comprises a CD3xCEA bispecific antibody having two identical heavy chains comprising the amino acid sequence of SEQ ID NO:3, a first light chain having the amino acid sequence of SEQ ID NO:4, and a second light chain having the amino acid sequence of SEQ ID NO:5.

[0020] In some embodiments, the bispecific antibody has a first light chain that is kappa and a second light chain that is lambda.

[0021] In another embodiment, the bispecific antibody has a first light chain that is lambda and a second light chain that is kappa.

[0022] In some embodiments, a portion of the first light chain is of the kappa type and at least a portion of the second light chain is of the lambda type. In some embodiments, the first light chain comprises at least a kappa constant region. In some embodiments, the first light chain further comprises a kappa variable region. In some embodiments, the first light chain further comprises a lambda variable region.

[0023] In some embodiments, the second light chain comprises at least a lambda constant region. In some embodiments, the second light chain further comprises a lambda variable region. In some embodiments, the second light chain further comprises a kappa variable region.

[0024] In some embodiments, the first light chain comprises a kappa constant region and a kappa variable region, and the second light chain comprises a lambda constant region and a lambda variable region.

[0025] Optionally, the bispecific antibody has an Fc domain that comprises one or more amino acid substitutions that reduce binding to activating Fc receptors and / or reduce effector function.

[0026] For example, the bispecific antibody has L234A and L235A substitutions. Additionally, the bispecific antibody has P329A, P329G, or P329R substitutions.

[0027] The bispecific antibody has an IgG isotype. The bispecific antibody is a human antibody. The bispecific antibody enables PD-L1-dependent T cell activation. In some embodiments, immune stimulation by the bispecific antibody occurs within and / or at the tumor. In some embodiments, immune stimulation by the bispecific antibody occurs outside the tumor. In some embodiments, immune stimulation by the bispecific antibody occurs in lymphoid organs or the lymphatic system.

[0028] The present disclosure provides a composition comprising a bispecific antibody described herein and a pharmaceutically acceptable carrier.

[0029] The present disclosure provides methods of reducing tumor cell proliferation and / or enhancing tumor cell killing, comprising contacting a cell with a composition comprising a bispecific antibody described herein.The present disclosure provides methods of treating cancer in a subject, comprising administering to the subject a composition comprising a bispecific antibody described herein.

[0030] In some aspects, the disclosure provides uses of compositions comprising the bispecific antibodies described herein for treating, preventing, or delaying the progression of a pathology. In some embodiments, the pathology is cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid tumor is or is derived from breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, renal cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary gastric cancer, prostate cancer, or a combination thereof.

[0031] The present invention further includes antibodies having an antigen-binding domain that binds to CD28, wherein the antigen-binding domain comprises: a 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); and a light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20 [AI3]; or a light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25 [AI13].

[0032] The antibodies are F(ab) fragments, F(ab')2 fragments, and Fv fragments or single chain Fv fragments. The antibodies are monospecific. The antibodies are monovalent.

[0033] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the field related to the present invention. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0034] Other features and advantages of the present invention will be apparent from and encompassed by the following detailed description, and the claims. [Brief explanation of the drawings]

[0035] [Figure 1] Mechanism of action enabled by PD-L1×CD28 bsAb. [Figure 2A] Figure 2A-C shows the concentration-dependent binding of AI3S79 / N to PD-L1-expressing HPAC cells (A), CD28-expressing Jurkat cells (B), and CD28- and PD-L1-double-negative TIB153 cells (C) preactivated with IFNγ. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3] Concentration-dependent blockade of the PD-1 / PD-L1 interaction as measured by reporter cell bioassay. The stronger the blockade of the PD-1 / PD-L1 axis, the higher the reporter cell luminescence. AI3S79 / N blocks the PD-1 / PD-L1 axis and, thanks to its agonistic CD28 arm, also enhances the reporter system, resulting in higher RLU than simple PD-L1 blockers. [Figure 4] T cell-dependent cytotoxicity of HPAC cells induced by AI3S79 / N in combination with CEAxCD3 bsAb. Effector cells (healthy donor PBMCs) were cocultured with PD-L1 / CEA double-positive HPAC cells at different E:T ratios (from left to right: 10:1, 3:1, 1:1, and 1:3) for 6 days. AI3S79 / N synergizes with CEAxCD3 to kill HPAC cells. In the absence of signal 1 (no CEAxCD3 bsAb added), no killing is induced. Dose range of CEAxCD3 with a fixed concentration of CD28 bsAb (2.5 μg / mL). [Figure 5] Upregulation of the T cell activation marker CD25 on human CD4+ and CD8+ T cells (top and bottom rows, respectively) collected at the end of the TDCC experiment shown in Figure 4. The combination of CEAxCD3 and PD-L1xCD28 bsAb induces stronger activation of both CD4+ and CD8+ T cells than CEAxCD3 treatment alone. [Figure 6] Proliferation of human CD4+ and CD8+ T cells (top and bottom rows, respectively) collected at the end of the TDCC experiment shown in Figure 4. The combination of CEAxCD3 and PD-L1xCD28 bsAb strongly increases the percentage of proliferating CD4+ and CD8+ T cells compared to CEAxCD3 treatment alone. [Figure 7A] Figures 7A-F show an in vivo efficacy study of AI3S79 / N in treating the subcutaneous syngeneic colorectal cancer model MC38-hPD-L1 in CD28 HuGEMM mice. (A) Experimental design of the in vivo efficacy study. (B) Mean tumor volume for the different treatment groups. (C) Comparison of tumor volumes at the end of the study (day 28 after treatment initiation). Tumor volume was set at 3000 mm3 for all tumors that grew above 3000 mm3. (D, E, and F) Tumor growth of individual mice belonging to the vehicle, atezolizumab, or AI3S79 / N groups, respectively. In contrast to atezolizumab, which only slowed tumor growth on average, AI3S79 / N as a single agent resulted in tumor regression in all mice, with 6 of 10 mice marked tumor-free at the end of the study. [Figure 7B]See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 8A] Figures 8A-B show (A) mouse body weight (tumor-corrected) after treatment with AI3S79 / N according to the experimental treatment plan in Figure 7A. (B) mouse cytokine secretion 6 hours after the first injection after treatment with AI3S79 / N according to the experimental treatment plan in Figure 7A. AI3S79 / N was well tolerated in immunocompetent huCD28 mice. [Figure 8B] See legend to Figure 8A. [Figure 9] Mean tumor volume in re-challenged mice. Tumor re-challenge of surviving mice from the experiment shown in Figure 7A with WT MC38 cells on the opposite flank. Re-challenge = surviving mice (n=8), untreated. Naive mice (n=8), untreated. AI3S79 / N induced immunological memory against MC38. [Figure 10] Figure 10A-B shows IL-2 release by freshly or high-density precultured PBMCs isolated from three donors after treatment with TGN1412 (A) or AI3S79 / N (B). In contrast to TGN1412, AI3S79 / N is not a superagonist. [Figure 11A] Figures 11A-B show (A) a scheme of an established T cell and DC mixed lymphocyte reaction (MLR), (B) IL-2 release induced by AI3S79 / N and PD-(L)1 blocking antibodies, including nivolumab and atezolizumab. [Figure 11B] See legend to Figure 11A. [Figure 12] Body weights (as percentage of day 0) of irradiated and PBMC-transplanted mice treated with dose response of TGN1412 or AI3S79 / N. AI3S79 / N was well tolerated in a mouse model susceptible to CD28-mediated CRS. [Figure 13] Proliferation of CD4+ and CD8+ T cells cultured in plates coated with either the wet or dry coating procedure. Each dot represents the average proliferation obtained in healthy PBMC donors. In contrast to TGN1412, AI3S79 / N is not a superagonist. [Figure 14] Figure 14A-B shows (A) killing of NLV-loaded tumor cells and (B) the corresponding expansion of NLV-specific T cells induced by AI3S79 / N dose-response (right). Two donors are shown. AI3S79 / N induced killing of NLV-loaded tumor cells and expansion of NLV-specific T cells in a dose-dependent manner. [Figure 15A] Figures 15A-C show in vivo efficacy studies of AI3S79 / N in combination with CEAxCD3 TCE in treating HPAC tumors subcutaneously implanted in PBMC-humanized NOG mice. (A) Experimental design of the in vivo efficacy study. (B) Mean tumor volume for different treatment groups. (C) Tumor growth of individual mice. Because two PBMC donors (D415 and D417) were used for humanization, mice are clearly identified as being humanized with D415 (dotted line) or D417 (solid line). AI3S79 / N synergized with TCE to induce regression or cessation of TV. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 16A] Figures 16A-B show an in vivo efficacy study of AI3S79 / N in combination with CEAxCD3 TCE in treating HPAF-II subcutaneous tumors in fully humanized BRGSF-HIS mice. (A) Mean tumor volume for different treatment groups. (B) Tumor growth of individual mice. Asterisks (*) indicate animals euthanized due to tumor ulceration (a surrogate for tumor volume endpoint). AI3S79 / N synergized with TCE to reduce tumor progression. [Figure 16B] See legend to Figure 16A. [Figure 17]AI3S79 / N concentrations (semi-logarithmic scale) after single (0.5 or 10 mg / kg) and repeated (2 × 10 mg / kg) IV administration in monkeys. The black dashed line represents the LLOQ value (0.02 μg / mL). [Figure 18] Serum cytokine concentrations in cynomolgus monkeys after single or repeated administration (IV) with AI3S79 / N. AI3S79 / N administration resulted in only a mild and transient release of IL-6. [Figure 19] CRP levels in animals administered single or repeated (n=2) injections of AI3S79 / N. AI3S79 / N treatment induced a mild and transient CRP release. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description of the Invention The present invention is based on bispecific antibodies (bsAbs) capable of immune checkpoint-dependent T cell activation and tumor cell killing. Specifically, the present invention is based on bsAb co-engagement of the immune checkpoint PD-L1, which is expressed inter alia on the surface of tumor cells, to mediate CD28 clustering and thus PD-L1-mediated T cell activation.

[0037] The bsAbs of the invention feature a single agonistic CD28 antigen-binding domain for monovalent costimulation of CD28 and a second antigen-binding domain capable of specifically and monovalently binding to PD-L1, preventing PD-L1 from binding to PD-1 expressed on T cells.

[0038] CD28 CD28 is an important costimulatory receptor expressed on the surface of T lymphocytes. It belongs to a subfamily of costimulatory molecules characterized by an extracellular variable immunoglobulin-like domain. Other members of this family of molecules include CTLA-4, ICOS, PD-1, and BTLA.

