Tumor-targeting agonist CD28 antigen-binding molecules

Abstract

The present invention relates to tumor-targeting bispecific agonist antigen-binding molecules characterized by monovalent binding to CD28, methods for their production, pharmaceutical compositions containing these antibodies, and methods of using them.

Description

[Technical Field]

[0001] The present invention relates to tumor-targeting bispecific agonist CD28 antigen-binding molecules characterized by monovalent binding to CD28, methods for their preparation, pharmaceutical compositions containing these molecules, and their use as immunomodulatory agents in the treatment of cancer. [Background technology]

[0002] Cancer immunotherapy is becoming an increasingly effective treatment option, capable of producing dramatic and durable responses in cancer types such as melanoma, non-small cell lung cancer, and renal cell carcinoma. This is primarily driven by the success of several immune checkpoint blockades, including anti-PD-1 (e.g., Merck's Keytruda; BMS's Opdivo), anti-CTLA-4 (e.g., BMS's Yervoy), and anti-PD-L1 (e.g., Roche's Tecentriq). While these agents are likely to serve as standard of care or as the backbone of combination therapies for many cancer types, only a small fraction of patients (<25%) benefit from such therapies. Furthermore, various cancers (e.g., prostate cancer, colorectal cancer, pancreatic cancer, sarcoma, non-triple-negative breast cancer) exhibit primary resistance to these immunomodulatory agents. Several reports have indicated that the absence of pre-existing antitumor T cells contributes to non-response or inadequate responses in some patients. In summary, despite the impressive anti-cancer effects of existing immunotherapies, there is a clear medical need to address the large cancer patient population and to develop therapies aimed at inducing and enhancing novel tumor-specific T cell responses.

[0003] CD28 is an established member of a subfamily of costimulatory molecules characterized by a paired V-set immunoglobulin superfamily (IgSF) domain linked to a single transmembrane and cytoplasmic domain containing key signaling motifs (Carreno and Collins, 2002). Other members of the subfamily include ICOS, CTLA-4, PD1, PD1H, TIGIT, and BTLA (Chen and Flies, 2013). CD28 expression is restricted to T cells and is common on the majority of all naive and antigen-experienced subsets, including those expressing PD-1 or CTLA-4. CD28 and CTLA-4 are highly homologous and compete for binding to the same B7 molecules, CD80 and CD86, which are expressed on dendritic cells, B cells, macrophages, and tumor cells (Linsley et al., 1990). The higher affinity of CTLA-4 for the B7 family of ligands allows it to outcompete CD28 for ligand binding and suppress effector T cell responses (Engelhardt et al., 2006). In contrast, PD-1 has been shown to inhibit CD28 signaling by partially dephosphorylating the cytoplasmic domain of CD28 (Hui et al., 2017). Ligation of CD28 by CD80 or CD86 on the surface of professional antigen-presenting cells is strictly required for the functional de novo priming of naive T cells, subsequent clonal expansion, cytokine production, target cell lysis, and the formation of long-lived memory. Binding of the CD28 ligand also promotes the expression of inducible costimulatory receptors such as OX-40, ICOS, and 4-1BB (reviewed in Acuto and Michel, 2003). Upon ligation of CD28, a disulfide-linked homodimer, the membrane-proximal YMNM motif and the distal PYAP motif, has been shown to form a complex with several kinases and adaptor proteins ( Boomer and Green, 2010 ).These motifs are important for the induction of IL2 transcription, which is mediated by CD28-dependent activation of NFAT, AP-1, and NFκB family transcription factors (Fraser et al., 1991; June et al., 1987; Thompson et al., 1989). However, additional, less well-characterized sites for phosphorylation and ubiquitination are found within the cytoplasmic domain of CD28. As reviewed by Esensten et al., 2016, CD28-initiated pathways play an important role in promoting the proliferation and effector function of conventional T cells. CD28 ligation also promotes the anti-inflammatory function of regulatory T cells. CD28 costimulates T cells, in part by enhancing signals from the T cell receptor, but has also been shown to mediate intrinsic signaling events (Acuto and Michel, 2003; Boomer and Green, 2010; June et al., 1987). Signals specifically triggered by CD28 regulate many important aspects of T cell function, including downstream protein phosphorylation and other post-translational modifications (e.g., PI3K-mediated phosphorylation), transcriptional changes (e.g., Bcl-xL expression), epigenetic changes (e.g., IL-2 promoter), cytoskeletal remodeling (e.g., microtubule-organizing center orientation), and altered glycolytic rates (e.g., glycolytic flux). CD28-deficient mice exhibit impaired responses to infectious pathogens, allograft antigens, graft-versus-host disease, contact hypersensitivity, and asthma (Acuto and Michel, 2003). Lack of CD28-mediated costimulation results in reduced T cell proliferation in vitro and in vivo, severe inhibition of germinal center formation and immunoglobulin isotype class switching, reduced T helper (Th) cell differentiation, and expression of Th2-type cytokines. CD4-dependent cytotoxic CD8+ T cell responses are also affected. Importantly, CD28-deficient naive T cells exhibited reduced proliferative responses, particularly at lower antigen concentrations. A growing body of literature supports the idea that engaging CD28 on T cells has anti-tumor potential.Recent evidence demonstrates that the anti-cancer effects of PD-L1 / PD-1 and CTLA-4 checkpoint inhibitors are CD28 dependent (Kamphorst et al., 2017; Tai et al., 2007). Clinical studies investigating the therapeutic efficacy of CTLA-4 and PD-1 blockade have shown very promising results in patients with advanced melanoma and other cancers. Furthermore, the infusion of genetically engineered T cells expressing an artificial chimeric T cell receptor containing an extracellular antigen recognition domain fused to an intracellular TCR signaling domain (CD3z) and an intracellular costimulatory domain (CD28 and / or 4-1BB domain) has shown high and durable response rates in B-cell and other cancers.

[0004] CD28 agonist antibodies can be divided into two categories: (i) CD28 superagonist antibodies and (ii) CD28 conventional agonist antibodies. Activation of naive T cells typically requires both T cell antigen receptor (TCR, signal 1) engagement and costimulatory signaling by CD28 (signal 2). CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies that can autonomously activate T cells without explicit T cell receptor engagement (Huenig, 2012). In rodents, CD28SA activates conventional regulatory T cells. CD28SA antibodies have been therapeutically effective in multiple models of autoimmunity, inflammation, and transplantation. However, a phase I study of the human CD28SA antibody TGN1412 resulted in a life-threatening cytokine storm in 2006. Follow-up studies suggest that the toxicity was caused by dosing errors due to differences in CD28 responsiveness of human T cells and T cells in preclinical animal models. TGN1412 is currently being reevaluated in an open-label, multicenter, dose-escalation study in patients with RA and patients with metastatic or unresectable advanced solid malignancies. Conventional agonist antibodies for CD28, such as clone 9.3, mimic the CD28 natural ligand and can enhance T cell activation only in the presence of a T cell receptor signal (signal 1). Published evidence indicates that the binding epitope of an antibody significantly influences whether an agonist antibody is a superagonist or a conventional agonist (Beyersdorf et al., 2005). The superagonist TGN1412 binds to a lateral motif on CD28, whereas the conventional agonist molecule 9.3 binds near the ligand-binding epitope. As a result of their different binding epitopes, superagonist and conventional agonist antibodies differ in their ability to form linear complexes of CD28 molecules on the surface of T cells. Specifically, TGN1412 can efficiently form linear arrays of CD28, presumably resulting in aggregated signaling components sufficient to overcome the threshold for T cell activation, whereas the conventional agonist 9.3 results in complexes with less linear structures.Attempts to convert conventional agonist binding factors based on the 9.3 clone have been previously published using recombinant bispecific single-chain antibodies against melanoma-associated proteoglycans and CD28 (Otz et al., 2009). The reported bispecific single-chain antibodies were reported to exert "superagonist" activity despite the use of the conventional CD28 agonist binding factor 9.3, based on the inherent tendency of bispecific single-chain antibodies to form multimeric constructs.

[0005] It has been found that better T cell activation can be achieved by combining a T cell bispecific antibody (TCB), such as a critical anti-CD3 bispecific antibody, i.e., CEA-TCB, with an agonistic anti-CD28 molecule. Given that CD28 is expressed at baseline on T cells in various tumor indications (Lavin et al., 2017; Tirosh et al., 2016; Zheng et al., 2017), and that activation of CD28 signaling enhances T cell receptor signaling, the combination of a TCB molecule with a tumor-targeted CD28 molecule is expected to act synergistically to induce potent and long-lasting antitumor responses. Therefore, we describe herein a novel tumor-targeted agonistic CD28 molecule that exhibits synergy with a TCB and requires CD28 binding monovalency for strict tumor-targeting dependency in the presence of a TCB signal.

[0006] Immunotherapy for solid tumors Treatment of solid tumors is an ongoing challenge, with little progress over the past few years. Treatment typically involves surgery combined with chemotherapy and / or radiation therapy. While a few new treatments have been developed recently, further improvements are still needed to increase survival rates and improve the quality of life of patients with solid tumors. Solid tumors rarely express a single tumor-specific antigen. In most solid tumors, it is more common to find tumor-associated antigens (TAAs) that are enriched on the tumor but also expressed at very low levels on normal tissues. TAAs are preferably presented on the surface of solid tumor cells or on cells in the tumor stroma. This is the case for many frequently targeted TAAs for solid tumors, including fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), folate receptor alpha (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and p95HER2. Additional TAAs include HER3, EpCAM, TPBG (5T4), mesothelin, MUC1, and PSMA. Thus, bispecific agonist CD28 antigen-binding molecules containing antigen-binding domains that specifically bind to tumor-associated antigens are primarily directed to the tumor surface or tumor microenvironment, specifically activating T cells in the vicinity of the tumor while systemic activation can be avoided.

[0007] Reasons for targeting CD28 agonism for B-cell malignancies Non-Hodgkin's lymphoma (NHL) is one of the leading causes of cancer death in the United States and Europe. Follicular lymphoma (FL) is indolent and progressive, with a median survival of 8–10 years, and patients with advanced clinical stages are usually incurable. Similarly, in 2%–3% of patients each year, the FL phenotype can transform into aggressive large cell lymphoma, a critical event in the course of the disease and associated with increased lymphoma-related mortality. Mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL) are more aggressive, with a median survival of only 6 months if untreated. Despite significant advances in immunotherapy that have prolonged progression-free survival, the lack of curative outcomes for many patients with both indolent and aggressive NHL subtypes remains an unmet medical need. Over the past few years, significant prolonged survival has been observed in DLBCL, particularly with the addition of the anti-CD20 monoclonal antibody rituximab (Rituxan®, MabThera®) to potent cytotoxic chemotherapy regimens. However, despite the intended curative nature of conventional treatments for previously untreated DLBCL, the majority of patients will ultimately relapse. Similarly, advanced FL remains largely incurable with current SoCs and is characterized by repeated relapses and increasingly shorter remissions. Currently, many new-generation monoclonal antibodies are in different preclinical and clinical stages of evaluation to further improve outcomes for NHL patients and overcome mechanisms of rituximab resistance. High-dose chemotherapy with autologous stem cell support or allogeneic stem cell transplantation offers a curative option for a small proportion (10%) of patients with relapsed / refractory (r / r) DLBCL and is associated with substantial treatment-related mortality. Other approaches currently under development for the treatment of NHL include molecularly targeted compounds such as venetoclax and BET inhibitors. Recently approved novel agents include lenalidomide, idelalisib, and copanlisib. Chimeric antigen receptor (CAR) T-cell therapy has been approved for the treatment of aggressive forms of r / r B-NHL, but this therapy is only available in limited settings and can be associated with fatal neurological events and cytokine release syndrome (CRS).Bispecific antibody constructs that redirect cytotoxic cell lysis toward malignant B cells are currently under development and have shown very promising efficacy against NHL. Chemotherapy-free treatments are envisioned for the future of NHL, likely based on bispecific antibodies or chimeric antigen receptor T cells (CAR T cells). CD28 agonists targeted against B-cell surface antigens in combination with immunotherapy could increase survival and / or cure rates for patients with B-cell malignancies without compromising their quality of life.

[0008] B cell surface antigens as targets for B cell malignancies A TAA associated with B-cell malignancies is a B-cell surface antigen. The human CD19 antigen is a 95-kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is classified as a type I transmembrane protein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. In normal cells, it is the most ubiquitously expressed protein in the B-lymphocyte lineage. CD19 expression is maintained in B-lineage cells that undergo neoplastic transformation; therefore, CD19 is useful in the diagnosis of leukemia and lymphoma using monoclonal antibodies (mAbs) and flow cytometry, as is the CD20 antigen. B-lineage leukemia and lymphoma rarely lose CD19 expression, and because it is not expressed in pluripotent stem cells, it has become a target for various immunotherapeutic agents, including immunotoxins. CD79 is a signaling component of the B-cell receptor, consisting of a covalent heterodimer containing CD79a (Igα, mb-1) and CD79b (Igβ, B29). CD79a and CD79b each contain an extracellular immunoglobulin (Ig) domain, a transmembrane domain, and an intracellular signaling domain, an immunoreceptor tyrosine-based activation motif (ITAM) domain, similar to other signaling proteins such as CD3 or activating Fcγ receptors. Thus, CD79a and CD79b are transmembrane proteins that constitute the signaling subunits of the B cell receptor (BCR). CD79b is a 39-kDa protein expressed exclusively on B cells and, in cooperation with CD79a, initiates the downstream signaling cascade of the BCR, leading to internalization of the BCR complex, its translocation to endosomes, and antigen presentation. In B cells, antigen-induced BCR clustering triggers tyrosine phosphorylation of the ITAMs of CD79a and CD79b by Src kinase. This leads to the recruitment and activation of a series of effector molecules belonging to the BCR signaling cascade, most notably SYK and BLNK. Further downstream, recruitment of PLCg2, Btk, and ERK promotes calcium flux and activates B cells, which are then ready to receive further co-activation signals that drive their proliferation and differentiation into memory or effector cells.During this process, B cells become robust APCs and release cytokines that can affect the outcome and quality of immune responses. In addition to its role in BCR signaling, the CD79 subunit is also essential for the transport and presentation of membrane-bound Ig from the endoplasmic reticulum to the cell surface. The average surface expression of CD79b on NHL is similar to that on normal B cells, but with a greater range. Given the expression of CD79b, it would be beneficial to generate therapeutic antibodies against the CD79b antigen that produce minimal or no antigenicity when administered to patients, especially for chronic treatment.

[0009] It has been found that better T cell activation can be achieved by combining a critical dose of an anti-CD3 bispecific antibody, i.e., a T cell bispecific antibody (TCB), such as a CD20 / CD3 bispecific antibody, with an agonistic anti-CD28 molecule. Given that CD28 is expressed at baseline on T cells in various tumor indications (Lavin et al., 2017; Tirosh et al., 2016; Zheng et al., 2017) and that activation of CD28 signaling enhances T cell receptor signaling, the combination of a T cell bispecific antibody and a bispecific agonistic CD28 antigen binding molecule targeting a B cell surface antigen is expected to act synergistically to induce potent and long-lasting antitumor responses. Therefore, we describe herein a novel bispecific agonistic CD28 antigen binding molecule targeting a B cell surface antigen that exhibits synergy with a TCB and requires monovalency of CD28 binding for strict tumor targeting dependence in the presence of a TCB signal.