[0039] In humans, CD28 is expressed on the cell surface of T lymphocytes as a disulfide-linked homodimer and is found on approximately 80% of human CD4+ T cells and 50% of CD8+ T cells.

[0040] Despite lacking intrinsic enzymatic activity, binding of CD28 by its ligands results in specific phosphorylation and transcriptional signaling that ultimately leads 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.

[0041] The major 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 differ in their expression patterns, multimeric state, and functionality. Because CD28 and CTLA-4 are highly homologous, they compete for the same ligands. However, because CTLA-4 binds to these ligands with higher affinity than CD28, CTLA-4 competes with CD28 for the ligands, ultimately suppressing T cell responses.

[0042] Several anti-CD28 monoclonal antibodies have been proposed for therapeutic targeting of CD28. A fraction of identified anti-CD28 antibodies, termed superagonist (SA) antibodies, were found to induce full activation of primary resting T cells through clustering of CD28 on the T cell surface, even in the absence of TCR ligation (signal 1). However, a first-in-human study of one such SA anti-CD28 antibody, TGN1412, resulted in severe inflammatory responses and chronic organ failure in all treated healthy volunteers. A cytokine storm, which was not predicted by either in vivo or in vitro preclinical safety studies, was responsible for these adverse events.

[0043] To minimize the side effects of anti-CD28 monoclonal antibodies, B7×anti-tumor-associated antigen (TAA) fusion proteins were proposed by Holliger et al. (Holliger et al. 1999). These were found to be equally effective but more specific than anti-CD28 monoclonal antibodies; i.e., B7×anti-CEA bispecific fusion proteins could activate T cells only in the presence of CEA-expressing cells.

[0044] By the same token, CD28 bsAbs cannot cluster CD28 on the surface of T cells by themselves but require the binding of a second target on the surface of another cell, and therefore CD28 bispecific antibodies cannot costimulate T cells by themselves.

[0045] In the context of the present invention, an agonistic anti-CD28 binding domain is paired with an anti-PD-L1 binding domain, resulting in a molecule capable of cross-linking T cells to cells expressing PD-L1.

[0046] Furthermore, even in the presence of PD-L1-positive cells that allow CD28 clustering on the surface of T cells, the full cytotoxic potential of T cells can only be unlocked in the presence of primary T cell stimulation via the TCR, in contrast to the bivalent superagonist CD28 monoclonal antibodies mentioned above.

[0047] Preclinical studies have shown the benefit of adding a costimulatory tumor-associated antigen × CD28 (TAA × CD28) bsAb for the treatment of solid tumors, enhancing the efficacy of bispecific T cell engagers or PD-(L)1 checkpoint inhibitors. Examples of agonistic TAAxCD28 bsAbs are described in WO 2019246514, WO 2020198009, WO 2020132066, WO 2020132024, WO 2020127618, WO 2021259890, WO 2021155071, and WO 2022040482, and some of these molecules are currently being tested in clinical trials (ClinicalTrials.gov Identifiers: NCT04590326, NCT03972657, NCT04626635, NCT05219513, NCT05585034).

[0048] PD-L1 Programmed cell death ligand-1 (PD-L1), also known as B7-H1 and CD274, is a transmembrane protein constitutively expressed in both hematopoietic and nonhematopoietic healthy tissues. It can also be expressed on tumor cells and tumor stroma. In cancer, expression of the inhibitory receptor PD-1 is considered a hallmark of exhausted T cells, which display a dysfunctional phenotype due to persistent antigenic and inflammatory stimuli. Furthermore, upregulation of PD-L1 in the tumor microenvironment has been shown to enable tumors to evade the host immune system by interacting with PD-1 on T cells. Multiple studies have reported that PD-L1 is expressed on either tumor cells or immune-infiltrating cells, or both, in various tumor tissues. Blocking the interaction between PD-1 and PD-L1 using monoclonal antibodies has proven successful in patients for various cancer indications and is widely believed to enhance antitumor T cell responses by reversing or preventing the onset of T cell exhaustion and by promoting the expansion of T cells during T cell priming in tumor-draining lymph nodes. However, despite the considerable improvements in patient outcomes achieved with PD-1 / PD-L1 checkpoint inhibitors, durable responses to these therapies are observed in only a minority of patients, and intrinsic or acquired resistance is common.

[0049] bispecific antibody The bsAb antibodies according to the present invention may be generated de novo or may be engineered from existing monospecific CD28 and PD-L1 antibodies.

[0050] The bsAbs of the present invention can be based on any of the different antibody formats previously described. Generally, IgG-like formats are preferred as they offer favorable properties such as long half-life and potentially reduced immunogenicity, although any other molecular bispecific format can also be used in the present invention.

[0051] Antibody heavy and light chain amino acid sequences identified by United States Adopted Names (USANs are available, for example, via the American Medical Association (https: / / www.ama-assn.org / ) or via the CAS registry).

[0052] Monospecific CD28 and PD-L1 binding variable domains can be selected de novo, for example, from phage display libraries, where phage are engineered to express human immunoglobulins or portions thereof, such as Fabs, single chain variable fragments (scFvs), or unpaired or paired antibody variable regions, and then engineered into a bispecific format. CD28 and PD-L1 variable domains can be isolated, for example, from phage display libraries that express antibody heavy and light chain variable regions as fusion proteins on the surface of bacteriophage M13 fused to the capsid protein pIII.

[0053] The antibody library is screened for binding to the CD28 antibody and PD-L1, and the resulting positive clones are further characterized. Such phage display methods for isolating human antibodies are established in the art. See, e.g., U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521404, 6,544731, 6,555313, 6,582,915, and 6,593,081. The resulting de novo variable region combinations are engineered into a bispecific format using methods known in the art and described herein.

[0054] Additionally, bispecific antibodies of the present invention can be produced using techniques including those disclosed in International Publication No. 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 structurally identical to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity, contributed by both the heavy and light chains. The light chain variable region can be from the lambda or kappa family and is preferably fused to lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide bonds.

[0055] However, it is also possible to obtain 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 referred to as IgGκλ antibodies or "κλ bodies" and are a new fully human bispecific IgG format. This κλ body format allows affinity purification of bispecific antibodies indistinguishable from standard IgG molecules, with characteristics indistinguishable from standard monoclonal antibodies, and is therefore preferred over previous formats.

[0056] In addition to the methods described above, bispecific antibodies of the present invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions to allow disulfide bond isomerization according to the method described in International Patent Publication No. 2011 / 131746. In this method, a first monospecific bivalent antibody and a second monospecific bivalent antibody are engineered to have certain substitutions in the CH3 domains that promote heterodimer stability, and the antibodies are incubated together under reducing conditions sufficient to allow cysteines in the hinge regions to undergo disulfide bond isomerization, thereby generating the bispecific antibody by Fab arm exchange.

[0057] 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.

[0058] 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. A full-length antibody heavy chain (HC) consists of the well-known heavy chain variable and constant domains, VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) consists of the well-known light chain variable and constant domains, VL and CL. A full-length antibody may lack the C-terminal lysine (K) in either one or both heavy chains.

[0059] The term "Fab arm" or "half molecule" refers to one heavy-light chain pair that specifically binds to an antigen.

[0060] Full-length bispecific antibodies of the present invention can be generated, for example, using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies by introducing substitutions into the heavy chain CH3 interface in each half molecule to support heterodimerization of two antibody half molecules with distinct specificities, either in vitro in a cell-free environment or using coexpression. The Fab arm exchange reaction is the result of disulfide bond isomerization 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 ​​in one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue in 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 can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding to a different epitope.

[0061] 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.

[0062] As used herein, "heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0063] Full-length bispecific antibodies can be generated using a "knob-in-hole" strategy (see, e.g., PCT Publication WO 2006 / 028936). 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 coexpression of the two antibodies, heterodimers are formed as a result of preferential interaction between the heavy chain with the "hole" and the heavy chain with the "knob." Exemplary CH3 substitution pairs that form knobs and holes are (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0064] Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions by substituting a positively charged residue on one CH3 surface and a negatively charged residue on the second CH3 surface, as described in U.S. Patent Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532. In another strategy, heterodimerization can 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 U.S. Patent Publication Nos. 2012 / 0149876 or 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.

[0065] Exemplary anti-cell surface antibodies that can be used to engineer bispecific molecules include, for example, anti-tumor associated antigen antibodies known in the art, such as pertuzumab and trastuzumab (HER-2); cetuximab, necitumumab, panitumumab, and amivantamab (EGFR); labetuzumab and civistamab (CEA); amatuximab (mesothelin); cordituzumab (Co atezolizumab, avelumab, and durvalumab (PD-L1); blinatumomab (CD19); brentuximab (CD30); daratumumab (CD38); gemtuzumab (CD33); tositumomab (CD22), or obinutuzumab, ocrelizumab, ofatumumab, rituximab, and ibritumomab (CD20).

[0066] Exemplary Bispecific Antibodies that Bind CD28 and PD-L1 Bispecific antibodies of the invention have one antigen-binding region specific for PD-L1 and a second antigen-binding region specific for CD28. Alternatively, however, the bispecific antibody is monovalent for PD-L1 and CD28. Bispecific antibodies share a common heavy chain.

[0067] In some embodiments, the heavy chain is a native heavy chain (i.e., does not contain any mutations). In some embodiments, the heavy chain comprises mutations compared to a native heavy chain. In some embodiments, the heavy chain is of the IgG14 type, containing different mutations to minimize effector function. Optionally, the bispecific antibody has light chains of different types. For example, one light chain is a kappa and the other is a lambda light chain (i.e., a k1 form). The different light chains allow for easy purification of the bispecific using kappa and lambda selective resins.

[0068] Exemplary PD-L1 antibodies from which the PD-L1 antigen-binding region may be derived include the S8 antibody, S9 antibody, S37 antibody, S14 antibody, S15 antibody, S17 antibody, S57 antibody, S58 antibody, S28 antibody, S30 antibody, S94 antibody, S23 antibody, S46 antibody, S71 antibody, and S79 antibody. Exemplary CD28 antibodies from which the CD28 antigen-binding region may be derived include the AI3 antibody, AI13 antibody, AI5 antibody, AI7 antibody, AI8 antibody, AI9 antibody, AI10 antibody, AI11 antibody, AI12 antibody, AI14 antibody, AI15 antibody, AI16 antibody, AI17 antibody, AI18 antibody, AI19 antibody, AI20 antibody, AI21 antibody, AI22 antibody, and AI23 antibody. Thus, reference to the antibody can be written, for example, as "S79xAI3" or "AI3xS79" or "AI3S79" or "S79AI3" to identify the first antigen-binding domain and the second antigen-binding domain.