[0010] Immunotherapy in multiple myeloma Multiple myeloma (MM) is one of the most common hematological malignancies, affecting approximately 75,000 new patients annually in the EU and the United States, with a significant unmet medical need. MM is characterized by terminally differentiated plasma cells secreting nonfunctional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide, as well as proteasome inhibitors such as carfilzomib or bortezomib, may remain the backbone of first-line therapy for MM (Moreau et al., 2016). However, these drugs do not specifically target diseased tumor cells, such as diseased plasma cells (PCs). Efforts have been made to selectively deplete plasma cells in MM. The lack of surface proteins that specifically mark plasma cells has hindered the development of antibody or cell-based therapies for MM. To date, there have been few successful biologics, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), with the caveat that both antigens are also expressed in other normal tissues, including hematopoietic and immune effector cells, potentially limiting their long-term clinical use. B-cell maturation antigen (BCMA), a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17), is expressed at significantly elevated levels in all patient MM cells but not in other normal tissues except normal plasma cells. BCMA-chimeric antigen receptor (CAR) T cells have already demonstrated significant clinical activity in patients with RRMM who have received at least three prior treatments, including proteasome inhibitors and immunomodulators. Further modalities, including anti-BCMA antibody-drug conjugates, have also achieved significant clinical responses in patients who have failed at least three prior therapies, including anti-CD38 antibodies, proteasome inhibitors, and immunomodulators (Cho et al., 2018). For example, one challenge with BCMA- or CD38-targeted therapies is the presence of high levels of soluble BCMA or CD38 in the serum of MM patients, which can reduce the amount of active drug a patient receives. An alternative could be a new target such as G protein-coupled receptor class C group 5 member D (GPRC5D), which is differentially expressed by plasma cells in multiple myeloma versus plasma cells from healthy donors and does not have a soluble form.GPRC5D has been reported to be associated with the prognosis and tumor burden of multiple myeloma patients (Atamaniuk, J. et al., 2012; and Cohen, Y. et al., 2013). GPRC5D is an orphan receptor with no known ligands and little known biology, both in men in general and in cancer specifically. The GPRC5D-encoding gene, mapped to chromosome 12p13.3, contains three exons and spans approximately 9.6 kb (Brauner-Osborne, H. et al., 2001). The first large exon encodes a seven-transmembrane domain. GPRC5D has been shown to be involved in keratinogenesis in animal hair follicles (Gao, Y. et al., 2016; and Inoue, S. et al., 2004). International Publication No. WO 2018 / 017786 A2 discloses GPRC5D-specific antibodies or antigen-binding fragments.

[0011] Rationale for targeting CD28 agonism to disease plasma cells in multiple myeloma CD28 agonism in multiple myeloma may exert distinct biological functions on each immune-mediated MM plasma cell. While CD28-mediated T cell coactivation is expected to drive antitumor responses, CD28 agonism on MM cells mediates prosurvival signaling through regulation of PI3K / Akt, FoxO3a, and Bimm, which has been described to induce chemotherapy resistance in multiple myeloma (Murray et al., 2014). Overexpression of CD28 on newly diagnosed multiple myeloma plasma cells has been described to correlate with worse clinical outcomes (Bahlis et al., 2007). However, while CD28 activation enhances myeloma cell survival, its activation inhibits myeloma cell proliferation. Agonizing CD28 in the presence of a strong immune cell-mediated response, such as bispecific T cell activation, can further enhance an efficient antitumor response. The present inventors provide herein a bispecific agonist CD28 antigen binding molecule that specifically binds to a human multiple myeloma (MM) cell surface antigen. In particular, the bispecific agonist CD28 antigen binding molecule according to the present invention, which targets a TAA selected from BCMA, CD38, and GPRC5D expressed on T cells, and CD28, has the efficacy to treat multiple myeloma as a single agent or in combination with other agents, such as T cell bispecific antibodies (TCBs) that target human MM cell surface antigens. Summary of the Invention

[0012] The present invention describes tumor-targeting bispecific agonist CD28 antigen-binding molecules that achieve tumor-dependent T cell activation and tumor cell killing without the need for multimerization. The bispecific CD28 antigen-binding molecules of the present invention are characterized by monovalent binding to CD28 and at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen (e.g., fibroblast activation protein (FAP) or carcinoembryonic antigen (CEA), CD19, or GPRC5D). Furthermore, they possess an Fc domain composed of a first subunit and a second subunit capable of stable association, each of which contains one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for the Fc receptor. This prevents Fc receptor-mediated cross-linking, and tumor-specific activation is achieved by cross-linking of at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen upon binding to that antigen.

[0013] Thus, the present invention provides a bispecific agonist CD28 antigen-binding molecule characterized by monovalent binding to CD28, comprising: (a) one antigen-binding domain capable of specifically binding to CD28; and (b) at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; The present invention provides a bispecific agonist CD28 antigen binding molecule comprising:

[0014] In one aspect, a bispecific agonist CD28 antigen-binding molecule as defined below is provided, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. In a particular aspect, the Fc domain composed of a first subunit and a second subunit capable of stable association is an IgG1 Fc domain. In one aspect, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one aspect, the Fc domain is an Fc domain of the human IgG1 subclass and comprises the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0015] In one embodiment, the antigen binding domain capable of specifically binding to CD28 comprises: (i) a heavy chain variable region (V) comprising heavy chain complementarity determining regions CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37, and CDR-H3 of SEQ ID NO: 38; H CD28) and a light chain variable region (V) comprising light chain complementarity determining regions CDR-L1 of SEQ ID NO: 39, CDR-L2 of SEQ ID NO: 40, and CDR-L3 of SEQ ID NO: 41. L CD28); or (ii) a heavy chain variable region (V) comprising CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25. L CD28) and

[0023] There is provided a bispecific agonist CD28 antigen binding molecule as defined herein above, comprising:

[0016] In one aspect, the antigen binding domain of the bispecific agonist CD28 antigen binding molecule capable of specifically binding to CD28 comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37 and CDR-H3 of SEQ ID NO: 38. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 39, CDR-L2 of SEQ ID NO: 40, and CDR-L3 of SEQ ID NO: 41 LCD28).

[0017] In another aspect, the antigen binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen binding molecule comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25 L CD28).

[0018] Furthermore, the antigen-binding domain capable of specifically binding to CD28 may comprise a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26. H CD28) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:27. L In accordance with the present invention, there is provided a bispecific agonist CD28 antigen-binding molecule as defined herein above, comprising a CD28 (antigen binding site) and a CD28 (antigen binding site).

[0019] In a further aspect, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51. H CD28) and a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. L A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0020] In another embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), or (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 51 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61 L CD28), or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (e) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (f) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 L CD28), or (g) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), or (h) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 43 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), or (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (j) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 LCD28), or (k) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0021] In one particular embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0022] In another particular embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0023] In a further aspect, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0024] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to carcinoembryonic antigen (CEA).

[0025] In one embodiment, the antigen-binding domain capable of specifically binding to CEA comprises: (i) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 188, CDR-H2 having the amino acid sequence of SEQ ID NO: 189, and CDR-H3 having the amino acid sequence of SEQ ID NO: 190; H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 191, CDR-L2 having the amino acid sequence of SEQ ID NO: 192, and CDR-L3 having the amino acid sequence of SEQ ID NO: 193. L CEA), or (ii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 180, CDR-H2 having the amino acid sequence of SEQ ID NO: 181, and CDR-H3 having the amino acid sequence of SEQ ID NO: 182; H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 183, CDR-L2 having the amino acid sequence of SEQ ID NO: 184, and CDR-L3 having the amino acid sequence of SEQ ID NO: 185. L CEA), or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 127, CDR-H2 having the amino acid sequence of SEQ ID NO: 128, and CDR-H3 having the amino acid sequence of SEQ ID NO: 129; H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 130, CDR-L2 having the amino acid sequence of SEQ ID NO: 131, and CDR-L3 having the amino acid sequence of SEQ ID NO: 132. L CEA), or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 507, CDR-H2 having the amino acid sequence of SEQ ID NO: 508, and CDR-H3 having the amino acid sequence of SEQ ID NO: 509 H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 510, CDR-L2 having the amino acid sequence of SEQ ID NO: 511, and CDR-L3 having the amino acid sequence of SEQ ID NO: 512. L CEA) and

[0023] The bispecific agonist CD28 antigen binding molecule described herein is provided, comprising:

[0026] In one embodiment, the antigen binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 133. H CEA) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 134. L In particular, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 186. H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 187 L CEA).

[0027] In another embodiment, the antigen-binding domain capable of specifically binding to CEA is (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 194 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 195 L CEA), or (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 196 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 197 L CEA), or (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 198 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 199 L CEA), or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 200 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 201 L CEA), or (e) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 202 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 203 L CEA), or (f) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 204 HCEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 205 L CEA), or (g) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 206 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 207 L CEA), or (h) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 208 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 209 L CEA), or (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 210 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 211 L CEA), or (j) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 212 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 213 L CEA) A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0028] In particular, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 200. H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 201 L CEA).

[0029] In yet another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP). In one aspect, the antigen-binding domain capable of specifically binding to FAP comprises (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. Hand a light chain variable region (V FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17. L (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. H and a light chain variable region (V FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9. L Provided herein is a bispecific agonist CD28 antigen binding molecule comprising a CD28 heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 12, a CD28 heavy chain variable region (VH2) comprising the amino acid sequence of SEQ ID NO: 13, and a CD28 antigen binding domain (VH3) comprising the amino acid sequence of SEQ ID NO: 14. In particular, the antigen binding domain capable of specifically binding to FAP comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 12, a CD28 heavy chain variable region (VH2) comprising the amino acid sequence of SEQ ID NO: 13, and a CD28 heavy chain variable region (VH3) comprising the amino acid sequence of SEQ ID NO: 14. H and a light chain variable region (V FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17. L In one embodiment, the antigen-binding domain capable of specifically binding to a FAP comprises (a) a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. H FAP) and a light chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19. L FAP), or (b) a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10. HFAP) and a light chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11. L In particular, the antigen-binding domain capable of specifically binding to FAP comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 18. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 19 L FAP).

[0030] In yet another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to epithelial cell adhesion molecule (EpCAM). In one aspect, the antigen-binding domain capable of specifically binding to EpCAM comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 515, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 516, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 517. H EpCAM) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 518, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 519, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 520. L In one aspect, a bispecific agonist CD28 antigen binding molecule described herein is provided, comprising an antigen-binding domain capable of specifically binding to EpCAM, the antigen-binding domain comprising: (a) a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 521; H EpCAM) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 522. L A bispecific agonist CD28 antigen-binding molecule is provided, comprising an antigen-binding domain capable of specifically binding to EpCAM. More specifically, the antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 521. HEpCAM) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 522 L Includes EpCAM.

[0031] In another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to HER3. In one aspect, the antigen-binding domain capable of specifically binding to HER3 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 523, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 524, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 525. H HER3) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 526, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 527, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 528. L In one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises a CD28 heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 529. H HER3) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 530. L A bispecific agonist CD28 antigen binding molecule is provided, comprising an antigen binding domain capable of specifically binding to HER3, the antigen binding domain comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 529. H HER3) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 530 L HER3).

[0032] In yet another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to CD30. In one aspect, the antigen-binding domain capable of specifically binding to CD30 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 531, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 532, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 533. H and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 534, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 535, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 536. L In one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises: (a) a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 537; and (b) a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 537. H CD30) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 538. L In particular, the antigen-binding domain capable of specifically binding to CD30 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 537. H CD30) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 538 L CD30).

[0033] Further provided is a bispecific agonist CD28 antigen-binding molecule, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to TBPG. In one aspect, the antigen-binding domain capable of specifically binding to TBPG comprises a heavy chain variable region (V) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 539, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 540, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 541.H TBPG), and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 542, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 543, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 544. L In one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises a CD28 antigen binding domain capable of specifically binding to TBPG, the CD28 antigen binding domain comprising: (a) a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 545; H TBPG) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 546. L A bispecific agonist CD28 antigen-binding molecule is provided, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 545. More specifically, the antigen-binding domain capable of specifically binding to TBPG is provided. H TBPG) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 546 L TBPG).

[0034] In a further aspect, the present invention provides a bispecific agonist CD28 antigen-binding molecule characterized by monovalent binding to CD28, comprising: (a) one antigen-binding domain capable of specifically binding to CD28, (b) at least one antigen-binding domain capable of specifically binding to a multiple myeloma (MM) cell surface antigen, and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit and the second subunit comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor. In one aspect, the multiple myeloma (MM) cell surface antigen is selected from the group consisting of CD38, BCMA, and GPRC5D.

[0035] Thus, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to GPRC5D. In one aspect, the antigen-binding domain capable of specifically binding to GPRC5D comprises: (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 563, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 564, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 565. H GPRC5D) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 566, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 567, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 568. L GPRC5D), or (b) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 579, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 580, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 581. H GPRC5D) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 582, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 583, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 584. L In one aspect, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 569. H GPRC5D) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 570. L GPRC5D). In particular, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 569. H GPRC5D) and a light chain variable region (V L GPRC5D).

[0036] In one aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to CD38. In one aspect, the antigen-binding domain capable of specifically binding to CD38 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 547, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 548, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 549. H and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 550, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 551, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 552. L In one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 553. H CD38) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 554. L In particular, the antigen-binding domain capable of specifically binding to CD38 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 553. H CD38) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 554 L CD38).

[0037] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to BCMA. In one aspect, the antigen-binding domain capable of specifically binding to BCMA comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 555, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 556, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 557. H BCMA) and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 558, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 559, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 560. L In one aspect, the antigen-binding domain capable of specifically binding to BCMA comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 559. H BCMA) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 560. L A bispecific agonist CD28 antigen binding molecule is provided, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 561. H BCMA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 562 L BCMA).

[0038] In a further aspect, the present invention provides a bispecific agonist CD28 antigen-binding molecule characterized by monovalent binding to CD28, comprising: (a) one antigen-binding domain capable of specifically binding to CD28; (b) at least one antigen-binding domain capable of specifically binding to a B cell surface antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit and the second subunit comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor. In one aspect, the B cell surface antigen is selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37.

[0039] Thus, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to CD19. In one aspect, the antigen-binding domain capable of specifically binding to CD19 comprises: (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 406, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 407, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 408. H and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 409, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 410, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 411. L (b) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 414, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 415, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 416 H and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 417, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 418, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 419. LIn one embodiment, the antigen-binding domain capable of specifically binding to CD19 comprises: (a) a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 412; H CD19) and a light chain variable region (V) comprising an amino acid sequence at least about 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 413. L CD19), or (b) a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 420. H CD19) and a light chain variable region (V) comprising an amino acid sequence at least about 95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 421. L In particular, the antigen-binding domain capable of specifically binding to CD19 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 412. H CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 413 L CD19).

[0040] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to CD79b. In one aspect, the antigen-binding domain capable of specifically binding to CD79b comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 422, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 423, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 424. H and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 425, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 426, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 427. LIn one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 428. H and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 429. L In particular, the antigen-binding domain capable of specifically binding to CD79b comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 428. H CD79b) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 429 L CD79b).

[0041] In a further aspect, there is provided a bispecific agonist CD28 antigen-binding molecule as defined herein above, wherein the antigen-binding domain capable of specifically binding to CD28 is a Fab fragment or a cross-Fab fragment.

[0042] In another aspect, (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; The bispecific agonist CD28 antigen binding molecule described herein is provided, comprising:

[0043] In another aspect, (a) a first Fab fragment capable of specifically binding to CD28; (b) a second Fab fragment capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; Including, Provided herein is a bispecific agonist CD28 antigen-binding molecule, in which a first Fab fragment capable of specifically binding to CD28 is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab fragment capable of specifically binding to a tumor-associated antigen, which is then fused at its C-terminus to the N-terminus of one of the Fc domain subunits.

[0044] In another aspect, (a) a first Fab fragment capable of specifically binding to CD28; (b) second and third Fab fragments capable of specifically binding to tumor-associated antigens; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; Including, Provided herein is a bispecific agonist CD28 antigen-binding molecule, in which a first Fab fragment capable of specifically binding to CD28 is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab fragment capable of specifically binding to a tumor-associated antigen, which is then fused at its C-terminus to the N-terminus of a first Fc domain subunit, and a third Fab fragment capable of specifically binding to a tumor-associated antigen is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit.

[0045] In a further aspect, (a) a Fab fragment capable of specifically binding to CD28; (b) a VH domain and a VL domain capable of specifically binding to a tumor-associated antigen; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; Including, Provided herein is a bispecific agonist CD28 antigen-binding molecule, in which a Fab fragment capable of specifically binding to CD28 is fused at its C-terminus to the N-terminus of a first Fc domain subunit, one of a VH domain and a VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of the first Fc domain subunit via a peptide linker, and the other of the VH domain and VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of a second Fc domain subunit via a peptide linker.