[0069] In some embodiments, the S79xAI3 bispecific antibody has a heavy chain 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); a lambda light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a kappa light chain having a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20.

[0070] In some embodiments, the S79xAI3 bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:16, and a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:21.

[0071] In some embodiments, the S79xAI3 bispecific antibody has a heavy chain variable and constant region comprising an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, a lambda light chain comprising the amino acid sequence of SEQ ID NO: 17, and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0072] In some embodiments, the S79xAI13 bispecific antibody comprises a heavy chain 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); a lambda light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a kappa light chain having a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0073] In some embodiments, the S79xAI13 bispecific antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO:16, and a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

[0074] In some embodiments, the S79xAI13 bispecific antibody has a heavy chain variable and constant region comprising an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; a lambda light chain comprising the amino acid sequence of SEQ ID NO: 17; and a kappa light chain comprising the amino acid sequence of SEQ ID NO: 27.

[0075] [Table 1] TIFF2025535352000003.tif246170TIFF2025535352000004.tif244170TIFF20255353520 00005.tif245170TIFF2025535352000006.tif245170TIFF2025535352000007.tif240170 TIFF2025535352000008.tif234170TIFF2025535352000009.tif233170TIFF20255353520 00010.tif234170TIFF2025535352000011.tif246170TIFF2025535352000012.tif233170

[0076] definition As used in this specification, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0077] As used herein, "TGN1412" refers to a superagonist (SA) anti-huCD28 antibody of the human IgG4 isotype described in WO2006050949, comprising the amino acid sequence of SEQ ID NOs: 1 and 2.

[0078] As used herein, "CEAxCD3" refers to the CEAxCD3 bispecific κλ body first described in WO2021053587 (incorporated herein by reference in its entirety), which comprises a common heavy chain of SEQ ID NO: 3, a kappa light chain of SEQ ID NO: 4, and a lambda light chain of SEQ ID NO: 5.

[0079] As used herein, "S79" refers to a high-affinity, human / cynomolgus / mouse cross-reactive, kappa-lambda-compatible (common dummy heavy chain) anti-human PD-L1 blocking antibody. It was first described in WO2022200389 (incorporated herein by reference in its entirety) and comprises the amino acid sequences of SEQ ID NOs: 9 and 16 for its variable heavy (VH) and variable light (VL) chains, respectively.

[0080] As used herein, "AI3" and "AI13" refer to human / cynomolgus cross-reactive, kappa-lambda-compatible (common dummy heavy chain) anti-human CD28 agonist antibodies. They were first described in WO2023170474, which is incorporated herein by reference in its entirety. AI3 comprises the amino acid sequences of SEQ ID NOs: 9 and 21 as its variable heavy chain (VH) and variable light chain (VL), respectively, while AI13 comprises the amino acid sequences of SEQ ID NOs: 9 and 26 as its variable heavy chain (VH) and variable light chain (VL), respectively.

[0081] As used herein, " / N" refers to the set of mutations (Leu234Ala+Leu235Ala+Pro329Ala) (i.e., LALAPA) introduced into the human IgG1 Fc portion of a given antibody to abrogate Fc-mediated effector function.

[0082] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0083] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" or "immunospecifically binds" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with, or does so with a lower affinity (K d >10 -6 Antibodies include polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, F ab , F ab’ , and F (ab’)2 fragments, scFv, and F ab Examples of antibodies that can be used include, but are not limited to, expression libraries. Antibodies with high affinity have an affinity of about 0.01 nM to 25 nM.

[0084] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other depending on the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, and others. Furthermore, in humans, light chains can be kappa or lambda chains.

[0085] As used herein, the term "monoclonal antibody" (MAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. mAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0086] The terms "antigen-binding region" or "antigen-binding site" or "binding portion" refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues from the N-terminal variable ("V" (variable)) regions of the heavy ("H" (heavy)) and light ("L" (light)) chains. Three highly divergent stretches within the V regions of the heavy and light chains, termed "hypervariable regions," are interposed between more conserved adjacent stretches known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs." Various methods for numbering the amino acid sequence of an antibody and identifying the complementarity-determining regions are known in the art. For example, see the Kabat numbering system (Kabat, EA, et al., Sequences of Protein of Immunological Interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (IMGT®, the international ImMunoGeneTics information system®, available online at http: / / www.imgt.org / ). The IMGT numbering system is routinely used and recognized in the art as a reliable and accurate system for determining amino acid positions in coding sequences, allelic alignment, and for easily comparing sequences in immunoglobulins (IG) and T-cell receptors (TR) from all vertebrate species.The accuracy and consistency of the IMGT data are based on the IMGT-ONTOLOGY, the first and so far unique ontology for immunogenetics and immunoinformatics (see Lefranc. MP et al., Biomolecules, 2014 Dec;4(4), 1102-1139). IMGT tools and databases run against the IMGT reference directory, which was constructed from a large repository of sequences. In the IMGT system, IG V-DOMAINS and IG C-DOMAINS are delimited, whenever appropriate, taking into account exon boundaries. Thus, with more sequences available for the IMGT database, the IMGT exon numbering system can reliably be used by those skilled in the art to determine amino acid positions in coding sequences and for allele alignment. Additionally, the correspondence between the IMGT unique numbering and other numbering (i.e., Kabat) is available in the IMGT Scientific Chart (see Lefranc. MP et al., Biomolecules, 2014 Dec;4(4), 1102-1139).

[0087] The terms "hypervariable region" or "variable region" typically refer to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include the V and VH sequences when numbered according to the amino acid residues from the "complementarity-determining regions" or "CDRs" (e.g., the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). L Around residues 24-34 (LI), 50-56 (L2), and 89-97 (L3) in V Hand / or residues from the "hypervariable loops" (e.g., around about 31-35 (H1), 50-65 (H2), and 95-102 (H3) in H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, H25, H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H36, H37, H38, H39, H40, H41, H42, H43, H44, H45, H46, H47, H48, H49, H50, H51, H52, L Residues 24–34 (LI), 50–56 (L2), and 89–97 (L3) in V H residues 26-32 (H1), 52-56 (H2), and 95-101 (H3) in (V1), and / or residues from the "hypervariable loops" VCDR (e.g., ... 26-32 (H1), 52-56 (H2), and 95-101 (H3) in (V1), and / or residues 26-32 (H1), 52-56 (H2), and 95-101 (H3) in (V1), and / or residues L Residues 27–38 (LI), 56–65 (L2), and 105–120 (L3) in V H Optionally, the antibody comprises the following points when numbered according to AHo; Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001)): V L 28, 36 (LI), 63, 74-75 (L2), and 123 (L3) in V H The gene has symmetric insertions at one or more of 28, 36 (HI), 63, 74-75 (H2), and 123 (H3).

[0088] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. Epitopes typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and typically possess specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of polypeptides. Depending on the design, an antibody or a single antibody arm may specifically bind to an antigen with a dissociation constant of ≦1 μM, e.g., ≦100 nM, preferably ≦10 nM, and more preferably ≦1 nM. In some embodiments, an antibody or a single antibody arm may bind to an antigen with a dissociation constant of greater than 20 nM.

[0089] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and "off rate constant" (K off Both the K and the K can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The ratio of α to β allows the cancellation of all parameters not related to affinity and gives the dissociation constant K d(See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies or single antibody arms of the invention have an equilibrium binding constant (K) as measured by an assay such as a radioligand binding assay or similar assay known to those skilled in the art. d ) is ≦1 μM, e.g., ≦100 nM, preferably ≦10 nM, more preferably ≦1 nM. In some embodiments, an antibody or single antibody arm can bind to an antigen with a dissociation constant of greater than 20 nM.

[0090] As used herein, the term "isolated polynucleotide" is intended to mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, and by reason of its origin, an "isolated polynucleotide" is (1) not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, (2) operably linked to a polynucleotide with which it is not linked in nature, or (3) not found in nature as part of a larger sequence. Polynucleotides in accordance with the invention include nucleic acid molecules encoding heavy chain immunoglobulin molecules and nucleic acid molecules encoding light chain immunoglobulin molecules described herein.

[0091] The term "isolated protein" as referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof; because of its origin or source of derivation, an "isolated protein" is (1) not associated with proteins found in nature, (2) free from other proteins from the same source, e.g., free from marine proteins, (3) expressed by cells from a different species, or (4) not naturally occurring.

[0092] The term "polypeptide" is used herein as a generic term to refer to naturally occurring proteins, fragments, or analogs of a polypeptide sequence. Naturally occurring protein fragments and analogs are therefore species of the polypeptide genus. Polypeptides according to the invention include the heavy and light chain immunoglobulin molecules described herein, as well as antibody molecules formed by combinations comprising heavy chain immunoglobulin molecules with light chain immunoglobulin molecules (e.g., kappa light chain immunoglobulin molecules), and vice versa, and fragments and analogs thereof.

[0093] As used herein, the term "naturally-occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory or elsewhere is naturally-occurring.

[0094] As used herein, the term "operably linked" refers to the positioning of the components so described being in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0095] As used herein, the term "control sequences" refers to polynucleotide sequences necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism in prokaryotes; in eukaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; generally, such control sequences include promoters and transcription termination sequences. The term "control sequences" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. As referred to herein, the term "polynucleotide" refers to a polymeric structure of nucleotides at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. This term includes single- and double-stranded forms of DNA.

[0096] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids may also be suitable building blocks for the polypeptides of the present invention. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0097] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[0098] Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0099] Conservative amino acid substitution refers to the interchangeability of residues with similar side chains.For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine.Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0100] As discussed herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are contemplated by the present invention, provided that the changes in amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% integrity. Specifically, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally classified into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine, etc. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine, etc. Other families of amino acids include (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to expect that the sole substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a framework region. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative, assays which are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present invention.

[0101] Preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity for forming protein complexes, (4) change binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs may include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in portions of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituting amino acid should not tend to disrupt helices present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized secondary and tertiary structure of polypeptides are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)), and Thornton et al. Nature 354:105 (1991).

[0102] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable marker, for example, by incorporation of a radiolabeled amino acid or by attachment to a polypeptide of a biotinyl moiety that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain circumstances, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labels include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphorus fluorophores), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. As used herein, the term "pharmaceutical or drug" refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0103] Other chemical terms herein are used in accordance with conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0104] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., more abundant, on a molar basis, than any other individual species in the composition); preferably, a substantially purified fraction is a composition in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present.

[0105] Generally, a substantially pure composition will contain greater than about 80%, more preferably greater than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. Most preferably, the target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0106] The term patient includes human and veterinary subjects.

[0107] antibody Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against a given target, such as CD47, a tumor-associated antigen, or other target, or against a derivative, fragment, analog, homolog, or ortholog thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0108] Antibodies are purified by well-known techniques, such as affinity chromatography using Protein A or Protein G, which primarily provide the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen, or epitope thereof, that is the target of the desired immunoglobulin can be immobilized on a column, and the immune-specific antibody purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0109] In some embodiments, the antibody of the present invention is a monoclonal antibody. Monoclonal antibodies are produced, for example, by using the procedures described in the Examples provided herein. Antibodies can also be produced, for example, by immunizing BALB / c mice with a combination of cell transfectants that express high levels of a given target on their surface. Hybridomas resulting from myeloma / B cell fusion are then screened for reactivity against the selected target.

[0110] Monoclonal antibodies are prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0111] The immunizing agent typically includes the protein antigen, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, e.g., polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0112] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

[0113] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0114] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0115] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0116] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of a murine antibody). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0117] The monoclonal antibodies of the present invention include humanized or human antibodies. These antibodies are suitable for administration to humans without eliciting an immune response against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that are composed primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulins. Humanization is performed, for example, according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, for example, residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. A humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0118] A fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including the CDRs, arises from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Monoclonal antibodies can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0119] In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368, 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[0120] Human antibodies can also be produced using transgenic non-human animals modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies (see PCT Publication WO 94 / 02602). Endogenous genes encoding immunoglobulin heavy and light chains in the non-human host are disabled, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals providing all of the desired modifications are then obtained as progeny by crossbreeding intermediate transgenic animals containing less than the full complement of modifications. An example of such a non-human animal is the Xenomouse™ mouse, as disclosed in PCT Publication WO 96 / 33735 and WO 96 / 34096. The animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from animals after immunization with an immunogen of interest, e.g., as polyclonal antibody preparations, or alternatively, from immortalized B cells derived from animals, such as hybridomas, that produce monoclonal antibodies. Additionally, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single-chain Fv (scFv) molecules.

[0121] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and the formation of transcripts of a rearranged immunoglobulin heavy chain locus (the deletion is effected by a targeting vector containing a gene encoding a selection marker), and producing a transgenic mouse from the embryonic stem cells whose somatic and germ cells contain the gene encoding the selection marker.

[0122] One method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding the heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding the light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses the antibody containing the heavy and light chains.

[0123] In a further improvement of this procedure, methods for identifying clinically relevant epitopes of immunogens and correlative methods for selecting antibodies that specifically bind with high affinity to the relevant epitopes are disclosed in PCT Publication WO 99 / 53049.

[0124] The antibodies can be expressed by vectors containing DNA segments encoding the single chain antibodies described above.

[0125] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0126] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpes virus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci. USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al. See, e.g., Wang, et al., Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995); and adeno-associated virus vectors (see, e.g., Kaplitt, M. et al., Nat. Genet. 8:148 (1994)).

[0127] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. Adenovirus vectors result in a shorter period of expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn is shorter than HSV vectors. The specific vector selected depends on the target cell and the condition being treated. Introduction can be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of modes of gene introduction include naked DNA, CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0128] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. Additionally, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on total body flow, called convection, has also proven effective in delivering large molecules to extended areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known administration routes.

[0129] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In this case, one of the binding specificities is for a target such as CD28 or any fragment thereof. The second binding target is any other antigen, preferably a cell surface protein, or a receptor or receptor subunit.

[0130] Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published May 13, 1993, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0131] The bispecific and / or monospecific antibodies of the present invention can be produced using any of a variety of art-recognized techniques, including those disclosed in co-pending application 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 structurally identical to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity, contributed by both the heavy and light chains. The light chain variable region can be from the lambda or kappa family and is preferably fused to lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide bonds. However, it is also possible to obtain 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 referred to as IgGκλ antibodies or "κλ bodies" and are a new fully human bispecific IgG format. This κλ body format allows affinity purification of bispecific antibodies indistinguishable from standard IgG molecules, with characteristics indistinguishable from standard monoclonal antibodies, and is therefore preferred over previous formats.

[0132] An essential step in this method is the identification of two antibody Fv regions (each composed of a variable light chain domain and a variable heavy chain domain) with different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the production of monoclonal antibodies and fragments thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference.) A fully human antibody is an antibody molecule in which both the light and heavy chain sequences, including CDR1 and CDR2, arise from human genes. The CDR3 region may be of human origin or engineered by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0133] Monoclonal antibodies are produced, for example, by immunizing animals with the target antigen or its immunogenic fragment, derivative, or variant. Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells. Various techniques for producing xenogeneic non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entireties.

[0134] Alternatively, antibodies are obtained by screening libraries containing antibody or antigen-binding domain sequences for binding to the target antigen, prepared, for example, in bacteriophage, as protein or peptide fusions with bacteriophage coat proteins expressed on the surface of assembled phage particles, with the encoding DNA sequences contained within the phage particle (i.e., "phage display libraries").

[0135] Hybridomas resulting from the myeloma / B cell fusion are then screened for reactivity against the target antigen. Monoclonal antibodies are prepared using the hybridoma method, for example, as described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0136] Although not strictly impossible, it is highly unlikely that different antibodies that have the same heavy chain variable domain but are directed against different antigens will be identified by chance.In fact, in most cases, the heavy chain contributes most to the antigen-binding surface and is the most variable in sequence.In particular, the CDR3 on the heavy chain is the most diverse CDR in sequence, length, and structure.Therefore, two antibodies that are specific for different antigens almost always have different heavy chain variable domains.

[0137] The method disclosed in co-pending application WO 2012 / 023053 overcomes this limitation and greatly facilitates the isolation of antibodies with the same heavy chain variable domain by using an antibody library in which the heavy chain variable domain is the same for all library members, and therefore diversity is limited to the light chain variable domain. Such libraries are described, for example, in co-pending applications WO 2010 / 135558 and WO 2011 / 084255, each of which is incorporated herein by reference in its entirety. However, because the light chain variable domain is expressed along with the heavy chain variable domain, both domains can contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and diverse lambda or kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach allows the identification of two antibodies that share a common heavy chain, but one with a lambda light chain variable domain and the other with a kappa light chain variable domain, which can be used as building blocks for the generation of bispecific antibodies in the full immunoglobulin format of the invention. Bispecific antibodies of the invention can be of different isotypes, and their Fc portions can be modified to alter their binding characteristics to different Fc receptors, thus modifying the antibody's effector functions and its pharmacokinetic properties. Numerous methods for modifying Fc portions have been described and can be applied to the antibodies of the invention. (See, e.g., Strohl, WR Curr Opin Biotechnol 2009(6):685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199 (filed January 9, 2009)). The methods of the invention can also be used to generate bispecific antibodies and antibody mixtures in the F(ab')2 format, which lack the Fc portion.

[0138] Coexpression of a common heavy chain and two different light chains in a single cell allows for the assembly of bispecific antibodies of the present invention. If all polypeptides were expressed at the same level and assembled equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, it is likely that different light chains are expressed at different levels and / or do not assemble with the same efficiency. Therefore, means of adjusting the relative expression of different polypeptides are used to compensate for their unique expression characteristics or different propensities to assemble with a common heavy chain. This adjustment can be achieved through promoter strength, the use of internal ribosome entry sites (IRES) characterized by different efficiencies, or other types of regulatory elements that act at the transcriptional or translational level and can also affect mRNA stability. Different promoters with different strengths can include the following: CMV (immediate-early cytomegalovirus viral promoter), EF1-1α (human elongation factor 1 α subunit promoter), Ubc (human ubiquitin C promoter), and SV40 (simian virus 40 promoter). Different IRES from mammalian and viral origins have also been described (see, e.g., Hellen CU and Sarnow P. Genes Dev 2001 15:1593-612). These IRES can vary greatly in their length and ribosome recruitment efficiency. Furthermore, activity can be further tuned by introducing multiple copies of the IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97:1536-1541). Regulation of expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expression. The examples provided herein demonstrate that controlling the relative expression of the different chains is important for maximizing the assembly and overall yield of bispecific antibodies.

[0139] Coexpression of a heavy chain and two light chains generates a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter must be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by using affinity chromatography media that specifically interact with kappa or lambda light chain constant domains, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multistep affinity chromatography purification approach is efficient and generally applicable to the antibodies of the present invention. This contrasts sharply with specific purification methods that must be developed and optimized for each bispecific antibody derived from a quadroma or other cell line expressing an antibody mixture. Indeed, if the biochemical characteristics of different antibodies in a mixture are similar, their separation using standard chromatographic techniques, such as ion exchange chromatography, may be difficult or even impossible.

[0140] Other suitable purification methods include those disclosed in co-pending application PCT / IB2012 / 003028 (filed October 19, 2012, published as WO 2013 / 088259, the contents of which are incorporated herein by reference in their entirety).

[0141] In another embodiment for producing bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0142] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 region of the antibody constant domains. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products, such as homodimers.

[0143] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The resulting bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0144] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. The fragments consist of a light-chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to the heavy chain variable domain (V H ) is included. Therefore, the V of one fragment H and V L The domain is a complementary V L and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0145] More than bivalent antibodies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0146] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates from the protein antigen of the present invention. Alternatively, the anti-antigenic arm of an immunoglobulin molecule can be combined with an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus cellular defense mechanisms on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a specific antigen. These antibodies possess an antigen-binding arm and an arm that binds a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the protein antigens described herein and also binds tissue factor (TF).

[0147] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (see WO 91 / 00360, WO 92 / 200373, and EP 03089). It is contemplated that antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0148] It may be desirable to modify the antibody of the invention with respect to effector function to enhance the antibody's effectiveness in, for example, treating cancer and / or other diseases and disorders associated with aberrant CD28 expression and / or activity. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0149] The present invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioactive isotope (i.e., a radioconjugate).

[0150] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diacin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and tricosene. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0151] Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. (See WO 94 / 11026).

[0152] Those skilled in the art will recognize that a wide variety of possible moieties can be coupled to the resulting antibodies of the invention (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0153] Coupling can be achieved by any chemical reaction that bonds two molecules, so long as the antibody and other moiety retain their respective activities. This linkage can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. However, covalent bonding is preferred. Covalent bonding can be achieved either by direct condensation of existing side chains or by the incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but rather is illustrative of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0154] Preferred linkers are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies by oligopeptide linkers.) Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. No. 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co., Cat. No. 2165-G), (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. No. 24510) (conjugated to EDC).