[0046] According to another aspect of the present invention, one or more isolated polynucleotides encoding the bispecific agonist CD28 antigen-binding molecules of the present invention are provided. The present invention further provides one or more vectors, particularly expression vector(s) comprising the isolated polynucleotide(s) of the present invention, and host cells comprising the isolated polynucleotide(s) or vector(s) of the present invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell. In another aspect, there is provided a method for producing the bispecific agonist CD28 antigen-binding molecules described herein, comprising culturing a host cell of the present invention under conditions suitable for expression of the bispecific agonist CD28 antigen-binding molecule. Optionally, the method also comprises recovering the bispecific agonist CD28 antigen-binding molecule. The present invention also encompasses bispecific agonist CD28 antigen-binding molecules produced by the methods of the present invention.

[0047] The present invention further provides a pharmaceutical composition comprising a bispecific agonist CD28 antigen binding molecule of the present invention and at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition is for use in the treatment of cancer.

[0048] The present invention also encompasses methods of using the bispecific agonist CD28 antigen-binding molecules and pharmaceutical compositions of the invention. In one aspect, the present invention provides a bispecific agonist CD28 antigen-binding molecule or pharmaceutical composition according to the invention for use as a medicament. In one aspect, a bispecific agonist CD28 antigen-binding molecule described herein is provided for use in (a) enhancing cell activation or (b) enhancing T cell effector function. In one aspect, a bispecific agonist CD28 antigen-binding molecule or pharmaceutical composition according to the invention is provided for use in treating a disease. In a specific aspect, the disease is cancer. In another aspect, a bispecific agonist CD28 antigen-binding molecule or pharmaceutical composition according to the invention is provided for use in treating cancer, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with a chemotherapeutic agent, radiation therapy, and / or other agent for use in cancer immunotherapy. In a further aspect, a bispecific agonist CD28 antigen-binding molecule or pharmaceutical composition for use in the treatment of cancer is provided, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with a T cell-activating anti-CD3 bispecific antibody. In yet another aspect, a bispecific agonist CD28 antigen-binding molecule or pharmaceutical composition for use in the treatment of cancer is provided, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with an anti-PD-L1 antibody or an anti-PD-1 antibody.

[0049] Also provided are the use of a bispecific agonist CD28 antigen-binding molecule according to the invention in the manufacture of a medicament for treating a disease, and a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific agonist CD28 antigen-binding molecule according to the invention or a composition comprising a bispecific agonist CD28 antigen-binding molecule according to the invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer. In one embodiment, a method is provided for (a) enhancing cell activation or (b) enhancing T cell effector function in an individual, comprising administering to the individual a bispecific agonist CD28 antigen-binding molecule according to the invention or a composition comprising a bispecific agonist CD28 antigen-binding molecule according to the invention in a pharmaceutically acceptable form. In another embodiment, a use of a bispecific agonist CD28 antigen-binding molecule according to the invention in the manufacture of a medicament for treating a disease, wherein the treatment comprises co-administration with a chemotherapeutic agent, radiation therapy, and / or other agent for use in cancer immunotherapy, is provided. In a further aspect, there is provided a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific agonist CD28 antigen-binding molecule of the invention or a composition comprising a bispecific agonist CD28 antigen-binding molecule of the invention in a pharmaceutically acceptable form, including co-administration with chemotherapy, radiation therapy, and / or other agents for use in cancer immunotherapy. In a further aspect, there is provided a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific agonist CD28 antigen-binding molecule of the invention or a composition comprising a bispecific agonist CD28 antigen-binding molecule of the invention in a pharmaceutically acceptable form, including co-administration with a T-cell-activating anti-CD3 bispecific antibody. In another aspect, there is provided a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific agonist CD28 antigen binding molecule of the invention, or a composition comprising a bispecific agonist CD28 antigen binding molecule of the invention in a pharmaceutically acceptable form, and co-administering an anti-PD-L1 antibody or an anti-PD-1 antibody.Also provided is a method for inhibiting tumor cell growth in an individual, comprising administering to the individual an effective amount of a bispecific agonist CD28 antigen-binding molecule according to the invention or a composition comprising a bispecific agonist CD28 antigen-binding molecule according to the invention in a pharmaceutically acceptable form, thereby inhibiting tumor cell growth. In any of the above aspects, the individual is preferably a mammal, particularly a human. [Brief explanation of the drawings]

[0050] [Figure 1]Schematic diagrams of the described molecules are shown in Figures 1A-1L. Figure 1A shows the CD28 agonist antibody CD28(SA) in its huIgG4 isoform (TGN1412). Figure 1B shows the CD28(SA) agonist antibody as a hu IgG1 PGLALA isotype ("Fc silent"). Bispecific FAP-CD28 antigen-binding molecules in 1+1, 1+2, 2+2, and 1+4 formats are shown in Figures 1C, 1D, 1E, and 1F, respectively. Bispecific CEA-CD28 antigen-binding molecules in 1+2, 2+2, and 1+1 formats are shown in Figures 1G, 1H, and 1J, respectively. Figure 1I shows a schematic diagram of a CD28 agonist antibody variant as a monovalent huIgG1 PGLALA isotype ("Fc silent"). Figure 1K shows a bispecific FAP-CD28 antigen-binding molecule in a 1+1 format, in which the FAP antigen-binding domain is presented as a VH domain and a VL domain, each fused to the C-terminus of one of the Fc domain subunits. Figure 1L illustrates a bispecific FAP-CD28 antigen-binding molecule in a 2+1 format, in which the CD28 antigen-binding domain is presented as a cross-Fab fused at its C-terminus to the N-terminus of one of the heavy chains of a "bivalent" FAP antibody. Figure 1M shows another bispecific FAP-CD28 antigen-binding molecule in a 1+1 format, in which the CD28 antigen-binding domain is presented as a cross-Fab fused at its C-terminus to the N-terminus of a Fab fragment that binds to the FAP. Figure 1N shows a trispecific FAP-CEA-CD28 antigen-binding molecule in a 1+1+1 format, in which the CD28 antigen-binding domain is represented as Fab fused at the C-terminus of both the light and heavy chains to the N-terminus of both the light and heavy chains of the anti-FAP antigen-binding domain on the huIgG1 PG-LALA Fc knob chain, and the anti-CEA cross-Fab fragment is part of the huIgG1 PG-LALA Fc hole chain. [Figure 2]Figures 2A, 2B, 2C, 2D, and 2E show the binding of CD28 agonist antibodies and FAP-CD28 antigen-binding molecules to human CD28 or human FAP on cells. Figure 2A shows the binding of CD28 (SA) in the IgG4 isoform to human CD28 (hu IgG1 PGLALA isotype), and the binding of different FAP-CD28 molecules to human CD28 (Figure 2B) and human FAP (Figure 2C) on cells. The median fluorescence intensity of binding of different CD28 agonist antibodies or anti-DP47 targeting molecules to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, engineered to stably overexpress human CD28) or 3T3 cells expressing human FAP (NIH / 3T3 cell line (ATCC CRL-1658)) was assessed by flow cytometry. SEM technical triplicates are shown. A comparison of FAP(4B9)-CD28(SA) antigen-binding molecules (molecules D, E, and F described in Example 1) is shown in Figure 2D (binding to human CD28) and Figure 2E (binding to human FAP). Figures 2F and 2G show the binding of FAP-CD28 antigen-binding molecules in various formats to CD28 and monovalent binding to FAP, respectively. Curves for FAP-CD28 CTF 1+1 (P1AE2236, molecule I), FAP-CD28 1+1 (P1AD4492, molecule C), FAP-CD28 H2T 1+1 (P1AE2021, molecule H), and two reference compounds, FAP-CD28(SA) 1+2 (P1AD9011, molecule E) and DP47, are shown. Figure 2 shows the median fluorescence intensity of binding of FAP-CD28 antibody or anti-DP47 antibody (negative control) to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61 engineered to stably overexpress human CD28) (Figure 2F) or 3T3 cells expressing human FAP (NIH / 3T3 cell line (ATCC CRL-1658)) (Figure 2G), as assessed by flow cytometry. Technical triplicates with SEM are shown. Figures 2H and 2I show binding of FAP-CD28 2+1 (P1AE5231, molecule G) to CD28 and FAP, respectively. [Figure 3]Alignments of the CD28(SA) variable domain and its mutants are shown in Figures 3A-3D. Alignments of the CD28(SA) VH domain and its mutants for removing cysteine ​​50 and reducing the affinity of the resulting anti-CD28 binding agent to different degrees are shown in Figures 3A and 3B. Notably, in VH mutants i and j, the CDRs of CD28(SA) were grafted from the IGHV1-2 framework onto the IGHV3-23 framework (Figure 3B). Figures 3C and 3D show alignments of the CD28(SA) VL domain and its mutants for reducing the affinity of the resulting anti-CD28 binding agent to different degrees. In mutant t, the CDRs were grafted onto the framework sequence of the trastuzumab (Herceptin) VL sequence. [Figure 4]Figures 4A-4C show the binding of affinity-reduced monospecific CD28 agonist antibody variants in a monovalent IgG format from supernatant to human CD28 on cells. Median fluorescence intensity binding to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61 engineered to stably overexpress human CD28) was assessed by flow cytometry, compared with a negative control (anti-DP47) and the original TGN1412. Binding curves for variants 1-10 are shown in Figure 4A, those for variants 11-22 are shown in Figure 4B, and those for variants 23-31 are shown in Figure 4C. Technical replicates are shown with SD. Figures 4D and 4E show the binding of FAP-targeted bispecific CD28 agonist antibody variants in a huIgG1 PG-LALA 1+1 format with selected affinity-reduced CD28 agonist antibody variants to human CD28 on cells. Binding curves for bispecific 1+1 constructs with variants 8, 11, 12, 15, 16, and 17 are shown in Figure 4D, and binding curves for bispecific 1+1 constructs with variants 19, 23, 25, 27, and 29 are shown in Figure 4E. Selected binders were chosen based on affinity for production in a 1+1 bispecific FAP-targeting format. Figures 4F and 4G show binding of the same FAP-targeting bispecific CD28 agonist antibody variants in a huIgG1 PG-LALA 1+1 format to a human FAP. Median fluorescence intensity values ​​assessed by flow cytometry for binding to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61 engineered to stably overexpress human CD28) or 3T3 cells expressing human FAP (NIH / 3T3 cell line (ATCC CRL-1658)) compared to negative controls (anti-DP47) and TGN1412 (molecule A) are provided. Technical triplicates with SEM are shown. The in vitro potency of selected FAP-targeting bispecific CD28 agonist antibody variants in the huIgG1 PG-LALA 1+1 format is illustrated in Figures 4H, 4I, and 4J.PBMC T cells were incubated with MCSP- and FAP-expressing MV3 melanoma cells for 5 days in the presence of increasing concentrations of FAP-CD28 constructs containing a limiting concentration of MCSP-TCB (5 pM, P1AD2189) and the indicated CD28 variant binders. Figure 4H shows CFSE dilution as a measure of T cell proliferation of CD8 T cells, assessed by flow cytometry. Error bars indicate SEM, and the graph shows representative results from technical triplicates from two donors. Figure 4I shows the correlation of KD (nM) of CD28 binder variants to potency by area under the curve (a) as a % of the parent TGN1412 clone (CD28(SA)). Figure 4J shows target cell killing at 90 hours. [Figure 5] Figures 5A-5D show the establishment of the mode of action of high-density (HD) preculture and CD28(SA). PBMC T cells were precultured at high density (HD) for 2 days or freshly used from PBMC isolation and stimulated with increasing concentrations of CD28(SA). CFSE dilution as a surrogate for T cell proliferation after 5 days of stimulation with CD28(SA) (molecule A, P1AE1975) is shown (Figure 5A) and cytokine secretion after 2 days of stimulation (Figure 5B). Figure 5C shows the percentage of FcγRIIb expression in PBMC monocytes and B cells, assessed by flow cytometry, before and after 2 days of HD PBMC preculture. Figure 5D: HD-precultured PBMCs were cocultured with CD28(SA) for 5 days in the presence or absence of an FcγRIIb blocking antibody or isotype control, and the percentage of CFSE dilution in CD4 T cells was assessed by flow cytometry. Graphs are representative of at least six donors (Figures 5A and 5B) and two donors (Figures 5C and 5D), each evaluated in independent experiments. Graphs show technical triplicates. Error bars indicate SEM. Statistical analysis was performed by Student's t-test. *** = p < 0.001. CD28(SA)IgG4 superagonism is dependent on cross-linking to FcγRIIb. [Figure 6]Figures 6A and 6B show T cell proliferation, i.e., CFSE dilution of CD4 T cells, after 5 days of stimulation with either the original Fc wild-type IgG4 CD28(SA) (P1AE1975) or CD28(SA) with the P329G-LALA mutation (P1AD9289). T cells were precultured at high density for 2 days. Graphs represent at least three independent experiments. Technical triplicates are shown. Fc silencing abolishes superagonism in TGN1412. Addition of a tumor-targeting moiety to Fc-silenced TGN1412 restores superagonism, which is then dependent on the presence of tumor targets. [Figure 7]Figures 7A, 7B, 7C, and 7D show a comparison of FAP-targeted CD28 agonists in different formats (2 + 2 and 1 + 2) with a superagonist (CD28(SA)) binding factor and a conventional agonist binding factor (9.3, CD28(CA)). FAP-targeted CD28 agonists with conventional CD28 agonist binding factors do not function as superagonists. PBMC T cells were cocultured with 3T3-huFAP cells (in the presence of FAP) for 5 days in the presence of increasing concentrations of FAP-CD28 formats with either a superagonist binding factor (SA, Figure 7A) or a conventional agonist binding factor (9.3, Figure 7B). T cell proliferation is shown. PBMC T cells were then cocultured with 3T3 WT cells (without FAP) for 5 days in the presence of increasing concentrations of FAP-CD28 formats containing superagonist binding factor (SA, Figure 7C) or conventional agonist binding factor (9.3, Figure 7D). CFSE dilution as a measure of T cell proliferation of CD8 T cells, assessed by flow cytometry 5 days after stimulation, is shown. The graph shows cumulative data from three donors in three independent experiments. Error bars indicate SEM. In the same experimental setup, cytokines were also measured from supernatants 2 days after coculture. Values ​​are shown in Figure 7E. The ability of various FAP-CD28 formats, either with superagonist CD28(SA) binding factor or conventional agonist binding factor (CD28(CA)), to induce killing of FAP-expressing RFP-MV3 melanoma cells was assessed over a 90-hour period by live-cell imaging using IncuCyte technology. All molecules, including FAP-TCB (P1AD4645), were used at 10 nM. [Figure 8] Figures 8A, 8B, and 8C show representative results from three donors, each with technical triplicates. Figure 8D shows cumulative results, expressed as area under the curve (AUC) at t = 90 h, for three donors from three independent experiments. Boxes represent the 25th to 75th percentiles, and whiskers represent minimum to maximum. Statistical analysis was performed by pairwise one-way ANOVA. ***: p < 0.001, ns: not significant. [Figure 9]A comparison of various formats of CEA-targeted CD28 agonists with superagonist and conventional agonist binding factors is shown in Figures 9A and 9B. The ability of various formats of CEA-CD28, using either the superagonist CD28 (SA) binding factor or the conventional agonist CD28 (CA) binding factor, to induce killing of CEA-expressing RFP+ MKN45 gastric cancer cells was assessed over a 90-hour period by live-cell imaging using IncuCyte technology. All molecules, including CEACAM5-TCB (P1AD5299), were used at 10 nM. Figure 9A shows representative results from one donor with technical triplicates. Figure 9B shows statistical analysis of the technical triplicates, expressed as the area under the curve (AUC) at t = 90 hours for one donor in a single experiment. Boxes represent the 25th to 75th percentiles, and whiskers represent minimum to maximum. Statistical analysis was performed by pairwise one-way ANOVA. ***=p<0.001 CEA-targeted CD28 agonists with conventional CD28 agonist binding agents have been shown not to behave in a superagonistic manner. [Figure 10] Figures 10A, 10B, and 10C show that targeting CD28 agonists with monovalent superagonistic binding factors is not functionally superagonistic. PBMC T cells were cocultured with 3T3-huFAP cells for 5 days in the presence of increasing concentrations of FAP-CD28 with a bivalent CD28 binding factor (P1AD9011, filled circles) or monovalent FAP-CD28 with respect to the CD28 binding factor (P1AD4492, open circles). Figure 10A shows the CFSE dilution of CD8 T cells. T cell activation was further assessed by flow cytometric detection of the activation markers CD69 (Figure 10B) and CD25 (Figure 10C). The mean fluorescence intensity (MFI) of CD69 and CD25 staining is shown 5 days after stimulation. Technical triplicates from one donor are shown, and error bars indicate SEM. TGN1412-like superagonism has been shown to require multivalent CD28 binding. [Figure 11]Figures 11A and 11B show that when combined with a T cell bispecific antibody (TCB), TCB-mediated effector function is supported by monovalent and bivalent CD28 binding of FAP-targeted agonist CD28 antigen binding molecules with comparable potency, but the monovalency of the CD28 binding agent is required to maintain tumor target dependence of the CD28 agonist in the presence of the TCB. In Figure 11A, PBMC T cells were incubated with MCSP-expressing and FAP-expressing MV3 melanoma cells for 90 hours in the presence of a combined limiting concentration of MCSP-TCB (5 pM, P1AD2189) and increasing concentrations (ranging from 0 to 10 nM) of FAP-CD28 (SA), with bivalent or monovalent binding to CD28, respectively. Target cell killing at 90 hours, assessed by live-cell imaging using IncuCyte technology, is shown. In Figure 11B, PBMC T cells were cocultured with FAP-negative CEA-expressing MKN45 gastric cancer cells (absent FAP) for 90 hours in combination with FAP-CD28, which has bivalent or monovalent CD28 binding, respectively, and in the presence of increasing concentrations (ranging from 0 to 10 nM) of CEACAM5-TCB (10 pM, P1AD5299). Target cell killing at 90 hours, as assessed by IncuCyte, is shown. Data show MKN45 target cell killing over time, from one donor in one experiment, technical triplicates; error bars indicate SEM. [Figure 12]Figures 12A and 12B show that FAP-CD28(SA), which has bivalent binding to CD28, loses FAP dependence when combined with T cell bispecificity. Figure 12A shows the absence of TCB. PBMC T cells were cocultured with CEA-expressing MKN45 and 3T3-huFAP ("FAP-present" condition, filled circles) or 3T3-WT ("FAP-absent" condition, open circles) in the presence of increasing concentrations of FAP-CD28(SA)2+1. Combination with TCB is shown in Figure 12B. PBMC T cells were cocultured with CEA-expressing MKN45 and 3T3-huFAP ("FAP-present" condition, filled circles) or 3T3-WT ("FAP-absent" condition, open circles) in the presence of limiting concentrations of CEACAM5-TCB (10 pM, P1AD5299) and increasing concentrations of FAP-CD282+1 SA. CD8 T cell proliferation after 5 days of stimulation is shown. Data are representative of two independent experiments using two donors. Results from one donor are shown, data points represent technical triplicates, and error bars indicate SEM. [Figure 13]Figures 13A, 13B, and 13C show the functionality of FAP-CD28(SA) antigen-binding molecules that bind monovalently to CD28 in different formats. Molecule C is the FAP-CD28(SA) classical 1 + 1 format (P1AD4492), molecule H is the FAP-CD28(SA) 1 + 1 "head-to-tail" (H2T) format (P1AE2021), molecule I is the FAP-CD28(SA) 1 + 1 format with a C-terminal fusion of a FAP binding agent (P1AE2236), and molecule G is the FAP-CD28(SA) 2 + 1 format (P1AD5231). As a reference, a bivalent CD28 antigen-binding molecule (P1AD9011) was used. PBMC T cells were incubated with MCSP- and FAP-expressing MV3 melanoma cells in the presence of a limiting concentration of MCSP-TCB (5 pM, P1AD2189) and increasing concentrations (range 0-10 nM) of FAP-CD28 in a given format. CFSE dilution is shown as a measure of T cell proliferation after 5 days, assessed by flow cytometry, for CD8 T cells (Figure 13A) and CD4 T cells (Figure 13B). Figure 13C shows MV3 cell killing over 84 hours in the presence of 5 pM MCSP-TCB alone compared with the combination of 5 pM MCSP-TCB with increasing concentrations of FAP-CD28 in various formats. Killing was assessed by live cell imaging using an IncuCyte system. All molecules were able to support TCB-mediated effector function. Graphs show cumulative data from three independent experiments and four donors. 10 pM MCSP-TCB; E:T 20; Statistics: Two-way ANOVA. Asterisks indicate the lowest concentration at which the add-on was significantly better than TCB alone: ​​*p≦0.05, **p≦0.01; ***p≦0.001. Error bars indicate SEM. [Figure 14]Target cell killing of the CEA-CD28 1+1 format in combination with TCB is shown in Figure 14. PBMC T cells were cocultured with CEA-expressing MKN45 gastric cancer cells for 90 hours in the presence of limiting concentrations of CEACAM5-TCB (10 pM, P1AD5299) combined with 2 nM CEA-CD28 (P1AE3127) or non-targeting CD28 (P1AD8944). Data show the killing of MKN45 target cells over time from one donor in one experiment. Killing was assessed by live cell imaging using an IncuCyte system. It has been shown that only the combination results in target cell killing; the molecules alone do not induce killing at the given concentrations. CEA-CD28 synergizes with CEACAM5-TCB. [Figure 15] Figure 15 shows that CEA-CD28 enhances CEA-TCB and CEACAM5-TCB, lowering the threshold for CEA expression in TCBs to induce T cell activation. PBMC T cells were incubated with increasing concentrations of either CEA-TCB (P1AD4646) or CEACAM5-TCB (P1AD5299) and a fixed concentration of CEA-CD28 (P1AE3127) in the presence of target cell lines with different CEA expression levels: (i) MKN45 (high expression, approximately 400,000 CEA-binding sites / cell), (ii) Lovo (moderate expression, approximately 60,000 CEA-binding sites / cell), and (iii) HT-29 (low expression, approximately 6,000 CEA-binding sites / cell). T cell proliferation was assessed as a surrogate for T cell activation by flow cytometry. [Figure 16] Binding of selected affinity-reduced CD28 binding agent variants in a bispecific CEA-targeted monovalent 1+1 format to CD28 on cells is shown in Figure 16. Median fluorescence intensity of binding of CEA-CD28 antibody or anti-DP47 antibody (negative control) to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC#CCL-61 engineered to stably overexpress human CD28) was assessed by flow cytometry. Technical triplicates with SEM are shown. [Figure 17]Figures 17A, 17B, and 17C show the functionality of selected affinity-reduced CD28 binding factor variants in a bispecific CEA-targeted monovalent 1+1 format. PBMC T cells were cocultured with CEA-expressing MKN45 gastric cancer cells in the presence of a limiting concentration of CEACAM5-TCB (10 pM, P1AD5299) in combination with 2 nM of CEA-CD28 1+1 molecules containing CD28 binding factor variants. CD8 T cell proliferation (Figure 17A) and CD4 T cell proliferation (Figure 17B) were assessed by CFSE dilution by flow cytometry after 5 days of coculture. Target cell killing was assessed after 90 hours of incubation (Figure 17C). Data represent MKN45 target cell killing over time from one donor in one experiment. All molecules are capable of supporting CEACAM5-TCB-mediated effector function. [Figure 18] Figure 18 shows the binding of the humanized CEA (A5B7) huIgG1 P329G LALA mutant to MKN-45 compared to the binding of the parent murine A5B7 antibody. The antibody was detected with a fluorescently labeled secondary antibody, and fluorescence was measured by flow cytometry. [Figure 19] Figures 19A-19C are schematic diagrams of recombinant proteins displaying different domains of the CEACAM5 protein used as antigens in a phage display campaign. Figure 19A shows the construct NABA-avi-His, which consists of the four Ig-like domains N, A1, B, and A2. Figure 19B shows the construct N(A2B2)A-avi-His, and Figure 19C shows the construct NA(B2)A-avi-His. [Figure 20] 20A and 20B show the VH and VL sequences, respectively, of the humanized CEA antibody A5H1EL1D, with randomized positions marked with an X. [Figure 21] Schematic diagrams of the phage vectors for the affinity maturation libraries are shown in Figure 21A (CDRH1 / H2 affinity maturation library), Figure 21B (CDRL1 / H2 affinity maturation library), and Figure 21C (CDRH3 / CDRL3 amplification library). [Figure 22]Figures 22A and 22B show alignments of the VH amino acid sequences (Figure 22A) and VL amino acid sequences (Figure 22B) of affinity matured humanized CEA (A5H1EL1D) antibody variants. [Figure 23] Figures 23A-23D show schematic diagrams of the bispecific CEA / CD28 antigen-binding molecules described in Example 11. Figure 23A shows a bispecific CEA-CD28 antigen-binding molecule in a 1+1 format, in which the CEA antigen-binding domain is presented as a cross-Fab (VH / VL swap), and the Fab fragment bearing the CD28 antigen-binding domain has a charge modification to support correct light chain pairing. The Fc domain has a knob to hole modification and a P329G LALA mutation to abolish binding to Fcγ receptors. In Figure 23B, the CD28 antigen-binding domain is presented as a cross-Fab (VH / VL swap), and the Fab fragment bearing the CEA antigen-binding domain contains a charge modification. Figure 23C shows a bispecific CEA-CD28 antigen-binding molecule in a 2+1 format, in which the CD28 antigen-binding domain is presented as a cross-Fab, and two Fab fragments bearing the CEA antigen-binding domain are fused to each other via their heavy chains (head to tail). Figure 23D illustrates a bispecific CEA-CD28 antigen-binding molecule in a 2+1 format, in which the CD28 antigen-binding domain is represented as a cross-Fab fused at its C-terminus to the N-terminus of one of the heavy chains of a "bivalent" CEA antibody ("classical" format). [Figure 24] Figure 24 shows that affinity-matured anti-CEA clone P002.139 exhibits improved binding to CEACAM5 on CEA-expressing MV3 cells. Binding of CEA-CD28 bispecific antibodies with either affinity-matured anti-CEA clone P002.139 or the parental A5H1EL1D clone is shown. Median fluorescence intensity of binding of CEA-CD28 bispecific antibodies or anti-DP47 antibodies (negative control) to MV3 cells engineered to express human CEACAM5 was assessed by flow cytometry. Technical replicates with SEM are shown. Graphs are representative of three independent experiments. [Figure 25]Figures 25A and 25B show that affinity-matured anti-CEA clone P002.139 exhibits improved functionality in an IL-2 reporter assay. Luminescence readouts are shown after 6 hours of co-incubation of MKN45 cells, IL-2 reporter cells, harboring either affinity-matured clone P002.139 or the parental clone A5H1EL1D, with 5 nM CEA-TCB and CEA-CD28. Figure 25A shows the dose response. The dotted line indicates the luminescence achieved by CEA-TCB alone. Figure 25B shows the area under the curve values ​​calculated from the data shown in Figure 25A. Technical replicates with SEM are provided. Graphs are representative of three independent experiments. [Figure 26] Figure 26 shows the study design for an efficacy study with bispecific CEA-CD28 antibodies in combination with CEA TCB (comparison of different CEA clones) in MKN45 xenografts in humanized mice. The design and different treatment groups are shown. [Figure 27] Figures 27A-27E show the results of an efficacy study using a combination of CEA-CD28 and CEA TCB in MKN45 xenografts in humanized mice. The mean tumor volume (Figure 27A) or tumor growth in individual mice for the four treatment groups is plotted on the y-axis (Figures 27B-27E). Figure 27B shows tumor growth in each individual mouse in the vehicle group, Figure 27C shows tumor growth in mice treated with CEA TCB alone, Figure 27D shows tumor growth in mice treated with CEA TCB and CEA(T84.66)-CD28(SA_variant15), and Figure 27E shows tumor growth in mice treated with CEA TCB and CEA(A5H1EL1D)-CD28(SA_variant15). Increased TCB-mediated tumor regression can be seen in the presence of both bispecific CEA-CD28 antibodies. [Figure 28] Figure 28 shows the study design for an efficacy study with bispecific CEA-CD28 antibodies in combination with CEACAM5 TCB in BXPC3 xenografts in humanized mice (comparison of different CD28 clones). The design and different treatment groups are shown. [Figure 29]Figure 29 shows the tumor growth kinetics (mean, +SEM) for all treatment groups, and the corresponding TGI values ​​for each treatment group are shown in Table 33 (Example 13.2). [Figure 30] Ex vivo Immuno-PD data are shown in Figures 30A-30D. Figure 30A shows representative dot plots (CD3 vs. CD45 and CD4 vs. CD8) of stained tumor single cell suspensions from each treatment arm. Summary of CD3, CD8, and CD4 T cell infiltration is shown in Figure 30B (CD3), Figure 30C (CD8), and Figure 30D (CD4), respectively. [Figure 31] Figure 31 shows the study design for an efficacy study with a bispecific CEA-CD28 antibody (CEA(A5H1EL1D)-CD28(SA_variant8)) in combination with CEA TCB in MKN45 xenografts in humanized mice. The design and various treatment groups are shown. [Figure 32] Figure 32 shows the tumor growth kinetics (mean, +SEM) for all treatment groups, and the corresponding TGI values ​​for each treatment group are shown below in Table 35 (Example 13.3). [Figure 33] Ex vivo Immuno-PD data are shown in Figures 33A and 33B. Figure 33A shows representative dot plots of stained tumor single cell suspensions from each treatment arm. A summary of CD3+ T cell infiltration is shown in Figure 33B. [Figure 34]Figures 34A-34D show schematic diagrams of the bispecific CD28 antigen-binding molecules described in Example 14. Figure 34A shows a bispecific EpCAM-CD28 antigen-binding molecule in a 1+1 format, where the CD28 antigen-binding domain is presented as a cross-Fab (VH / VL swap), and the Fab fragment containing the EpCAM antigen-binding domain has a charge modification to support correct light chain pairing. The Fc domain has a knob to hole modification and a P329G LALA mutation to abolish binding to Fcγ receptors. Figure 34B shows a Fab containing the CD28 antigen-binding domain with a charge modification, and a Fab containing the HER3 antigen-binding domain with a cross-Fab (VH / VL swap). Figure 34C shows a bispecific CD30-CD28 antigen-binding molecule in a 1+1 format, where the Fab molecule containing the CD28 antigen-binding domain contains a charge modification, and the Fab containing the CD30 antigen-binding domain with a cross-Fab (VH / VL swap). Figure 34D shows a bispecific TPBG-CD28 antigen-binding molecule in a 1+1 format, where the Fab molecule with the CD28 antigen-binding domain contains a charge modification and the Fab with the TPBG (5T4) antigen-binding domain is represented as a cross-Fab (VH / VL swap). [Figure 35] Figures 35A-35C relate to the functional characterization of the EpCAM-CD28 bispecific antigen-binding molecule. Figure 35A shows that EpCAM-CD28 (molecule 14A) binds to human CD28 on CD28-expressing CHO-k1 cells, as assessed by flow cytometry. Figure 35B shows binding to EpCAM on HT29 cells, as assessed by flow cytometry. Anti-DP47 served as a negative control for nonspecific binding of the antibody compound to the cells. Dots represent technical replicates. Figure 35C shows that EpCAM-CD28 (P1AE9051) enhances T cell responses to anti-CD3 stimulation in an IL-2 reporter assay. IL-2 reporter cell activation, measured by luminescence readout, after 6 hours of co-incubation with HT-29 cells in the presence of a suboptimal concentration of anti-CD3 IgG (10 nM) and increasing concentrations of EpCAM-CD28 is shown. Dots represent technical replicates. [Figure 36] Figures 36A-36C relate to the functional characterization of the HER3-CD28 bispecific antigen binding molecule. Figure 36A shows that HER3-CD28 (P1AF0151) binds to human CD28 on CD28-expressing CHO-k1 cells, as assessed by flow cytometry. Figure 36B shows the binding of HER3-CD28 to HER3 on T-47D cells, as assessed by flow cytometry. Anti-DP47 served as a negative control for nonspecific binding of the antibody compound to the cells. Dots represent technical replicates. Figure 36C shows that HER3-CD28 (P1AF0151) enhances T cell responses to anti-CD3 stimulation in an IL-2 reporter assay. IL-2 reporter cell activation, measured by luminescence readout, after 6 hours of co-incubation with T-47D cells in the presence of a suboptimal concentration of anti-CD3 IgG clone OKT3 (10 nM) and increasing concentrations of HER3-CD28, is shown. Dots represent technical replicates. [Figure 37]Figures 37A-37C show schematic diagrams of bispecific CD28 antigen binding molecules targeting multiple myeloma (MM) cell surface antigens, as described in Example 16. Figure 37A shows a bispecific GPRC5D-CD28 antigen binding molecule in a 1+1 format, where the CD28 antigen binding domain is presented as a cross-Fab (VH / VL swap), and the Fab fragment bearing the GPRC5D antigen binding domain has a charge modification to support correct light chain pairing. The Fc domain has a knob to hole modification and a P329G LALA mutation to abolish binding to Fcγ receptors. In Figure 37B, the Fab bearing the CD28 antigen binding domain contains a charge modification, and the Fab bearing the CD38 antigen binding domain is presented as a cross-Fab (VH / VL swap). Figure 37C shows a bispecific BCMA-CD28 antigen binding molecule in a 1+1 format, where the Fab molecule with the CD28 antigen binding domain is represented as a cross-Fab (VH / VL swap) and the Fab with the BCMA antigen binding domain contains a charge modification. Figure 37D illustrates an anti-GPRC5D / anti-CD3 bispecific antibody (GPRC5D TCB) in a 2+1 format, where the Fab molecule with the GPCR5D antigen binding domain contains a charge modification and the Fab with the CD3 antigen binding domain is represented as a cross-Fab (VH / VL swap). [Figure 38] Figures 38A-38F relate to the binding of bispecific antigen-binding molecules targeting CD28 and multiple myeloma (MM) cell surface antigens to cells (Example 17.1). Binding of the bispecific antigen-binding molecules to either human CD28 on CHO huCD28 cl45 cells (Figures 38A and 38E), human CD38 on OCI-Ly18 cells (Figure 38B), human BCMA (B cell maturation antigen, Figure 38C) on IM-9 cells, and human GPRC5D on CHO huGPRC5D L2 cells expressed on the indicated cell lines (Figures 38D and 38F) is shown. Median relative fluorescence (MFI) from duplicates with SD is shown. EC50 values ​​for binding were calculated using GraphPad Prism and are included in Table 38. [Figure 39]Figures 39A-39F show T cell activation of bispecific antigen binding molecules targeting CD28 and multiple myeloma (MM) cell surface antigens assessed by IL-2 reporter assay. IL-2 reporter cell assays are shown after 5 and 22 hours of incubation, as determined by luminescence. IL-2 reporter effector and GPRC5D-expressing target cells were incubated at an effector-to-target ratio (E:T) of 5:1. GPRC5D-TCB was added at a fixed final assay concentration of 1 nM, and the indicated MM-targeted CD28 bispecific antigen binding molecules were titrated as indicated. Representative dose-response curves are shown in Figure 39A (after 5 hours of incubation) and Figure 39B (after 22 hours) for CD38-CD28, Figure 39C (after 5 hours) and Figure 39D (after 22 hours) for BCMA-CD28, and Figure 39E (after 5 hours) and Figure 39F (after 22 hours of incubation) for GPRC5D-CD28. [Figure 40] Figures 40A-40C show the boost of T cell-mediated lysis of the GPRC5D-expressing MM cell line NCI-H929 in the presence of 0.2 nM of the indicated CD28 bispecific molecules CD38-CD28 (Figure 40A), BCMA-CD28 (Figure 40B), and GPRC5D-CD28 (Figure 40C). Lysis was determined after 22 hours of co-incubation of human pan T cells with MM tumor target cells at a final E:T ratio of 1:1. Technical replicates with SD are shown. EC50 values ​​and areas under the curve for tumor cell lysis were calculated using GraphPad Prism and are shown in Table 39. [Figure 41] Figure 41A shows a schematic diagram of the bispecific CD19-CD28 antigen-binding molecules in a 1+1 format described in Example 18, in which the VH and VL domains of the Fab containing the CD19 antigen-binding domain have been swapped with each other (VH / VL cross-Fab), and the Fab containing the CD28 antigen-binding domain has specific amino acids in the CH1 and CL domains swapped (charge variant) to allow better pairing with the light chain. Figure 41B shows the corresponding molecule in which the CD19 antigen-binding domain has been replaced with a CD79b antigen-binding domain (anti-CD79b cross-Fab). [Figure 42]Figure 42 relates to the determination of kinetic and thermodynamic parameters of CD79b (polatuzumab) in the construct CD79b(huMA79b.v28)-CD28(v15)1+1. Soluble recombinant CD79b-His was captured on a CM5 chip via an anti-penta-His antibody, and the bispecific CD79b(huMA79b.v28)-CD28(v15)1+1 was used as the analyte. The smoothed line represents a global fit of the data to a 1:1 interaction mode. [Figure 43] Figure 43A shows the median fluorescence intensity (MFI) of binding of CD19-CD28 variant 15 (P1AE9040) to four different B cell lines expressing different levels of CD19. Binding was assessed by flow cytometry. Technical replicates with SEM are shown. Figure 43B shows FACS staining of CD19 on four different B cell lines (MFI). [Figure 44] Binding of CD19-CD28 with varying CD28 affinities to human CD19 and CD28 on cells is shown in Figures 44A and 44B. Median fluorescence intensity (MFI) is shown for binding to CHOk1-CD28 cells (Figure 44A) and CD19 on Nalm6 B cells (Figure 44B). Dots represent technical replicates with SEM. Corresponding EC50 values ​​are shown in Table 42 (CHOk1-CD28) and Table 43 (Nalm6) in Example 20. Binding was assessed by flow cytometry. [Figure 45] Figures 45A-45D show that CD19-CD28v15 enhances CD20-TCB in an IL-2 reporter assay in the presence of different B cell lines. IL-2 reporter cell activation measured by luminescence readout (LUM) after 6 hours of co-incubation with different B cell lines in the presence of suboptimal concentrations of CD20-TCB and increasing concentrations of CD19-CD28v15 is shown. Dots represent technical replicates with SEM. The suboptimal CD20-TCB concentration varies depending on the target cell line: 10 nM for Nalm6, 0.05 nM for RCK8, WSU DLCL2, and Z138. [Figure 46]Figure 46 shows that CD19-CD28 with varying CD28 affinity enhances CD20-TCB-mediated T cell activation. IL-2 reporter cell activation measured by luminescence readout (LUM) after 6 hours of co-incubation with Nalm6 B cells in the presence of a suboptimal concentration of CD20-TCB (10 nM) and increasing concentrations of CD19-CD28 v15 is shown. Dots represent technical replicates with SEM. The activation status of PBMC-derived T cells after co-culture with CD20-expressing target cells (Nalm6) (E:T ratio 5:1) and CD19-CD28 was assessed in the absence or presence of CD20-TCB. [Figure 47] The activity of CD19-CD28 in the absence or presence of TCR signals is shown in Figure 47. CD69 expression on PBMC-derived CD4 T cells after 48 hours of co-incubation with Nalm 6 cells and increasing concentrations of CD19-CD28v15 in the presence or absence of 10 nM CD20-TCB is shown. Dots represent technical triplicates with SEM. [Figure 48] Figures 48A-48D illustrate that CD19-CD28 alone does not induce cytokine secretion in PBMCs. Cytokine release in total PBMCs after 48 hours of co-culture with CD19-CD28 molecules in the presence or absence of CD20-TCB is shown. Bars represent the mean + SEM of technical triplicates. Data are representative of two donors. Cytokine secretion was assessed by Bio-Plex Pro Human Cytokine 17 plex Assay. IFNγ (Figure 48A), IL-2 (Figure 48B), IL-10 (Figure 48C), and TNF (Figure 48D) are shown. [Figure 49]Figures 49A and 49B show functional data for CD79b-CD28, which enhances CD20-TCB in an IL-2 reporter assay in the presence of Z138 B cells. Figure 49A shows the median fluorescence intensity (MFI) of binding to CD79b on Z138 B cells. Figure 49B shows that CD79b-CD28 enhances CD20-TCB in an IL-2 reporter assay in the presence of Z138 B cells. IL-2 reporter cell activation, measured by luminescence readout (LUM), after 6 hours of co-incubation with different B cell lines in the presence of suboptimal concentrations of CD20-TCB and increasing concentrations of CD79b-CD28 is shown. Dots represent technical replicates with SEM. [Figure 50] Figure 50 shows the study design for an efficacy study using a bispecific CD19-CD28 antibody (comparison of two different CD28 clones) in NALM6 xenografts in humanized mice. The design and different treatment groups are shown. [Figure 51] Figures 51A-51D show the results of an efficacy study with CD19-CD28 in NALM6 xenografts in humanized mice. The mean tumor volume (Figure 51A) or tumor growth in individual mice for the three treatment groups is plotted on the y-axis (Figures 51B-51D). Figure 51B shows tumor growth in individual mice in the vehicle group, Figure 51C shows a mouse treated with CD19-CD28 (variant 15), and Figure 51D shows a mouse treated with CD19-CD28 (variant 8). It can be seen that CD19-CD28 (variant 8) as a single agent induced stronger tumor growth inhibition compared to CD19-CD28 (variant 15). DETAILED DESCRIPTION OF THE INVENTION