[0155] The above linkers contain components with different attributes, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, allowing for the formation of conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. Specifically, sulfo-NHS can enhance the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC) when used with sulfo-NHS forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0156] The antibodies disclosed herein can also be formulated as immunoliposomes. Antibody-containing liposomes can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556.

[0157] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to liposomes via a disulfide-interchange reaction as described by Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0158] How to use It will be understood that administration of therapeutic entities according to the present invention will be administered with suitable carriers, excipients, and other agents that are incorporated into the formulation to provide improved mobility, delivery, tolerability, and the like. Numerous suitable formulations can be found in the following formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975), in particular Chapter 87 therein by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be suitable in treatments and therapies according to the present invention, provided that the active ingredient therein is not inactivated by the formulation, the formulation is physiologically compatible, and can be tolerated for the route of administration. Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals" Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al., "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein for additional information regarding formulations, excipients, and carriers well known to pharmaceutical chemists.

[0159] Therapeutic formulations of the invention comprising antibodies of the invention are used to treat or alleviate symptoms associated with cancer, including, by way of non-limiting example, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral cavity and pharyngeal cancer, endometrial cancer, and / or melanoma. The invention also provides methods for treating or alleviating symptoms associated with cancer. Treatment regimens are carried out by identifying a subject, e.g., a human patient, suffering from (or at risk of developing) cancer using standard methods.

[0160] Efficacy of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody provides a clinical benefit.

[0161] Methods for screening antibodies with the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known in the art.

[0162] Antibodies directed against targets such as CD28, PD-L1, or combinations thereof (or fragments thereof) can be used in methods known in the art related to the localization and / or quantification of these targets, e.g., for use in measuring the levels of these targets in appropriate physiological samples, for use in diagnostic methods, for use in protein imaging, etc. In certain embodiments, antibodies specific for any of these targets, or derivatives, fragments, analogs, or homologs thereof (which contain the antigen-binding domain derived from the antibody), are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0163] Antibodies of the present invention can be used to isolate specific targets using standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies of the present invention (or fragments thereof) can also be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the effectiveness of a given therapeutic regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol, examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include: 125 I, 131 I, 35 S, or 3 H is one example.

[0164] The antibodies of the present invention, including polyclonal antibodies, monoclonal antibodies, humanized antibodies, and fully human antibodies, can be used as therapeutic agents. Such agents are generally used to treat or prevent diseases or pathologies associated with the abnormal expression or activation of a given target in a subject. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and generally has an effect resulting from its binding to the target. Administration of the antibody can suppress, inhibit, or interfere with the signaling function of the target. Administration of the antibody can suppress, inhibit, or interfere with the binding of the target to the endogenous ligand to which it naturally binds. Administration of the antibody can activate, stimulate, or enhance the signaling function of the target.

[0165] A therapeutically effective amount of an antibody of the present invention generally relates to the amount necessary to achieve a therapeutic goal. As noted above, this may be a binding interaction between the antibody and its target antigen, which, in certain cases, interferes with the function of the target. In some embodiments, administration of the antibody may activate, stimulate, or enhance the signaling function of the target. In some embodiments, the antibody may suppress, inhibit, or interfere with the binding of the target to its endogenous ligand, activating, stimulating, or enhancing the signaling function of another target. The amount required to be administered further depends on the binding affinity of the antibody for its specific antigen and the rate at which the administered antibody is depleted from the free volume of the subject to which it is administered. A typical therapeutically effective dose range for an antibody or antibody fragment of the present invention may be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical administration frequencies may range, for example, from twice daily to once weekly.

[0166] The antibodies or fragments thereof of the present invention can be administered in the form of pharmaceutical compositions for the treatment of various diseases and disorders. Principles and considerations involved in the preparation of such compositions, as well as guidance in the selection of components, are provided, for example, in Remington: The Science and Practice of Pharmacy, 19th ed. (Alfonso R. Gennaro, et al., editors), Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0167] When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein binding domain is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation can also contain two or more active compounds necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can contain an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0168] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions.

[0169] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0170] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0171] The antibodies according to the present invention can be used as agents for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is polyclonal, more preferably monoclonal. The intact antibody or a fragment thereof (e.g., F ab , scFv, or F (ab)2) is used. With respect to probes or antibodies, the term "labeled" is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, blood and fractions or components of blood, including serum, plasma, or lymph, are included in the use of the term "biological sample." That is, the detection methods of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in in vitro and in vivo biological samples. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, J.R.Crowther (Ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0172] Pharmaceutical Composition The antibodies of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0173] 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 application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0174] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), 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 the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0175] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent, and with one or combination of the above-listed ingredients as required, and then filter sterilization.Generally, dispersion is prepared by incorporating this active compound into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from the above-listed ones.For the sterile powder that is used to prepare sterile injectable solution, preparation method is vacuum drying and freeze-drying, thereby obtaining the powder of active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0176] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the liquid carrier is applied orally and swished in the mouth and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavor.

[0177] 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, eg, a gas such as carbon dioxide, or a nebulizer.

[0178] Systemic administration can also be via 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, surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, the active compound is generally formulated into ointments, salves, gels, or creams known in the art.

[0179] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0180] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0181] It is particularly advantageous to formulate oral or parenteral compositions into unit dosage forms for ease of administration and uniform dosage.As used herein, unit dosage form refers to a physically discrete unit suitable as a unit dose for the subject to be treated.Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The specifications of the unit dosage form of the present invention are determined and directly depend on the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the technical field of compounding such active compounds for the treatment of individuals.

[0182] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0183] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0184] Example 1: Expression and purification of a bispecific antibody with 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.

[0185] Here, the two light chains were cloned into the vector pNoviκHλ, which had been previously generated to enable simultaneous expression of one heavy chain, one kappa light chain, and one lambda light chain, as described in U.S. Patent No. 20120184716 and International Publication No. 2012023053 (each of which is incorporated herein by reference in its entirety). Expression of the three genes was driven by the human cytomegalovirus promoter (hCMV), and the vector also contained a glutamine synthetase (GS) gene, which enabled the selection and establishment of stable cell lines. The common VH and VL genes of anti-CD28 IgG and anti-PD-L1 IgG were cloned into the vector pNoviκHλ for transient expression in mammalian cells. Expi293 cells were cultured in suspension in an appropriate Erlenmeyer flask with the appropriate number of cells and volume of culture medium. 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. Depending on the antibody concentration, the supernatants were collected 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, a CaptureSelect™ FcXL affinity matrix (Thermo Fisher Scientific) was washed with PBS and then added to the clarified supernatant. After overnight incubation at +4°C and 20 rpm, the supernatant was centrifuged at 2000 g for 10 min, the flow-through was saved, and the resin was washed twice with PBS. The resin was then transferred to an Amicon Pro column, and a solution containing 50 mM glycine at pH 3.5 was used for elution. Several elution fractions were generated, neutralized with Tris-HCl pH 7.4, and pooled. The pool containing total human IgG (the bispecific antibody and the 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™ KappaXL affinity matrix (Thermo Fisher Scientific) for 30 minutes at room temperature and 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 matrix (Thermo Fisher Scientific) and the same process as the kappa purification step. Alternatively, purification was based on a two-step purification process using only CaptureSelect™ KappaXL affinity matrix and CaptureSelect™ Lambda Fab affinity matrix. All elution fractions were pooled and desalted against His-NaCl pH 6.0 formulation buffer using a 50 kDa Amicon Ultra centrifugal filter unit (Merck Millipore). The final product was quantified using a Nanodrop.

[0186] Purified bispecific antibodies were analyzed by electrophoresis under denaturing and reducing conditions using an Agilent 2100 Bioanalyzer equipped with a Protein 80 kit, as described by the manufacturer (Agilent Technologies). Aggregation levels were determined by SEC-UPLC. All samples were tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories). Table 2 summarizes the PD-L1xCD28κλ bodies produced.

[0187] [Table 2]

[0188] Example 2: In vitro characterization of PD-L1 x CD28 bispecific antibodies To demonstrate the binding of PD-L1×CD28κλ bodies to target cells ( Fig. 1 ), a series of flow cytometry-based experiments were performed.

[0189] Examples of cells that can be used include PD-L1 positive cell lines such as the pancreatic adenocarcinoma epithelial cell line HPAC, CD28 positive cell lines such as leukemic Jurkat T cells, and PD-L1 and CD28 double negative cell lines such as leukemic TIB153 cells.

[0190] Cells were harvested, checked for viability, and counted. 200,000 cells were incubated with increasing concentrations of antibodies diluted in FACS buffer (PBS 2% BSA, 0.1% NaN3) for 15 minutes at 4°C. Cells were washed twice with cold FACS buffer and re-incubated with the appropriate anti-human IgG secondary antibody for another 15 minutes at 4°C. Cells were washed twice with cold FACS buffer and resuspended in 150 μl of FACS buffer containing the appropriate viability marker. Antibody binding to live cells was measured by flow cytometry using a Cytoflex Platform (Beckman Coulter). Data were analyzed with FlowJo™ v10 software (BD Life Sciences), and dose-response binding curves were generated using GraphPad Prism 9 software.

[0191] The binding curves of an exemplary PD-L1xCD28 bsAb AI3S79 / N of the invention obtained using HPAC, Jurkat, and TIB153 cells are shown in Figure 2A, Figure 2B, and Figure 2C, respectively.

[0192] AI3S79 / N binds to cells expressing either PD-L1 (Figure 2A) or CD28 (Figure 2B). The absence of a binding signal on TIB-153 suggests that all binding arms of the present invention are specific for their designated targets (Figure 2C).

[0193] The affinity of AI3S79 / N for CD28, PD-L1, Fc gamma receptor (FcγR), and neonatal Fc receptor (FcRn) from human, cynomolgus monkey, and mouse species was determined by Octet® surface plasmon resonance (Tables 3, 4, 5, 6, and 7).

[0194] [Table 3]

[0195] The extracellular domains of human and cynomolgus CD28 are 100% identical, and therefore, the KD values ​​of AI3S79 / N for human and cynomolgus CD28 are comparable (Table 3). This data demonstrated that AI3S79 / N is cross-reactive with cynomolgus CD28 (KD is approximately 54 nM for both human and cynomolgus CD28) and PD-L1 (KD is approximately 0.3 nM for humans and approximately 1.8 nM for cynomolgus). The anti-PD-L1 arm of AI3S79 / N is also cross-reactive with mouse PD-L1 (KD is approximately 0.3 nM for humans and approximately 0.79 nM for mice). However, the anti-CD28 arm of AI3S79 / N is not cross-reactive with mouse CD28. In fact, it exhibits low affinity for mouse CD28 and is therefore not considered cross-reactive.