[0051] definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa.

[0052] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and scaffold antigen-binding proteins.

[0053] As used herein, the term "antigen-binding domain that binds to a tumor-associated antigen" or "moiety capable of specifically binding to a tumor-associated antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding domain is capable of activating signal transduction via its target cell antigen. In a particular embodiment, the antigen-binding domain can direct an entity (e.g., a CD28 antibody) that binds to a target site, e.g., a specific type of tumor cell, or tumor stroma that bears the antigenic determinant. Antigen-binding domains capable of specifically binding to a target cell antigen include antibodies and fragments thereof as further defined herein. Furthermore, antigen-binding domains capable of specifically binding to a target cell antigen include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565).

[0054] With respect to an antigen-binding molecule, i.e., an antibody or a fragment thereof, the term "antigen-binding domain that binds to a target cell antigen" refers to a portion of the molecule that contains a region that specifically binds to and is complementary to a part or all of an antigen. An antigen-binding domain capable of specific antigen binding can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Specifically, an antigen-binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In another embodiment, an "antigen-binding domain capable of specific binding to a tumor-associated antigen" may be a Fab fragment or a cross-Fab fragment. In another embodiment, an "antigen-binding domain capable of specific binding to a tumor-associated antigen" may be a Fab fragment or a cross-Fab fragment. As used herein, the terms "first," "second," and "third" with respect to antigen-binding domains, etc., are used for the convenience of distinguishing when two or more of each type of moiety are present. The use of these terms is not intended to imply a particular order or orientation of the moieties, unless explicitly indicated as such.

[0055] As used herein, the term "antigen binding domain that binds to a B cell surface antigen" or "moiety capable of specifically binding to a B cell surface antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant on the B cell surface. In one aspect, an antigen binding domain is capable of activating signaling via its target cell antigen. In particular aspects, an antigen binding domain is capable of targeting an entity to which it is bound (e.g., a CD28 agonist) to a target site, e.g., on a B cell. Antigen binding domains capable of specifically binding to a B cell surface antigen include antibodies and fragments thereof as further defined herein. Furthermore, antigen binding domains capable of specifically binding to a B cell surface antigen include scaffold antigen binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565).

[0056] The term "antigen-binding domain that binds to a multiple myeloma (MM) cell surface antigen" or "moiety capable of specifically binding to a multiple myeloma (MM) cell surface antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant on a multiple myeloma (MM) cell. In one embodiment, an antigen-binding domain is capable of activating signaling through its target cell antigen. In particular embodiments, an antigen-binding domain is capable of targeting an entity to which it is bound (e.g., a CD28 agonist) to a target site, e.g., on an MM cell. Antigen-binding domains capable of specifically binding to a multiple myeloma (MM) cell surface antigen include antibodies and fragments thereof as further defined herein. Furthermore, antigen-binding domains capable of specifically binding to a B-cell surface antigen include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565).

[0057] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0058] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, excluding possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of a monoclonal antibody preparation. Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0059] The term "monospecific" antibody, as used herein, refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific for a different antigenic determinant. However, a bispecific antigen-binding molecule may also contain an additional antigen-binding site that binds to an additional antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells or the same cell. Thus, the term "bispecific" according to the present invention may also include trispecific molecules, such as a CD28 antibody and a bispecific molecule comprising two antigen-binding domains directed against two different target cell antigens.

[0060] The term "monovalent" as used herein means that within an antigen-binding molecule specific for one different antigenic determinant, there is a specific number of binding sites specific for one different antigenic determinant. Thus, the terms "bivalent," "tetravalent," and "hexavalent" mean that there are two, four, and six binding sites specific for a particular antigenic determinant in the antigen-binding molecule, respectively. In certain embodiments of the present invention, bispecific antigen-binding molecules according to the present invention can be monovalent for a particular antigenic determinant, meaning that they have only one binding site for that antigenic determinant. Alternatively, they can be bivalent or tetravalent for a particular antigenic determinant, meaning that they have two or four binding sites for that antigenic determinant, respectively.

[0061] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also called a variable heavy domain or a heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL) (also called a variable light domain or a light chain variable domain) followed by a light chain constant domain (CL) (also called a light chain constant region). The heavy chain of an antibody may be assigned to one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and some of these may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody may be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0062] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994), and also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments containing two antigen-binding domains that may be bivalent or bispecific; see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003), and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc. (Waltham, Massachusetts), see, e.g., U.S. Pat. No. 6,248,516 B1).Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0063] Papain digestion of an intact antibody yields two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment containing a light chain fragment containing the variable light (VL) domain and constant domain of the light chain (CL), and a variable heavy (VH) domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) in the constant domains retain a free thiol group. Pepsin treatment yields an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0064] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two possible chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention. On the other hand, the variable regions of the Fab heavy and light chains are swapped, i.e., the crossover Fab molecule contains a peptide chain composed of a light chain variable (VL) domain and a heavy chain constant domain (CH1), and a peptide chain composed of a heavy chain variable domain (VH) and a light chain constant domain (CL). This crossover Fab molecule is a cross-Fab (VLVH)On the other hand, when the constant regions of the Fab heavy and light chains are replaced, the crossover Fab molecule contains a peptide chain consisting of a heavy chain variable domain (VH) and a light chain constant domain (CL), and a peptide chain consisting of a light chain variable domain (VL) and a heavy chain constant domain (CH1). This crossover Fab molecule is called a crossover Fab. (CLCH1) It is also called.

[0065] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker are arranged in one of the following orders from N- to C-terminus: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL. The linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. Furthermore, these single-chain Fab molecules may be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering).

[0066] A "crossover single-chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker are arranged in one of the following orders from N- to C-terminus: (a) VH-CL-linker-VL-CH1 and (b) VL-CH1-linker-VH-CL. Together, VH and VL form an antigen-binding site that specifically binds to a given antigen, and the linker is a polypeptide of at least 30 amino acids. Furthermore, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain, according to the Kabat numbering system).