[0196] As shown in Table 4, AI3S79 / N exhibits comparable affinity for human and cynomolgus monkey FcRn (KD: approximately 7.1 nM for human and approximately 8.6 nM for cynomolgus monkey).

[0197] [Table 4]

[0198] As shown in Table 5, AI3S79 / N binds to human CD64 with low affinity (KD approximately 804 nM) and does not bind to other human FcγRs (CD32a R167, CD32a H167, CD32b, CD16a V158, CD16a F158, CD16b).

[0199] [Table 5]

[0200] The LALAPA mutation introduced into AI3S79 / N silenced the Fc of this molecule. The absence of binding of AI3S79 / N to FcγRs prevents Fc-mediated effector functions (i.e., ADCP or ADCC).

[0201] AI3S79 / N binds to cynomolgus CD64 with low affinity (KD approximately 183 nM) and does not bind to other cynomolgus FcγRs (CD32a, CD32b, CD16), as shown in Table 6. As in humans, the LALAPA mutation in AI3S79 / N is expected to interfere with Fc-mediated effector functions (i.e., ADCP, ADCC, and CDC) in cynomolgus monkeys.

[0202] [Table 6]

[0203] As shown in Table 7, AI3S79 / N does not bind to any of the mouse FcγRs tested (CD64, CD32b, CD16).

[0204] [Table 7]

[0205] Example 3: PD-1 / PD-L1 Blockade Bioassay The ability of AI3S79 / N to block the PD-1 / PD-L1 interaction was assessed using the PD-1 / PD-L1 blocking bioassay, a biologically relevant MOA-based assay that measures the potency of antibodies designed to block the PD-1 / PD-L1 interaction (Promega, J1250).

[0206] The kit consists of two cell lines: (1) artificial antigen-presenting cells (aAPCs) based on engineered CHO-K1 cells that express both PD-L1 and proteins designed to activate cognate TCRs in an antigen-independent manner on their cell surface, and (2) Jurkat T cells stably expressing human PD-1 and NFAT-inducible luciferase. When the two cell types are cocultured, PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-mediated luciferase activity. Addition of antibodies that block either PD-1 or PD-L1 releases an inhibitory signal, resulting in TCR signaling and NFAT-mediated luciferase activity.

[0207] As shown in Figure 3, because the reporter cells also express CD28 on their cell surface, AI3S79 / N not only induces luminescence through blocking PD-1 / PD-L1 binding but also further enhances it through CD28 costimulation, resulting in a higher RLU signal than simple PD-(L)1 blockers (i.e., atezolizumab or bivalent PD-L1 mAbs based on the anti-PD-L1 arm S79). Due to the lack of a PD-(L)1 blocking arm, the CD28 monovalent bsAb cannot induce the reporter system by itself and does not induce a luminescent signal.

[0208] This reporter assay confirms that in the presence of T cell signal 1 (provided by aAPC) and PD-L-1, the PD-L1xCD28 bispecific antibody of the invention can enhance T cell responses by inhibiting PD-1 / PD-L1 interaction, while still delivering costimulatory signal 2 to the T cell.

[0209] Example 4: T-cell dependent cellular cytotoxicity (TDCC) mediated by PD-L1 x CD28 bispecific antibodies TDCC of PD-L1 and CEA double-positive cell lines The T cell-dependent cytotoxicity (TDCC) of PD-L1 / CEA double-positive cell lines induced by the PD-L1xCD28 bispecific antibody of the present invention was evaluated in combination with a CEAxCD3 bsAb using human PBMCs as effector cells.

[0210] Target cells were washed twice with PBS and then detached with trypsin or cell dissociation solution. After a centrifugation step, cells were resuspended in assay medium, adjusted to the required concentration, and seeded into 96-well plates.

[0211] Effector cells were human peripheral blood mononuclear cells (PBMCs) isolated from buffy coats derived from healthy human donors using SepMate™ Tubes (Stemcell Technologies) with Lymphoprep™ buffer (Stemcell Technologies).

[0212] For the TDCC assay, PBMCs were added to target cells at different final E:T ratios (10:1, 3:1, 1:1, and 1:3). A dose range of CEA x CD3 and a fixed dose of the PD-L1 x CD28 antibody of the present invention (2.5 μg / mL) were added to pre-seeded target cells and effector cells. Single-agent AI3S79 / N was used as a negative control (no CEA x CD3 = no T cell signal 1). Target cell killing is assessed by quantifying the number of viable, adherent cells in the culture using Promega's CellTiter-Glo® (G7570) after 6 days of incubation at 37°C and 5% CO2. TDCC curves for each E:T ratio (Figure 4) were plotted using GraphPad Prism 9.

[0213] The PD-L1 × CD28 bsAb synergized with the CEA × CD3 bsAb to kill PD-L1 / CEA double-positive HPAC target cells, particularly at low E:T ratios (Figure 4, right panel). At optimal E:T ratios (i.e., 10:1), the CD3 bsAb consistently induced target cell killing. With decreasing E:T ratios, its activity declined to the point where it was unable to completely induce target cell killing as a single agent (i.e., E:T ratio = 1:3). However, along the E:T ratio gradient, the addition of AI3S79 / N enhanced the activity of the CD3 bsAb, and specific lysis as high as 50% was observed even at the unfavorable E:T ratio of 1:3. Importantly, in the absence of CEA×CD3, PD-L1×CD28 bsAb alone did not induce killing, highlighting the importance of primary T cell stimulation (signal 1) for the activity of PD-L1×CD28 bsAb.

[0214] Upregulation of T cell activation markers during killing of PD-L1 / CEA-expressing tumor cells induced by the combination of CEA×CD3 and PD-L1×CD28 bsAb The killing of CEA-positive tumor cells induced by the CEAxCD3 bsAb is based on T cell activation. The activation state of T cells can be further increased by CD28 costimulation. Therefore, the ability of PD-L1xCD28κλ bodies to enhance T cell activation in the presence of appropriate signals was quantified by flow cytometry using antibodies that recognize specific T cell activation markers, such as CD25 (a late activation marker).

[0215] To assess the activation status of T cells at the end of the killing assay (detailed in Example 4a), the following procedure was applied: Suspended cells (containing both CD4+ and CD8+ T cells) were transferred to a new V-bottom 96-well plate. The supernatant was removed by centrifugation, and the cells were washed twice with cold FACS buffer (PBS 2% BSA, 0.1% NaN3) before incubation with Fc-blocking reagent (BD Biosciences) for 15 minutes at 4°C. After washing twice with FACS buffer, the cells were incubated with the following antibodies for 15 minutes at 4°C: anti-CD8-PerCP-Cy 5.5 (BioLegend), anti-CD25-PE (BioLegend), and anti-CD4-APC (ThermoFisher). The cells were washed and analyzed by flow cytometry using a Cytoflex Platform (Beckman Coulter). Data were analyzed using FlowJo™ v10 software (BD Life Sciences). The results of quantification of T cell activation from the TDCC experiments shown in FIG. 4 are shown in FIG.

[0216] T cell activation was measured by quantifying the late activation marker CD25 on the surface of both CD4+ and CD8+ T cells (top and bottom rows in Figure 5, respectively) at different E:T ratios. Compared with CEAxCD3 treatment alone, the combination of CEAxCD3 with AI3S79 / N activated both CD4+ and CD8+ T cells to a greater extent, resulting in much brighter CD25 staining for the combination treatment (up to 47-fold higher MFI signal for the combination compared with CD3 bsAb alone). Synergy between the CD3 and CD28 bsAbs was best observed at high E:T ratios, but even at the unfavorable 1:3 E:T ratio, T cells showed brighter CD25 staining (13-fold higher MFI for the combo compared with CD3 bsAb alone at the highest antibody concentration).

[0217] Effect of CD28 costimulation mediated by PD-L1×CD28 bsAb on T cell proliferation in the presence of CEA×CD3 bsAb AI3S79 / N was analyzed for its ability to enhance the effect of CEA×CD3 on inducing T cell proliferation in the presence of PD-L1 / CEA-positive tumor target cells. Freshly isolated human PBMCs were stained and washed using the CellTrace Violet Cell Proliferation Kit (ThermoFischer Scientific) according to the manufacturer's instructions and co-cultured with target cells at different E:T ratios in the presence of a range of doses of CEA×CD3 and a fixed dose of 2.5 μg / mL of PD-L1×CD28 bsAb. After co-culture, effector cells were harvested, washed, and stained with appropriate viability markers to exclude dead cells and with anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (BioLegend, 301032) to identify the target population. T cell proliferation rates were calculated by measuring the level of CellTrace Violet staining intensity on live CD4+ or CD8+ T cells by flow cytometry using a CytoFLEX (Beckman Coulter). Data were evaluated using FlowJo software and plotted using GraphPad Prism (Figure 6).

[0218] The percentages of proliferative CD4+ and CD8+ T cells at different E:T ratios are shown in the top and bottom rows of Figure 6, respectively. The ability of the CEA x CD3 bsAb to induce T cell proliferation was enhanced by the addition of AI3S79 / N, with CD4 T cells being more positively affected by the combination treatment than CD8 T cells. Depending on the T cell activation state, T cell proliferation is best observed at high E:T ratios; however, even at the unfavorable 1:3 E:T ratio, T cells exhibited a higher proliferative potential when treated with the combination (up to 35-fold higher percentage of proliferative T cells with the combo compared to the CD3 bsAb alone).

[0219] Example 5: In vivo efficacy study of AI3S79 / N in treating MC38-hPD-L1 engrafted into CD28 HuGEMM mice This efficacy study aimed to evaluate the antitumor efficacy of AI3S79 / N as a monotherapy. The experimental design is summarized in Figure 7A. Briefly, MC38-hPD-L1 HuCELL (MC38 cells constitutively expressing human PD-L1 on their cell surface; Crown Bioscience) were subcutaneously implanted into CD28 HuGEMM mice (an immunocompetent chimeric mouse model engineered to express humanized CD28 instead of mouse CD28; Crown Bioscience). When the mean tumor volume approached 100 mm3 (7 days after implantation), mice were randomized and treated weekly with vehicle, atezolizumab [5 mg / kg, bivalent anti-PD-L1 antibody], or AI3S79 / N [10 mg / kg, PD-L1 × CD28, monovalent binding of each target] for a total of three injections (Figure 7A). Tumors were measured twice weekly with digital calipers until the experimental endpoint (tumor volume = 3000 mm3). Tumor volume was calculated using the formula (length × width2) × 0.5.