[0067] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V) linked using a short linker peptide of 10 to about 25 amino acids. H ) and light chain (V L ) variable region fusion proteins. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and H N-terminus and V L The scFv antibody can be linked to the C-terminus of a full-length antibody, or vice versa. This protein retains the specificity of the original antibody, although the constant regions have been removed and a linker has been introduced. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments contain a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0068] "Scaffold antigen-binding proteins" are known in the art; for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains. See, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one aspect of the invention, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (e.g., CTLA-4, lipocalins, anticalins), protein A-derived molecules such as the Z-domain (affibodies), A-domain (avimers / maxibodies) of protein A, serum transferrin (transbodies), engineered ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAbs), variable domains of antibody heavy chains (nanobodies, aVHs), VHs, ... NAR Fragments, fibronectin (adnectin), C-type lectin domain (tetranectin); variable domain of novel antigen receptor β-lactamase (V NAR fragments), human gamma-crystallin or ubiquitin (affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies, e.g., proteins from the knottin family, peptide aptamers, and fibronectin (adnectins). CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is primarily a cytotoxic T-lymphocyte-associated antigen. +It is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to antibody CDRs may be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as ebibodies (e.g., U.S. Patent No. 7,166,697 B1). Ebibodies are approximately the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with many loops at the open end of a conical structure that can be engineered to bind to different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297 B1, and U.S. Patent Application Publication No. 20070224633. Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. Domains consist of three helical bundles of approximately 58 amino acids. Libraries are created by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and European Patent Application Publication No. 1641818 A1. Avimers are multidomain proteins derived from the A-domain scaffold family. Native domains of approximately 35 amino acids conform to defined disulfide-bonded structures. Diversity is generated by shuffling the natural variation exhibited by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the adhesion of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. A single ankyrin repeat can be engineered to bind different target antigens by randomizing residues in the first α-helix and β-turn of each repeat. The binding interface can be increased by increasing the number of modules (affinity maturation method). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US Patent Application Publication No. 20040132028 A1. Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domains were derived from the variable domain of an antibody heavy chain from camelids (nanobodies or V. H Furthermore, the term single domain antibody refers to an antibody that contains an autonomous human heavy chain variable domain (aVH) or a shark-derived VH. NARThese include fragments. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins consist of a backbone with the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, WO 2005056764, and U.S. Patent No. 6,818,418 B1. Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop that is inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins that are 25-50 amino acids long and contain 3-4 cysteine ​​bridges; examples of microproteins include KalataBI, conotoxins, and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0069] An "antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of the reference molecule to its antigen by 50% or more in a competitive assay; conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0070] The term "antigen-binding domain" refers to a portion of an antigen-binding molecule that specifically binds to and is complementary to a part or all of an antigen. When an antigen is large, an antigen-binding molecule may bind only to a specific portion of the antigen, which portion is called an epitope. An antigen-binding domain may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0071] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of noncontiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or within the extracellular matrix (ECM). Proteins useful as antigens of the present invention can be any native form of the protein from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When referring to a particular protein of the present invention, the term encompasses the "full-length," unprocessed protein and any form of the protein obtained from cellular processing. The term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants.

[0072] "Specifically bind" means that the binding is antigen-selective and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known in the art, such as surface plasmon resonance (SPR) technology (analyzed using a BIAcore device) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, molecules that bind to an antigen have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 M).

[0073] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to specific binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be expressed by the dissociation constant (Kd), which is the ratio of the desorption rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities can involve different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by methods common in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0074] "Tumor-associated antigen" or TAA, as used herein, refers to an antigenic determinant displayed on the surface of target cells, e.g., cells within a tumor, such as cancer cells, cells of the tumor stroma, malignant B lymphocytes, or melanoma cells. In certain embodiments, the target cell antigen is an antigen on the surface of a tumor cell. In one embodiment, the TAA is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), folate receptor alpha (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), p95HER2, EpCAM, HER3, CD30 or TPBG (5T4), CD19, CD79b, CD20, CD22, CD37, CD38, BCMA, and GPRC5D. In one particular aspect, the TAA is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), folate receptor alpha (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and p95HER2. In another particular aspect, the TAA is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), EpCAM, HER3, CD30, or TPBG (5T4). In one particular aspect, the tumor-associated antigen is fibroblast activation protein (FAP) or carcinoembryonic antigen (CEA). In one aspect, the TAA is a B-cell surface antigen selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37, in particular CD19 and CD79b. In one embodiment, the TAA is a multiple myeloma (MM) cell surface antigen selected from the group consisting of CD38, BCMA, and GPRC5D.

[0075] The term "fibroblast activation protein (FAP)," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any naturally occurring FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed FAPs and any form of FAP that results from processing in cells. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO: 2) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence of His-tagged human FAP ECD is shown in SEQ ID NO: 135. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO: 136) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NO: 137 shows the amino acid sequence of His-tagged mouse FAP ECD. SEQ ID NO: 138 shows the amino acid sequence of His-tagged cynomolgus monkey FAP ECD. Preferably, the anti-FAP binding molecules of the invention bind to the extracellular domain of FAP.

[0076] The term "carcinoembryonic antigen (CEA)," also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), refers to any naturally occurring CEA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CEA is set forth in UniProt Accession No. P06731 (version 151, SEQ ID NO: 3). CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462, 1965; Berinstein NL, J Clin Oncol., 20:2197-2207, 2002). Originally classified as a protein expressed exclusively in fetal tissues, CEA has now been identified in several healthy adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urinary tracts, as well as cells of the colon, cervix, sweat glands, and prostate (Nap et al., Tumor Biol., 9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992). Epithelial-derived tumors and their metastases contain CEA as a tumor-associated antigen. The presence of CEA itself does not imply transformation into cancerous cells, but indicates its distribution. In healthy tissues, CEA is generally expressed on the apical surface of cells (Hammarstroem S., Semin Cancer Biol. 9(2):67-81(1999)), making it inaccessible to antibodies in the bloodstream. In contrast to healthy tissues, CEA is expressed throughout cancerous cells (Hammarstroem S., Semin Cancer Biol. 9(2):67-81(1999)). This change in expression pattern makes CEA more accessible to antibody binding within cancerous cells. Furthermore, CEA expression increases in cancerous cells. Furthermore, increased CEA expression increases cell-cell adhesion, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003).The prevalence of CEA expression in various tumor components is generally very high. In agreement with published data, our own analyses performed on tissue samples confirmed its high prevalence, approximately 95% in colon cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC co-expressing HER3), and 40% in breast cancer, with lower expression in small cell lung cancer and glioblastoma.

[0077] CEA is rapidly cleaved from the cell surface and enters the bloodstream either directly from tumors or via the lymphatic system. Because of this property, serum CEA levels have been used as a clinical marker for cancer diagnosis and screening for cancer recurrence, particularly colorectal cancer (Goldenberg D M., The International Journal of Biological Markers, 7:183-188, 1992; Chau I., et al., J Clin Oncol., 22:1420-1429, 2004; Flamini et al., Clin Cancer Res; 12(23):6985-6988, 2006).

[0078] The term "epithelial cell adhesion molecule (EpCAM)" refers to any native EpCAM from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed EpCAM and any form of EpCAM resulting from processing in cells. The term also encompasses naturally occurring variants of EpCAM, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse, and / or cynomolgus monkey EpCAM. The amino acid sequence of human EpCAM is set forth in UniProt (www.uniprot.org) Accession No. P16422 (version 167, SEQ ID NO: 68) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_002345.2. The amino acid sequence of mouse EpCAM is shown in UniProt (www.uniprot.org) accession number Q99JW5 (version 111, SEQ ID NO: 75) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_032558.2. Epithelial cell adhesion molecule (EpCAM), also known as tumor-associated calcium signaling substance 1 (TACSTD1), 17-1A, and CD326, is a type I, approximately 40 kDa, transmembrane glycoprotein frequently overexpressed in cancers of epithelial origin and by cancer stem cells, and is therefore a molecule of significant interest for therapy and diagnosis. The extracellular domain of EpCAM can be cleaved to yield a soluble extracellular domain molecule, EpEX, and an intracellular molecule, EpICD. EpICD has been shown to associate with other proteins to form nuclear complexes that upregulate the expression of genes that promote cell proliferation. EpCAM may also be involved in epithelial-to-mesenchymal cell transition (EMT) and contribute to the formation of large metastases.

[0079] "CD30" or "TNFRSF8" is a member of the tumor necrosis factor receptor superfamily. It is characteristically expressed in certain hematopoietic malignancies, including anaplastic large cell lymphoma and Hodgkin's lymphoma, among others. The variable expression of CD30 on both normal and malignant lymphoid cells has focused research efforts on understanding the pathogenesis of CD30 upregulation, its contribution to lymphopoiesis via anti-apoptotic mechanisms, and its effects on cell survival. Given the restriction of CD30 to certain tumor types, a logical extension of this has been to attempt to exploit it as a therapeutic target. CD30 is a 120 kD transmembrane glycoprotein receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily, possessing intracellular, transmembrane, and extracellular domains. The amino acid sequence of human CD30 is set forth in UniProt Accession No. P28908 (SEQ ID NO: 472).

[0080] The term "TPBG" refers to trophoblast glycoprotein, also known as "5T4." TBPG is a leucine-rich transmembrane glycoprotein involved in cell adhesion. In adults, this protein is highly expressed in many tumor cells and is associated with poor clinical outcomes in many cancers. Unless otherwise specified, this term refers to any native TPBG from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human TPBG is set forth in UniProt Accession No. Q13641 (SEQ ID NO: 473).

[0081] The term "FolR1" refers to folate receptor alpha, which has been identified as a potential prognostic and therapeutic target in several cancers. Unless otherwise specified, this term refers to any native FolR1 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human FolR1 is set forth in UniProt Accession No. P15328 (SEQ ID NO: 139), mouse FolR1 has the amino acid sequence set forth in UniProt Accession No. P35846 (SEQ ID NO: 140), and cynomolgus monkey FolR1 has the amino acid sequence set forth in UniProt Accession No. G7PR14 (SEQ ID NO: 141). FolR1 is an N-glycosylated protein expressed on the plasma membrane of cells. FolR1 has high affinity for folic acid and several reduced folate derivatives and mediates the delivery of the physiological folate, 5-methyltetrahydrofolate, into cells. FOLR1 is overexpressed in a large proportion of ovarian cancers, as well as many uterine, endometrial, pancreatic, renal, lung, and breast cancers. However, FOLR1 expression in normal tissues is restricted to the apical membranes of kidney proximal tubules, lung alveolar pneumocytes, bladder cancer, testicular cancer, choroid plexus, and thyroid epithelial cells, making it a desirable target for FOLR1-directed cancer therapy. Recent studies have identified that FolR1 expression is particularly high in triple-negative breast cancer (Necela et al. PloS One 2015, 10(3), e0127133).

[0082] The term "melanoma-associated chondroitin sulfate proteoglycan (MCSP)," also known as chondroitin sulfate proteoglycan 4 (CSPG4), refers to any naturally occurring MCSP derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human MCSP is set forth in UniProt Accession No. Q6UVK1 (version 103, SEQ ID NO: 142). MCSP is a highly glycosylated integral membrane chondroitin sulfate proteoglycan consisting of an N-linked 280 kDa glycoprotein component and a 450 kDa chondroitin sulfate proteoglycan component expressed on the cell membrane (Ross et al., Arch. Biochem. Biophys. 1983, 225:370-38). MCSP is more widely distributed in many normal and transformed cells. In particular, MCSP is found in almost all basal cells of the epidermis. MCSP is differentially expressed in melanoma cells and has been found to be expressed in more than 90% of benign nevi and melanoma lesions analyzed. MCSP has also been found to be expressed in tumors of non-melanocytic origin, including basal cell carcinoma, various tumors of neural crest origin, and breast cancer.

[0083] The term "epidermal growth factor receptor (EGFR)," also known as the proto-oncogene c-ErbB-1 or receptor tyrosine-protein kinase erbB-1, refers to any native EGFR from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human EGFR is set forth in UniProt Accession No. P00533 (version 211, SEQ ID NO: 143). The proto-oncogene "HER2" (human epidermal growth factor receptor 2) encodes a protein tyrosine kinase (p185HER2) that is related to and somewhat homologous to the human epidermal growth factor receptor. HER2 is also known in the art as c-erbB-2 and is sometimes referred to as the rat homolog, neu. Amplification and / or overexpression of HER2 is associated with multiple human malignancies and appears to be integrally involved in the progression of 25-30% of human breast and ovarian cancers. Furthermore, the degree of amplification is inversely correlated with observed median patient survival (Slamon, DJ et al., Science 244:707-712 (1989)). The amino acid sequence of human HER2 is shown in UniProt Accession No. P04626 (version 230, SEQ ID NO: 144). As used herein, the term "p95HER2" refers to the carboxy-terminal fragment (CTF) of the HER2 receptor protein, also known as "611-CTF" or "100-115 kDa p95HER2." The p95HER2 fragment is generated intracellularly by translation initiation of HER2 mRNA at codon position 611 of the full-length HER2 molecule (Anido et al., EMBO J 25;3234-44 (2006)). It has a molecular weight of 100-115 kDa and is expressed in the cell membrane, where it can form homodimers maintained by intermolecular disulfide bonds (Pedersen et al., Mol Cell Biol 29, 3319-31 (2009)). An exemplary sequence of human p95HER2 is given in SEQ ID NO: 145.

[0084] "HER3" or "ErbB3" (human epidermal growth factor receptor 3), like other members of the ErbB receptor tyrosine kinase family, consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains four subdomains (I-IV). Subdomains I and III are leucine-rich and are primarily involved in ligand binding. Subdomains II and IV are cysteine-rich and most likely contribute to protein conformation and stability through disulfide bond formation. Subdomain II also contains a dimerization loop required for dimer formation. The cytoplasmic domain contains a juxtamembrane segment, a kinase domain, and a C-terminal domain. While there is no evidence that overexpression, constitutive activation, or mutations of ErbB3 alone are oncogenic, the protein, most critically associated with ErbB2 as a heterodimerization partner, is involved in promoting growth, proliferation, chemoresistance, and invasion and metastasis (https: / / en.wikipedia.org / wiki / ERBB3-cite_note-pmid8632008-18). ErbB3 is associated with resistance to targeted therapy in many cancers. The amino acid sequence of human HER3 is shown in UniProt accession number P21860 (version 224, SEQ ID NO: 471).

[0085] As used herein, "B cell surface antigen" refers to an antigenic determinant displayed on the surface of B lymphocytes, particularly malignant B lymphocytes (in which case the antigen is also referred to as a "malignant B cell surface antigen"). Several B cell surface antigens are of interest for immunotherapy of hematological malignancies. In one aspect, the B cell surface antigen is selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37.

[0086] The term "CD19" refers to the B lymphocyte antigen CD19, also known as B lymphocyte surface antigen B4 or T cell surface antigen Leu-12, and includes any native CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CD19 is set forth in Uniprot Accession No. P15391 (version 160, SEQ ID NO: 434). This term encompasses "full-length," unprocessed human CD19, as well as any form of human CD19 resulting from processing in cells, so long as the antibodies described herein bind to it. CD19 is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, early developmental B cells (i.e., immature B cells), mature B cells through terminal differentiation to plasma cells, and malignant B cells. CD19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, and some null acute lymphoblastic leukemias. Expression of CD19 on plasma cells further suggests that it may be expressed on differentiated B-cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0087] "CD79b" refers to the B-cell antigen receptor complex-associated protein β chain, also known as Ig-β or B-cell-specific glycoprotein B29, and unless otherwise specified, includes any native CD79b from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD79b is set forth in Uniprot Accession No. P40259 (version 180, SEQ ID NO: 435). CD79b is a 39 KDa protein expressed exclusively on B cells and, in conjunction with CD79a, initiates a signaling cascade downstream of the BCR, leading to internalization of the BCR complex, its translocation to endosomes, and antigen presentation. CD79 (composed of the subunits CD79a and CD79b) is a heterodimeric signaling component of the B cell receptor that is ubiquitously expressed in mature B cell lymphomas and is located on the cell surface by the earliest committed B cell precursors before the expression of immunoglobulin μ. The term "CD79b" encompasses "full-length," unprocessed CD79b, as well as any form of CD79b that results from intracellular processing. The term also encompasses naturally occurring variants of CD79b, such as splice variants or allelic variants.

[0088] "CD20" refers to the B lymphocyte antigen CD20, also known as B lymphocyte surface antigen B1 or leukocyte surface antigen Leu-16, and includes any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CD20 is set forth in Uniprot Accession No. P11836 (version 149, SEQ ID NO: 436). CD20 is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD expressed on pre-B lymphocytes and mature B lymphocytes. The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this emerging protein family are characterized by common structural features and similar intron / exon splice boundaries and display unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is localized to 11q12 within a cluster of family members. Alternative splicing of this gene results in two transcript variants that encode the same protein. The term "CD20" encompasses "full-length," unprocessed CD20, as well as any form of CD20 that results from intracellular processing. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants.

[0089] "CD22" refers to the B cell receptor CD22, also known as B lymphocyte cell adhesion molecule or SIGLEC2, and unless otherwise specified, includes any native CD22 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD22 is set forth in Uniprot Accession No. P20273 (version 209, SEQ ID NO: 437). CD22 is a member of the SIGLEC family of lectins and is found on the surface of mature B cells, but is less abundant on some immature B cells. Thus, CD22 is a 130-150 kDa B cell-restricted cell surface phosphoglycoprotein that can regulate the generation of B lymphocyte antigen receptor (BCR)-mediated signals as well as BCR-independent signals. The term "CD22" encompasses "full-length," unprocessed CD22, as well as any form of CD22 obtained from intracellular processing. The term also encompasses naturally occurring variants of CD22, such as splice variants or allelic variants.

[0090] "CD37" refers to the leukocyte antigen CD37, also known as tetraspanin-26 (Tspan-26), and, unless otherwise specified, includes any native CD37 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD37 is set forth in Uniprot Accession No. P11049 (version 162, SEQ ID NO: 438). CD37 expression is restricted to cells of the immune system, being most abundant on mature B cells, with lower expression found on T cells and myeloid cells. The glycoprotein CD37 is a member of the transmembrane 4 superfamily and regulates both humoral and cellular immune responses. The term "CD37" encompasses "full-length," unprocessed CD37, as well as any form of CD37 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD37, such as splice variants or allelic variants.

[0091] As used herein, "multiple myeloma (MM) cell surface antigen" refers to an antigenic determinant displayed on the surface of multiple myeloma (MM) cells. Several MM cell surface antigens are of interest for immunotherapy of multiple myeloma. In one aspect, the MM cell surface antigen is selected from the group consisting of CD38, BCMA, and GPRC5D.

[0092] The term "CD38," also known as cluster of differentiation 38 or cyclic ADP-ribosyl hydrolase, is a glycoprotein found on the surface of many immune cells (white blood cells), including CD4+, CD8+, B lymphocytes, and natural killer cells. CD38 also functions in cell adhesion, signal transduction, and calcium signaling. Under normal conditions, CD38 is expressed at relatively low levels on myeloid and lymphoid cells and some non-hematopoietic tissues. In contrast, normal plasma cells and multiple myeloma (MM) cells have high levels of CD38 expression, making CD38 an attractive target for targeting cell surface molecules in MM. As used herein, CD38 refers to any CD38 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CD38 is shown in UniProt (www.uniprot.org) accession number P28907 (SEQ ID NO: 474).

[0093] The term "BCMA" refers to B-cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRS17), a type III transmembrane protein lacking a signal peptide and containing a cysteine-rich extracellular domain. BCMA is expressed at significantly elevated levels in all patient MM cells but not in other normal tissues except normal plasma cells. Together with two related receptors, the TNFR superfamily B-cell activating factor receptor (BAFF-R) and the transmembrane activator and calcium regulator and cyclophilin ligand interactor (TACI), BCMA critically regulates B-cell proliferation and survival, as well as maturation and differentiation into plasma cells. These three functionally related receptors support the long-term survival of B cells at different stages of development by binding to their cognate ligands, BAFF and / or APRIL. As used herein, BCMA refers to any BCMA protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human BCMA is set forth in UniProt (www.uniprot.org) Accession No. Q02223 (SEQ ID NO: 475).

[0094] The term "GPRC5D" refers to G protein-coupled receptor class C group 5 member D, a target identified from plasma cells of multiple myeloma using RNA sequencing. GPRC5D has been reported to be associated with poor prognosis and tumor burden in multiple myeloma patients. Unless otherwise specified, GPRC5D refers to any GPRC5D protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human GPRC5D is set forth in UniProt (www.uniprot.org) accession number Q9NZD1 (SEQ ID NO: 476).

[0095] The term "CD28" (cluster of differentiation 28, Tp44), unless otherwise specified, refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides costimulatory signals necessary for T cell activation and survival. T cell stimulation by CD28 in addition to the T cell receptor (TCR) can provide a potent signal for the production of various interleukins. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is set forth in UniProt (www.uniprot.org) accession number P10747 (SEQ ID NO: 1).

[0096] An "agonist antibody" refers to an antibody that has agonist function for a given receptor. Generally, when an agonist ligand (factor) binds to a receptor, the tertiary structure of the receptor protein changes, activating the receptor (if the receptor is a membrane protein, cell growth signals, etc., are usually transduced). If the receptor is a dimer-forming type, an agonist antibody can dimerize the receptor at an appropriate distance and angle, thus acting in the same way as the ligand. An appropriate anti-receptor antibody can mimic the receptor dimerization caused by the ligand, and therefore can be an agonist antibody.

[0097] A "CD28 agonist antigen-binding molecule" or "CD28 conventional agonist antigen-binding molecule" is an antigen-binding molecule that mimics the CD28 natural ligand (CD80 or CD86) in its role of enhancing T cell activation in the presence of a T cell receptor signal ("signal 2"). T cells require two signals to be fully activated. Under physiological conditions, "signal 1" results from the interaction of the T cell receptor (TCR) molecule with a peptide / major histocompatibility complex (MHC) complex on an antigen-presenting cell (APC), and "signal 2" is provided by the engagement of a costimulatory receptor, such as CD28. CD28 agonist antigen-binding molecules can costimulate T cells (signal 2). CD28 agonist antigen-binding molecules can also induce T cell proliferation and cytokine secretion in combination with molecules specific for the TCR complex, but CD28 agonist antigen-binding molecules cannot fully activate T cells without further stimulation of the TCR. However, there exists a subclass of CD28-specific antigen-binding molecules, so-called CD28 superagonist antigen-binding molecules. "CD28 superagonist antigen-binding molecules" are CD28 antigen-binding molecules that can fully activate T cells without further TCR stimulation. CD28 superagonist antigen-binding molecules can induce T cell proliferation and cytokine secretion without prior T cell activation (Signal 1).

[0098] The term "variable domain" or "variable region" refers to the domain of an antibody heavy or light chain that is involved in binding of the antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0099] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antigen-binding variable domain, e.g., "complementarity-determining regions" (CDRs), that are hypervariable in sequence and determine antigen-binding specificity. Generally, an antigen-binding domain comprises six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0100] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR designations can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature. Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. Those skilled in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence without reliance on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in antibody variable regions are in accordance with the Kabat numbering system.

[0101] As used herein, the term "affinity maturation" in relation to an antigen-binding molecule (e.g., an antibody) refers to an antigen-binding molecule that is derived from a reference antigen-binding molecule, for example, by mutation, and binds to the same antigen as the reference antibody, preferably to the same epitope, and has a higher affinity for the antigen than the reference antigen-binding molecule. Affinity maturation generally involves modifying one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, the affinity-matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule.

[0102] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0103] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or may comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0104] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0105] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0106] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from that of the original antibody to generate properties according to the invention, particularly in terms of C1q binding and / or Fc receptor (FcR) binding.

[0107] A "human" antibody is an antibody having an amino acid sequence that corresponds to that of an antibody derived from a non-human source, whether produced by a human or human cell, or utilizing the human antibody repertoire or other human antibody-encoding sequences. This definition of human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0108] The term "CH1 domain" refers to a portion of an antibody heavy chain polypeptide extending from approximately EU position 118 to EU position 215 (EU numbering system according to Kabat). In one embodiment, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 477). Typically, a segment having the amino acid sequence of EPKSC (SEQ ID NO: 480) then connects the CH1 domain to the hinge region.

[0109] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 domains in a wild-type antibody heavy chain (e.g., from about position 216 to about position 230, or from about position 226 to about position 230 according to the EU numbering system of Kabat). Hinge regions of other IgG subclasses can be determined by aligning with the hinge region cysteine ​​residues of the IgG1 subclass sequence. Hinge regions are usually dimeric molecules consisting of two polypeptides with identical amino acid sequences. Hinge regions generally contain up to 25 amino acid residues and are flexible, allowing the associated target binding sites to move independently. The hinge region can be subdivided into three domains: the upper, middle, and lower hinge domains (see, e.g., Roux, et al., J. Immunol. 161 (1998) 4083).

[0110] In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO:481), wherein X is either S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO:482), wherein X is either S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO:483), wherein X is either S or P.

[0111] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. An IgG Fc region contains the IgG CH2 domain and the IgG CH3 domain.

[0112] The "CH2 domain" of a human IgG Fc region typically extends from about the amino acid residue at EU position 231 to about the amino acid residue at EU position 340 (EU numbering system according to Kabat). In one embodiment, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 478). The CH2 domain is unique in that it is not tightly paired with other domains. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc region. It has been hypothesized that the carbohydrates provide a surrogate for domain-domain pairing and help stabilize the CH2 domains. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chain is attached to the CH2 domain. The CH2 domain of the present invention may be a native sequence CH2 domain or a variant CH2 domain.

[0113] A "CH3 domain" refers to a portion of an antibody heavy chain polypeptide comprising a stretch of residues C-terminal to the CH2 domain in the Fc region, extending from approximately EU position 341 to EU position 446 (EU numbering system according to Kabat). In one aspect, the CH3 domain has the amino acid sequence GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO:479). A CH3 region of the invention may be a native-sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protrusion" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such mutant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0114] The "knob-into-hole" technique is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a further specific embodiment, the Fc domain subunit containing the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0115] A "region equivalent to the Fc region of an immunoglobulin" is intended to include allelic variants of naturally occurring immunoglobulin Fc regions and variants having alterations that result in substitutions, additions, or deletions, but that do not substantially reduce the ability of the immunoglobulin to mediate effector function (e.g., antibody-dependent cellular cytotoxicity). For example, one or more amino acids may be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants may be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie, JU et al., Science 247:1306-10 (1990)).

[0116] The term "wild-type Fc domain" refers to an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions, and native human IgG4 Fc regions, as well as naturally occurring variants thereof. Wild-type Fc regions are set forth in SEQ ID NO: 484 (IgG1, Caucasian allotype), SEQ ID NO: 485 (IgG1, Afro-American allotype), SEQ ID NO: 486 (IgG2), SEQ ID NO: 487 (IgG3), and SEQ ID NO: 488 (IgG4).

[0117] The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the "wild-type" (human) Fc domain amino acid sequence by at least one "amino acid mutation." In one embodiment, the variant Fc region has at least one amino acid mutation compared to a native Fc region, e.g., about 1 to about 10 amino acid mutations, and in one embodiment, about 1 to about 5 amino acid mutations in the native Fc region. In one embodiment, the (variant) Fc region has at least about 95% homology to the wild-type Fc region.

[0118] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0119] Fc receptor binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), which are dedicated cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and other cellular targets (e.g., tumor cells) coated with the corresponding antibodies by antibody-dependent cellular cytotoxicity (ADCC) (see, e.g., Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0120] Cross-linking of receptors to the Fc region of IgG antibodies (FcγR) triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as control of immune complex clearance and antibody production. In humans, three classes of FcγR have been characterized, these are:

[0121] FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modifications within the Fc region at at least one of amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbering according to the EU index of Kabat) reduce binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with IgG1 and IgG4 reduced binding to FcγRI by 103-fold and eliminated human mononuclear cell responses to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624).

[0122] FcγRII (CD32) binds complexed IgG with moderate to low affinity and is widely expressed. The receptor can be divided into two subtypes: FcγRIIA and FcγRIIB. FcγRIIA is found primarily on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and has been found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function in suppressing immunoglobulin production and further isotype switching, for example, to the IgE class. On macrophages, FcγRIIB plays a role in inhibiting phagocytosis, as mediated by FcγRIIA. On eosinophils and mast cells, the B form may help suppress the activation of these cells by binding IgE to its respective receptor. Decreased binding to FcγRIIA has been found, for example, for antibodies comprising an IgG Fc region with mutations in at least one of amino acid residues E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index of Kabat).

[0123] FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two species. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Decreased binding to FcγRIIIA has been observed, for example, for antibodies containing an IgG Fc region with a mutation at least in one of the following amino acid residues: E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (numbering according to the EU index of Kabat).

[0124] Mapping of the binding site on human IgG1 for Fc receptors, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604.

[0125] The term "ADCC" or "antibody-dependent cellular cytotoxicity" refers to an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or derivative thereof containing an Fc region specifically binds, typically via a protein portion N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as either a decrease in the number of target cells lysed in a given time period with a given concentration of antibody in the medium surrounding the target cells, due to the mechanism of ADCC defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period, due to the mechanism of ADCC. The decrease in ADCC is compared to unengineered ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, an amino acid substitution that reduces ADCC, which is a reduction in ADCC mediated by an antibody containing its Fc domain, is relative to ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Application WO 2006 / 082515 or PCT Application WO 2012 / 130831). For example, the ability of an antibody to trigger the early steps that mediate ADCC is investigated by measuring binding of the antibody to cells expressing Fcγ receptors, such as cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which constitutively express FcγRIIIA). Specifically, binding to FcγR on NK cells is measured.

[0126] An "activating Fc receptor" is an Fc receptor that, upon binding by the Fc region of an antibody, triggers signaling events that stimulate the receptor-containing cell to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0127] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). The ectodomain is usually the part of the protein that initiates contact with a surface that results in signal transduction.

[0128] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n ,(SG4) n or G4 (SG4) n and a peptide linker, wherein "n" is generally a number from 1 to 5, typically from 2 to 4, and in particular 2, i.e., the peptide is selected from the group consisting of GGGGS (SEQ ID NO: 146), GGGGSGGGGS (SEQ ID NO: 147), SGGGGSGGGG (SEQ ID NO: 148) and GGGGSGGGGSGGGG (SEQ ID NO: 149), but also includes the sequences GSPGSSSSGS (SEQ ID NO: 150), (GS) (SEQ ID NO: 151), (GS) (SEQ ID NO: 152), GSGSGSGS (SEQ ID NO: 153), GSGSGNGS (SEQ ID NO: 154), GGSGSGSG (SEQ ID NO: 155), GGSGSG (SEQ ID NO: 156), GGSG (SEQ ID NO: 157), GGSGNGSG (SEQ ID NO: 158), GGNGSGSG (SEQ ID NO: 159) and GGNGSG (SEQ ID NO: 160). Peptide linkers of particular interest are (G4S) (SEQ ID NO: 146), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 147), (G4S)3 (SEQ ID NO: 151) and (G4S)4 (SEQ ID NO: 152).

[0129] The term "amino acid," as used herein, refers to the group of naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0130] By "fused" or "connected" is meant that the components (e.g., the polypeptide and ectodomain of the TNF ligand family member) are linked by a peptide bond directly or via one or more peptide linkers.

[0131] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, SAWI, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and the source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, CA), or may be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A (which may alternatively be purchased as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) to, with, or against a given amino acid sequence B is calculated as follows:100 times the ratio X / Y (X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in an alignment of A and B in that program, and Y is the total number of amino acid residues in B). It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0132] In certain embodiments, amino acid sequence variants of the CD28 antigen-binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the CD28 antigen-binding molecules. Amino acid sequence variants of the CD28 antigen-binding molecules can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics (e.g., antigen binding). Target sites for substitutional mutagenesis include HVRs and framework regions (FRs). Conservative substitutions are provided in Table B under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC). [Table A]

[0133] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0134] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0135] The term "amino acid sequence variant" includes substantial variants in which there is an amino acid substitution in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modified (e.g., improved) specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antigen-binding molecule and / or will substantially retain the specific biological properties of the parent antigen-binding molecule. Exemplary substitution variants are affinity-matured antibodies, which may be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, variant antigen-binding molecules in which one or more HVR residues are mutated, phage-displayed, and screened for a specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs as long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. A useful method for identifying antibody residues or regions that can be targeted for mutation is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antigen complex may be used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as candidates for substitution or removed. Mutants may be screened to determine whether they possess the desired properties.