[0220] In contrast to atezolizumab, which produced only limited tumor growth inhibition compared with vehicle controls, AI3S79 / N resulted in tumor regression in all mice (Figure 7B). Comparison of tumor volumes at the end of the study (day 28 after treatment initiation) highlights the statistically stronger antitumor activity of AI3S79 / N compared with atezolizumab (Figure 7C). Tumor growth kinetics for individual mice are shown for vehicle (Figure 7D), atezolizumab (Figure 7E), and AI3S79 / N (Figure 7F). Notably, six of ten mice treated with AI3S79 / N were considered tumor-free at the end of the study (day 28), and two additional mice had small, regressing tumors. Normal weight gain was observed after the treatment cycle, and no clinical signs of toxicity were observed (Figure 8A). Cytokine secretion was measured 6 hours after the first injection of either vehicle, atezolizumab (5 mg / kg), or AI3S79 / N (10 mg / kg) (Figure 8B). Low cytokine concentrations were detected in mice treated with AI3S79 / N, consistent with those measured in atezolizumab-treated mice, suggesting a lack of immune system hyperactivation. Overall, AI3S79 / N was well tolerated in immunocompetent huCD28 mice, where it acts as a fully mouse cross-reactive antibody.

[0221] Because the majority of AI3S79 / N-treated mice survived primary tumor challenge with MC38-huPD-L1 (8 / 10 mice) (experimental protocol shown in Figure 7A), surviving animals were re-challenged with MC38 WT cells in the contralateral flank on day 59 (Figure 9). In contrast to naive mice, all of which succumbed to MC38 WT tumors, none of the re-challenged mice developed tumors, suggesting that AI3S79 / N induced immunological memory against MC38 cells.

[0222] Example 6: AI3S79 / N is not a CD28 superagonist The AI3 anti-CD28 arm was previously shown to not be a superagonist in WO 2023170474. To exclude superagonist activity in the context of the bsAbs of the present invention, AI3S79 / N was tested in two different in vitro safety assays for its ability to induce T cell proliferation or T cell-mediated cytokine release in the absence of signal 1.

[0223] Wet and dry plate-coated T cell proliferation assay (Stebbings assay) The method was adapted from Stebbings et al. (2007). Briefly, 96-well polypropylene plates were coated overnight at 4°C or room temperature with 100 μl of antibody solution (wet coating) diluted to 10 μg / ml in PBS, followed by 50 μL of antibody solution in an unsealed, class II laminar flow cabinet (dry coating, allowing for evaporation of the buffer). After either coating procedure, the plates were washed twice with PBS. In parallel, PBMCs isolated from buffy coats obtained from healthy donors were stained using the CellTrace Violet Cell Proliferation Kit (ThermoFischer Scientific) according to the manufacturer's instructions. 100,000 stained PBMCs were added to the 96-well plate in a final volume of 200 μL / well and incubated at 37°C + 5% CO2 for 6 days. Cells were then harvested and stained for flow cytometry evaluation using anti-CD4-APC (ThermoFischer, 17-0049-41) and anti-CD8-PerCP-Cy5.5 (BioLegend, 301032), as detailed in Examples 2 and 4c. The proliferation rates of live CD4+ and CD8+ T cells were calculated by measuring the level of CellTrace Violet staining by flow cytometry using a CytoFLEX (Beckman Coulter), and results were evaluated by FlowJo software for both coating procedures. The anti-CD3 antibody OKT3 and the CD28 SA antibody TGN1412 served as positive controls, while the background proliferation rate of T cells was determined in the presence of an isotype control antibody.

[0224] As shown in Figure 13, the TGN1412 analog induced proliferation of both CD4+ and CD8+ subtype T cells under both wet and dry coating conditions, confirming its superagonist characteristics. In contrast, the AI3 anti-CD28 arm, neither as a monoclonal antibody (mAb AI3 / N) nor as part of the PD-L1xCD28 bsAb of the present invention (AI3S79 / N), induced proliferation of resting CD4+ and CD8+ T cells under both experimental conditions (wet and dry coating).

[0225] Pre-culture of PBMCs at high cell density to increase the sensitivity of T cell responses (recovery protocol) To confirm the lack of superagonism of AI3N79 / N in a more physiologically relevant setting, AI3S79 / N was tested according to the "RESTORE" protocol (Romer et al. 2011). PBMCs from healthy donors were first pre-cultured at high density (HDP) to induce functional maturation of both monocytes and T cells, and then cultured at normal density in the presence of soluble antibody.

[0226] Under these conditions, the TGN1412 analog induced dose-dependent secretion of IL-2 in all three donors tested, although to different degrees, reflecting donor variability (Figure 10A). As expected, the superagonist activity of TGN1412 was not apparent when PBMCs were cultured at high density and not subjected to a preculture step (fresh PBMCs). Importantly, AI3S79 / N did not induce any IL-2 secretion, even when added to HDP PBMCs (Figure 10B).

[0227] Example 7: T cell activation in the mixed lymphocyte reaction in the presence of T cell superantigens The mixed lymphocyte reaction (MLR) is an in vitro assay in which immune cells from two individuals are co-cultured to induce the "non-self" recognition required for allogeneic T cell activation and proliferation. In this assay, immune checkpoint inhibitors (ICIs), such as anti-PD1 or anti-PD-L1 mAbs, enhance the MLR, as measured by increased cytokine secretion. To assess the ability of AI3S79 / N to enhance T cell responses in the presence of appropriate stimuli, we developed a variant of the MLR in which CD4+ T cells (responders) and monocyte-derived DCs (stimulators) were co-cultured in the presence of the Staphylococcus aureus T cell superantigen SEA (Figure 11A).

[0228] As expected, both atezolizumab and nivolumab enhance IL-2 secretion in this MLR assay (Figure 11B). The profiles of the two antibodies are not identical, likely reflecting the fact that atezolizumab blocks only the PD-L1 / PD-1 interaction, whereas nivolumab can block both the PD-L1 / PD-1 and PD-L2 / PD-1 interactions by binding to PD-1. S79 mAb, a bivalent monoclonal antibody with the same anti-PD-L1 arm present in AI3S79 / N, induces IL-2 secretion at levels comparable to atezolizumab. Notably, AI3S79 / N is the most active molecule in this assay because it not only blocks PD-L1 but also provides a costimulatory signal to CD4+ T cells, resulting in better T cell activation (Figure 11B).

[0229] Example 8: In vivo safety evaluation in a humanized mouse model susceptible to CD28-mediated cytokine release syndrome We evaluated the in vivo safety of AI3S79 / N using a humanized mouse model susceptible to CD28-mediated CRS. Briefly, nonobese diabetic (NOD) scid gamma (NSG)-major histocompatibility complex (MHC) I / II double knockout (KO) mice were irradiated and transplanted with human PBMCs preselected for sensitivity to an anti-CD28 superagonist Ab. Six days later, mice were administered the Ab. A TGN1412 analog was used as a positive control. As a negative control, mice were injected with phosphate-buffered saline (PBS) vehicle. Mice were observed daily for body weight monitoring and CRS score assessment. Mice that reached endpoint based on body weight (>20%) or CRS score (>3) were euthanized. As shown in Figure 12, treatment with a range of doses of AI3S79 / N was tolerated by these mice, in contrast to mice receiving 2 mg / kg TGN1412, which all showed significant weight loss after drug administration on day 6.

[0230] Example 9: In vitro single agent activity of AI3S79 / N in the CMV recall assay To assess the single-agent activity of AI3S79 / N in a physiologically relevant in vitro context, tumor cells were transfected with an NLV peptide (HLA-A peptide derived from CMV lower matrix protein pp65). *In this assay, tumor cells are artificially loaded with a pool of CMV-derived peptides containing the 02-restricted epitope. These tumor cells are then co-cultured with PBMCs from donors known to contain NLV-specific T cells. When these T cells again encounter NLV peptides presented in the context of MHC molecules, they become activated, begin to proliferate, and specifically kill NLV-loaded target cells, a process predicted to be amplified by the PD-L1 x CD28 bsAb providing signal 2 for T cell activation. Killing is quantified by measuring remaining viable target cells using the CellTiter-Glo® luminescent cell viability assay, while killing is quantified by measuring remaining viable target cells using fluorescently labeled HLA-A. * The expansion of NLV-specific T cells was quantified using 02-NLV tetramers.

[0231] The PD-L1-positive MDA-MB-231 cell line was first loaded with a pool of CMV-derived peptides and then cocultured with two different PBMC donors containing CMV-reactive T cells. For both donors, AI3S79 / N-induced dose-dependent killing of NLV-loaded target cells was observed (Figure 14A). Correspondingly, AI3S79 / N-induced dose-dependent expansion of NLV-specific T cells was also observed (Figure 14B). Importantly, when target cells were not loaded with CMV peptides and therefore unable to provide T cell signal 1, they were not killed, and T cells did not proliferate in the presence of AI3S79 / N (non-CMV-loaded control on both panels).

[0232] Example 10: In vivo efficacy study in combination with T cell engager (TCE) in a TCE-resistant model In vivo efficacy study of AI3S79 / N in combination with CEA x CD3 TCE in the treatment of HPAC tumors transplanted into PBMC-humanized NOG mice The synergistic effect between AI3S79 / N and CEAxCD3 observed in vitro (Example 4) was confirmed in vivo in a model in which the human pancreatic adenocarcinoma epithelial cell line HPAC was subcutaneously implanted into PBMC-humanized NOG mice (Figure 15A). Briefly, 3x106 HPAC cells were subcutaneously implanted into NOG mice (Taconic). The average tumor volume was 100 mm 3 When it reaches 10 x 10 6 Mice were humanized intravenously with PBMC cells. Two donors were used, and each group contained four mice per donor for a final total of eight mice per group. Treatment was initiated when the mean tumor volume was 300 mm 3 The study began on day 15, approaching tumor volume, and consisted of three injections of vehicle, CEAxCD3 (3 mg / kg), AI3S79 / N (5 mg / kg), or a combination of both CEAxCD3 and AI3S79 / N (administered at 3 mg / kg or 1 mg / kg, respectively), on days 15, 21, and 28. The endpoint of the study (tumor volume = 1500 mm) was 3 Tumors were measured three times a week by digital calipers until the onset of GvHD symptoms or until the appearance of GvHD symptoms. Tumor volume was calculated using the formula (length × width). 2 ) × 0.5.

[0233] The experiment had to be stopped due to the appearance of GvHD symptoms on day 31. Before that, and from day 24 onwards, the mean tumor volume in the combination group began to decrease (Fig. 15B), and tumor regression or cessation was observed in all mice treated with the combination (Fig. 15C), suggesting that AI3S79 / N synergized with CEAxCD3 to induce a strong antitumor response.

[0234] In vivo efficacy study of AI3S79 / N in combination with CEA×CD3 TCE in the treatment of HPAF-II tumors in fully humanized BRGSF-HIS mice The synergy between AI3S79 / N and CEAxCD3 was further evaluated in a second model in which the human pancreatic adenocarcinoma cell line HPAF-II was subcutaneously implanted into fully humanized BRGSF-HIS mice. Briefly, 1.5x10 6CEA / PD-L1 HPAF-II cells were transplanted into hFlt-3L-boosted BRGSF-HIS mice (genOway). Each group contained two mice for each of the six hematopoietic stem cell donors used, for a total of 12 mice per group. Treatment began when the mean tumor volume reached 65 mm (day 8 post-transplant). Mice received three doses of vehicle, CEA x CD3 (10 mg / kg), or the combination of CEA x CD3 + AI3S79 / N (10 + 10 mg / kg) on ​​days 8, 11, and 14. The endpoint of the experiment (tumor volume = 1500 mm 3 Tumors were measured 2-3 times a week using a digital caliper until tumor volume reached 1000 m / s. Tumor volume was calculated using the formula (length × width). 2 ) × 0.5.

[0235] In contrast to CEAxCD3 alone, which showed no antitumor activity in this model, the combination of CEAxCD3+AI3S79 / N resulted in reduced tumor progression (Figure 16A). Tumor growth kinetics in individual mice are shown in Figure 16B.

[0236] Example 11: Single and repeat dose PK and tolerability studies in cynomolgus monkeys A study was conducted in cynomolgus monkeys to investigate the PK and tolerability of AI3S79 / N after single and repeated intravenous injections. The study included three groups: (a) a single iv injection of AI3S79 / N at 0.5 mg / kg, (b) a single iv injection of AI3S79 / N at 10 mg / kg, and (c) repeated (n=2) iv injections of AI3S79 / N at 10 mg / kg, followed by histopathological examination.

[0237] Pharmacokinetics A validated generic pharmacokinetic assay based on Meso-Scale Discovery (MSD) technology was used to quantify serum levels of AI3S79 / N in cynomolgus monkey serum. Briefly, biotinylated anti-human CH2 was coated onto a streptavidin MSD plate to capture AI3S79 / N from the sample. Detection was enabled by SulfoTag-conjugated anti-human CH2. Signals were acquired using a Meso Sector S600 instrument, and concentrations were extrapolated against a standard curve of AI3S79 / N. Upon quantification of AI3S79 / N in the samples, pharmacokinetic data evaluation was performed according to standard non-compartmental analysis using SAS software version 9.4.

[0238] The AI3S79 / N concentration versus time curves obtained after bioanalytical testing are shown in Figure 17. AI3S79 / N PK was close to dose proportional, with mean C max , AUC inf , and AUC 0-168h Values ​​were 13-23 times higher after administration of 10 mg / kg compared with 0.5 mg / kg.

[0239] In monkeys treated twice, the mean C max and AUC 0-168h Accumulation of AI3S79 / N was observed after repeated injections at 10 mg / kg, as values ​​were 1.4- and 1.6-fold higher on day 8 compared to day 1, respectively.

[0240] Tolerability Single and repeated intravenous administration of AI3S79 / N was well tolerated. No mortality or abnormal clinical signs were observed throughout the study. No treatment-related changes were reported in body weight, food intake, clinical pathology, or postmortem examination.

[0241] Quantification of serum cytokines (IFNγ, TNFα, IL-2, IL-6, IL-8, and IL-10) using MesoScale Discovery (MSD) was performed in samples collected throughout the study from selected groups ( FIG. 18 ). No increases in IFNγ, TNFα, IL-2, IL-8, and IL-10 levels were observed after single or repeated treatment with AI3S79 / N at both dose levels. A transient increase in IL-6 was observed at 4 or 8 hours after the first dose. The increase in IL-6 levels after the second dose of AI3S79 / N was less severe than after the first dose for all three monkeys.

[0242] C-reactive protein (CRP) levels in the serum of treated monkeys were measured by ELISA (Life Diagnostics Inc.). A transient, minimal increase in CRP was observed in most animals after the first injection, beginning 8 hours after administration, peaking at 24 hours, and resolving approximately 4–5 days later. In animals receiving two doses, a similar transient increase in CRP occurred after the second injection in only one animal, and a delayed, lower maximum level occurred in another, suggesting a lower AI3S79 / N-associated increase in this marker after repeated injections (Figure 19).

[0243] Overall, changes in serum biomarkers were limited to IL-6 and CRP, were mild in magnitude, and were transient: within 24–48 h (IL-6) or 2–3 days (CRP), levels returned to pre-treatment levels for all animals not receiving anti-inflammatory medication.

[0244] In conclusion, single and repeated intravenous administration of AI3S79 / N was well tolerated. No clinical signs or abnormalities in food intake were observed, and there were no significant treatment-related changes in body weight, clinical pathology, or anatomic pathology.

[0245] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0246] References Correnti, Colin E., George S. Laszlo, Willem J. de van der Schueren, Colin D. Godwin, Ashok Bandaranayake, Melanie A. Busch, Chelsea J. Gudgeon, et al. 2018. “Simultaneous Multiple Interaction T-Cell Engaging(SMITE)Bispecific Antibodies Overcome Bispecific T-Cell Engager(BiTE)Resistance via CD28 Co-Stimulation”. Leukemia 32(5):1239-43. https: / / doi.org / 10.1038 / s41375-018-0014-3. Holliger, Philipp, Oliver Manzke, Mary Span, Robert Hawkins, Bernd Fleischmann, Liu Qinghua, Jurgen Wolf, Volker Diehl, Olivier Cochet, and Greg Winter. 1999. “Carcinoembryonic Antigen(CEA)-Specific T-Cell Activation in Colon Carcinoma Induced by Anti-CD3 x Anti-CEA Bispecific Diabodies and B7 x Anti-CEA Bispecific Fusion Proteins”.Cancer Research 59(12):2909-16. Romer,Paula S.,Susanne Berr,Elita Avota,Shin-Young Na,Manuela Battaglia,Ineke ten Berge,Hermann Einsele,and Thomas Hunig.2011.「Preculture of PBMCs at High Cell Density Increases Sensitivity of T-Cell Responses,Revealing Cytokine Release by CD28 Superagonist TGN1412」.Blood 118(26):6772-82.https: / / doi.org / 10.1182 / blood-2010-12-319780. Skokos,Dimitris,Janelle C.Waite,Lauric Haber,Alison Crawford,Aynur Hermann,Erica Ullman,Rabih Slim,et al.2020.「A Class of Costimulatory CD28-Bispecific Antibodies That Enhance the Antitumor Activity of CD3-Bispecific Antibodies」.Science Translational Medicine 12(525):eaaw7888.https: / / doi.org / 10.1126 / scitranslmed.aaw7888. Stebbings,Richard,Lucy Findlay,Cherry Edwards,David Eastwood,Chris Bird,David North,Yogesh Mistry,et al.2007.「Cytokine Storm」in the Phase I Trial of Monoclonal Antibody TGN1412:Better Understanding the Causes to Improve PreClinical Testing of Immunotherapeutics」.The Journal of Immunology 179(5):3325-31.https: / / doi.org / 10.4049 / jimmunol.179.5.3325. Waite,Janelle C.,Bei Wang,Lauric Haber,Aynur Hermann,Erica Ullman,Xuan Ye,Drew Dudgeon,et al.2020.「Tumor-Targeted CD28 Bispecific Antibodies Enhance the Antitumor Efficacy of PD-1 Immunotherapy」.Science Translational Medicine 12(549):eaba2325.https: / / doi.org / 10.1126 / scitranslmed.aba2325.

Claims

1. below: a first antigen-binding domain that binds to programmed death-ligand 1 (PD-L1), 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; and ii. a first light chain variable region having a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

15. a first antigen-binding domain comprising: a second antigen-binding domain that binds to CD28, 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. and a second antigen-binding domain comprising: A bispecific antibody comprising:

2. the second antigen-binding domain comprising: a second light chain variable region, a) CDR1 comprising the amino acid sequence of SEQ ID NO: 18; CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and having a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or b) CDR1 comprising the amino acid sequence of SEQ ID NO: 23; CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and having a CDR3 comprising the amino acid sequence of SEQ ID NO: 25; Second light chain variable region 2. The bispecific antibody of claim 1 , comprising:

3. 10. The bispecific antibody of claim 9, wherein the first and second heavy chain variable regions comprise the amino acid sequence of SEQ ID NO:

9.

4. 10. The bispecific antibody of claim 9, wherein the first and second heavy chains comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

5. 2. The bispecific antibody of claim 1 , wherein the first light chain variable region of portion ii comprises the amino acid sequence of SEQ ID NO:

16.

6. 2. The bispecific antibody of claim 1 , wherein the first light chain of portion ii comprises the amino acid sequence of SEQ ID NO:

17.

7. 1. the second light chain variable region of portion (a) comprises the amino acid sequence of SEQ ID NO: 21; 2. The second light chain variable region of portion (b) comprises the amino acid sequence of SEQ ID NO: 26; The bispecific antibody of claim 2.

8. 1. The second light chain of portion (a) comprises the amino acid sequence of SEQ ID NO: 22; 2. The second light chain of part (b) comprises the amino acid sequence of SEQ ID NO: 27; The bispecific antibody of claim 2.

9. 10. The bispecific antibody of claim 1, wherein the first light chain is kappa and the second light chain is lambda.

10. 10. The bispecific antibody of claim 1, wherein the first light chain is lambda and the second light chain is kappa.

11. An Fc domain comprising one or more amino acid substitutions that reduce binding to activating Fc receptors and / or reduce effector function.

2. The bispecific antibody of claim 1 , comprising:

12. 12. The bispecific antibody of claim 11 , wherein the amino acid substitutions comprise L234A and L235A substitutions.

13. 12. The bispecific antibody of claim 11 , wherein the amino acid substitution comprises a P329A, P329G, or P329R substitution.

14. 10. The bispecific antibody of claim 1, wherein the antibody has an IgG isotype.

15. 10. The bispecific antibody of claim 1, wherein the antibody is human.

16. 10. The bispecific antibody of any one of the preceding claims, wherein the composition enables PD-L1-dependent T cell activation.

17. A composition comprising a bispecific antibody according to any one of the preceding claims.

18. 20. A method of reducing tumor cell proliferation and / or enhancing killing of said tumor cells, comprising contacting said cells with the composition of claim 17.

19. 20. A method of treating cancer in a subject, comprising administering to the subject the composition of claim 17.

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