[0136] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. Exemplary insertions include CD28 antigen-binding molecules having N- or C-terminal fusions to polypeptides that increase the serum half-life of the CD28 antigen-binding molecule.

[0137] In certain embodiments, the CD28 antigen binding molecules provided herein are modified to increase or decrease the degree of glycosylation of the antibody. Glycosylation variants of the molecule can be conveniently obtained by modifying the amino acid sequence to create or remove one or more glycosylation sites. When the agonist ICOS-binding molecule contains an Fc domain, the carbohydrate attached to the Fc domain can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally linked to Asn297 in the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in agonistic ICOS-binding molecules can be performed to generate variants with specific improved properties. In one aspect, agonistic ICOS-binding molecule variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function; see, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) or U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Additional variants of the CD28 antigen-binding molecules of the present invention include those with bisected oligosaccharides, e.g., those in which the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.Such antibody variants may have improved CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0138] In certain embodiments, it may be desirable to generate cysteine-engineered variants of the CD28 antigen-binding molecules of the present invention, e.g., "thioMAbs" in which one or more residues of the molecule are replaced with cysteine ​​residues. In particular embodiments, the substituted residues occur at accessible sites of the molecule. By replacing these residues with cysteine, reactive thiol groups are located at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (e.g., drug moieties or linker-drug moieties) and generate immunoconjugates. In particular embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antigen-binding molecules may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0139] In certain embodiments, the CD28 antigen binding molecules provided herein can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when more than one polymer is attached, the polymers may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization may be determined based on constraints, including but not limited to, the particular property or function of the antibody to be improved, whether the bispecific antibody derivative will be used under specified conditions, etc. In another aspect, conjugates of antibodies and nonproteinaceous moieties are provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may have any wavelength, including but not limited to, wavelengths that are not harmful to normal cells but that heat the nonproteinaceous moiety to temperatures that kill cells proximal to the antibody nonproteinaceous moiety.In another embodiment, immunoconjugates of the CD28 antigen binding molecules provided herein can be obtained. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, a cytotoxic agent.

[0140] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by their base sequence, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically presented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules may be linear or circular. Additionally, the term nucleic acid molecule encompasses both sense and antisense strands, and both single- and double-stranded forms. Furthermore, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include those containing derivatized sugar or phosphate backbone linkages or chemically modified residues, such as modified nucleotide bases. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for directing expression of antibodies of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified.For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2 101 823 B1).

[0141] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that originally contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive- and negative-stranded forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0142] A nucleic acid or polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with alternative nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either individually between residues in the reference sequence or in one or more consecutive groups within the reference sequence. In practical terms, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0143] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide that contains a specific sequence of nucleic acid elements that allows for transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector contains, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, expression cassettes of the invention comprise a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0144] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and induce expression of an operably associated specific gene in a target cell. This term includes a vector as a self-replicating nucleic acid structure and a vector integrated into the genome of a host cell into which the vector is introduced. The expression vector of the present invention comprises an expression cassette. The expression vector enables stable transcription of large amounts of mRNA. Once the expression vector is inside the target cell, the protein encoded by the ribonucleic acid molecule or gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0145] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the original transformed cell and progeny derived from the original transformed cell, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the present invention. Host cells are any type of cell line that can be used to produce the bispecific antigen-binding molecules of the present invention. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, to name just a few, yeast cells, insect cells and plant cells, but also cells contained in transgenic animals, transgenic plants or cultured plant or animal tissue.

[0146] An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which the drug is administered.

[0147] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective, at the necessary dosage and for the necessary period of time, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0148] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0149] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0150] A "pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

[0151] The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products, including information regarding the indications, uses, dosages, administration, concomitant therapy, contraindications and / or warnings regarding such therapeutic products.

[0152] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course in the treated individual and can be carried out prophylactically or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of the condition, and improving or improving prognosis. In some embodiments, the molecules of the invention are used to delay the onset of disease or slow the progression of the disease.

[0153] The term "combined treatment" or "co-administration" as used herein encompasses combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of an antibody as reported herein may occur before, simultaneously with, and / or after administration of one or more additional therapeutic agents, preferably one or more antibodies.

[0154] By "B cell proliferative disorder" is meant a disease in which the number of B cells in a patient is increased compared to the number of B cells in a healthy subject, particularly a disease in which an increased number of B cells is the cause or characteristic of the disease.

[0155] The term "hematological cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Accordingly, the term cancer, as used herein, refers to proliferative diseases such as carcinomas, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, and leukemias. In particular, the term cancer refers to B-cell proliferative disorders. In one aspect, the cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL).

[0156] The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Accordingly, the term cancer as used herein refers to proliferative diseases such as carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In particular, the term "cancer" refers to lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, alveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal cancer, colon cancer, breast cancer, uterine cancer, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, Cancers include adenocarcinoma, adrenal gland cancer, soft tissue sarcoma, cancer of the urethra, penile cancer, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, cancer of the renal pelvis, mesothelioma, hepatocellular carcinoma, bile duct cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory forms of any of the above cancers), or a combination of one or more of the above cancers. In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer is a hematological cancer, particularly a leukemia, most particularly acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

[0157] Bispecific agonist CD28 antigen-binding molecules of the present invention The present invention provides novel bispecific agonist CD28 antigen-binding molecules with particularly advantageous properties, such as manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, a broader dosage range that can be administered to patients, and thereby potentially enhanced efficacy. The novel bispecific agonist CD28 antigen-binding molecules contain an Fc domain composed of a first subunit and a second subunit capable of stable association, which contain one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for Fc receptors (Fc silent), thereby avoiding nonspecific cross-linking via Fc receptors. Instead, they contain at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen, such as fibroblast activation protein (FAP) or carcinoembryonic antigen (CEA), which causes cross-linking at tumor sites. Thus, tumor-specific T cell activation is achieved.

[0158] A bispecific agonist CD28 antigen binding molecule having monovalent binding to CD28, (a) one antigen-binding domain capable of specifically binding to CD28; and (b) at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; Provided herein is a bispecific agonist CD28 antigen binding molecule comprising:

[0159] In one aspect, there is provided a bispecific agonist CD28 antigen-binding molecule as defined herein above, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. In a particular aspect, the Fc domain composed of a first subunit and a second subunit capable of stable association is an IgG1 Fc domain. The Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or reduce or eliminate effector function. In one aspect, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one aspect, the Fc domain is an Fc domain of the human IgG1 subclass and comprises amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index). In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, comprising an Fc domain composed of a first and a second subunit capable of stable association, wherein the first subunit comprises the amino acid sequence of SEQ ID NO: 176 and the second subunit comprises the amino acid sequence of SEQ ID NO: 177.

[0160] In one embodiment, the antigen binding domain capable of specifically binding to CD28 comprises: (i) a heavy chain variable region (V) comprising heavy chain complementarity determining regions CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; H CD28) and a light chain variable region (V) comprising light chain complementarity determining regions CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25. L CD28), or (ii) a heavy chain variable region (V) comprising CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37, and CDR-H3 of SEQ ID NO: 38 H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 39, CDR-L2 of SEQ ID NO: 40, and CDR-L3 of SEQ ID NO: 41. L CD28) and

[0023] There is provided a bispecific agonist CD28 antigen binding molecule as defined herein above, comprising:

[0161] In one aspect, the antigen binding domain of the bispecific agonist CD28 antigen binding molecule capable of specifically binding to CD28 comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37 and CDR-H3 of SEQ ID NO: 38. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 39, CDR-L2 of SEQ ID NO: 40, and CDR-L3 of SEQ ID NO: 41 L CD28).

[0162] In another aspect, the antigen binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen binding molecule comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25 L CD28).

[0163] Furthermore, the antigen-binding domain capable of specifically binding to CD28 may comprise a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26. H CD28) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:27. L In accordance with the present invention, there is provided a bispecific agonist CD28 antigen-binding molecule as defined herein above, comprising a CD28 (antigen binding site) and a CD28 (antigen binding site).

[0164] In another embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 54 L CD28) CDR, or (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 HA light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 27 L CD28) CDR, or (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 51 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 61 L CD28) CDR, or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 53 L CD28) CDR, or (e) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 54 L CD28) CDR, or (f) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 59 L CD28) CDR, or (g) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 27 L CD28) CDR, or (h) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 43 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 27 L CD28) CDR, or (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 53 L CD28) CDR, or (j) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 59 L CD28) CDR, or (k) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H A light chain variable region (V) comprising the CDRs of CD28 and the amino acid sequence of SEQ ID NO: 27 L CD28 CDR A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0165] In one embodiment, the antigen binding domain capable of specifically binding to CD28 comprises the CDRs of a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. H CD28) and the CDR (V) of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 54 L In another aspect, the bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen-binding molecule comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 489, CDR-H2 of SEQ ID NO: 490, and CDR-H3 of SEQ ID NO: 491. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 492, CDR-L2 of SEQ ID NO: 493, and CDR-L3 of SEQ ID NO: 494 L CD28).

[0166] In another embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises the CDRs (V H CD28) and the CDR (V) of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 53 L In another aspect, the bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen-binding molecule comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 495, CDR-H2 of SEQ ID NO: 496, and CDR-H3 of SEQ ID NO: 497. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 498, CDR-L2 of SEQ ID NO: 499, and CDR-L3 of SEQ ID NO: 500 L CD28).

[0167] In another embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises the CDRs (V H CD28) and the CDR (V) of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 L In another aspect, the bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen binding molecule comprises a heavy chain variable region (VH1) comprising CDR-H1 of SEQ ID NO: 501, CDR-H2 of SEQ ID NO: 502, and CDR-H3 of SEQ ID NO: 503. H CD28) and a light chain variable region (V) comprising CDR-L1 of SEQ ID NO: 504, CDR-L2 of SEQ ID NO: 505, and CDR-L3 of SEQ ID NO: 506 L CD28).

[0168] In a further aspect, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51. H CD28) and a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. L A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0169] In another embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), or (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 51 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61 L CD28), or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (e) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (f) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 L CD28), or (g) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), or (h) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 43 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), or (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (j) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 L CD28), or (k) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28) A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0170] In one embodiment, the antigen binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 26. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L In one embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 26. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47, which binds to CD28 with reduced affinity compared to an antigen-binding domain comprising H and a light chain variable region (V) comprising CDR-H1, CDR-H2, and CDR-H3 of CD28 and the amino acid sequence of SEQ ID NO: 54. L In one embodiment, the heavy chain variable region (V) comprises the amino acid sequence of SEQ ID NO: 26. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L The antigen-binding domain may be capable of specifically binding to CD28 with lower affinity than an antigen-binding domain comprising a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47. H CD28) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:54. L CD28).

[0171] In one particular embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0172] In another particular embodiment, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0173] In a further aspect, the antigen-binding domain capable of specifically binding to CD28 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42. H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L

[0004] Bispecific agonist CD28 antigen-binding molecules are provided, comprising:

[0174] CEA-targeted bispecific agonist CD28 antigen-binding molecule In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to carcinoembryonic antigen (CEA).

[0175] In one embodiment, the antigen-binding domain capable of specifically binding to CEA comprises: (i) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 188, CDR-H2 having the amino acid sequence of SEQ ID NO: 189, and CDR-H3 having the amino acid sequence of SEQ ID NO: 190; H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 191, CDR-L2 having the amino acid sequence of SEQ ID NO: 192, and CDR-L3 having the amino acid sequence of SEQ ID NO: 193. L CEA), or (ii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 180, CDR-H2 having the amino acid sequence of SEQ ID NO: 181, and CDR-H3 having the amino acid sequence of SEQ ID NO: 182; HCEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 183, CDR-L2 having the amino acid sequence of SEQ ID NO: 184, and CDR-L3 having the amino acid sequence of SEQ ID NO: 185. L CEA), or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 127, CDR-H2 having the amino acid sequence of SEQ ID NO: 128, and CDR-H3 having the amino acid sequence of SEQ ID NO: 129; H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 130, CDR-L2 having the amino acid sequence of SEQ ID NO: 131, and CDR-L3 having the amino acid sequence of SEQ ID NO: 132. L CEA), or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 507, CDR-H2 having the amino acid sequence of SEQ ID NO: 508, and CDR-H3 having the amino acid sequence of SEQ ID NO: 509 H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 510, CDR-L2 having the amino acid sequence of SEQ ID NO: 511, and CDR-L3 having the amino acid sequence of SEQ ID NO: 512. L CEA)

[0023] In one embodiment, a bispecific agonist CD28 antigen binding molecule as described herein is provided, comprising:

[0176] In a specific embodiment, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 188, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 189, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 190. H CEA) and a light chain variable region (V) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 191, CDR-L2 having the amino acid sequence of SEQ ID NO: 192, and CDR-L3 having the amino acid sequence of SEQ ID NO: 193. L CEA).

[0177] In particular, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 133. H CEA) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 134. L In one embodiment, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 133. H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 134 L CEA).

[0178] In another embodiment, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 186. H CEA) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 187. L In one embodiment, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 186. H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 187 L CEA).

[0179] In another embodiment, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 513. H CEA) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 514. L In one embodiment, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 513. HCEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 514 L CEA).

[0180] In another embodiment, the antigen-binding domain capable of specifically binding to CEA is (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 194 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 195 L CEA), or (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 196 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 197 L CEA), or (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 198 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 199 L CEA), or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 200 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 201 L CEA), or (e) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 202 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 203 L CEA), or (f) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 204 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 205 L CEA), or (g) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 206 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 207 L CEA), or (h) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 208 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 209 L CEA), or (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 210 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 211 L CEA), or (j) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 212 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 213 L CEA) A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0181] In particular, the antigen-binding domain capable of specifically binding to CEA comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 200. H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 201 L CEA).

[0182] FAP-targeted bispecific agonist CD28 antigen-binding molecule In yet another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP).

[0183] In one embodiment, the antigen binding domain capable of specifically binding to a FAP comprises: (a) a heavy chain variable region (V) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; H and a light chain variable region (V FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17. L FAP), or (b) a heavy chain variable region (V) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; Hand a light chain variable region (V FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9. L FAP) and

[0023] The bispecific agonist CD28 antigen binding molecule described herein is provided, comprising:

[0184] In particular, the antigen-binding domain capable of specifically binding to FAP comprises a heavy chain variable region (V) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. H and a light chain variable region (V FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17. L In one embodiment, the antigen-binding domain capable of specifically binding to a FAP comprises (a) a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. H FAP) and a light chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19. L FAP), or (b) a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10. H FAP) and a light chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11. L A bispecific agonist CD28 antigen-binding molecule is provided, which comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 18. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 19 L FAP).

[0185] EpCAM targeting bispecific agonist CD28 antigen binding molecule In yet another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to epithelial cell adhesion molecule (EpCAM).

[0186] In one aspect, the antigen-binding domain capable of specifically binding to EpCAM comprises a heavy chain variable region (VH1) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 515; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 516; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 517. H EpCAM) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 518, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 519, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 520. L In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, comprising an antigen binding domain capable of specifically binding to EpCAM, and a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 521. H EpCAM) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 522. L A bispecific agonist CD28 antigen-binding molecule is provided, comprising an antigen-binding domain capable of specifically binding to EpCAM, the antigen-binding domain comprising: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 521; H EpCAM), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 522 L Includes EpCAM.

[0187] HER3-targeted bispecific agonist CD28 antigen-binding molecule In another embodiment, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to Her3.

[0188] In one embodiment, the antigen-binding domain capable of specifically binding to HER3 comprises a heavy chain variable region (VH1) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 523; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 524; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 525. H HER3) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 526, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 527, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 528. L In one aspect, the antigen-binding domain capable of specifically binding to HER3 comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 529. H HER3) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 530. L A bispecific agonist CD28 antigen-binding molecule is provided, comprising an antigen-binding domain capable of specifically binding to HER3, the antigen-binding domain comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 529. H HER3) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 530 L HER3).

[0189] CD30-targeted bispecific agonist CD28 antigen-binding molecule In another embodiment, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to CD30.

[0190] In one aspect, an antigen-binding domain capable of specifically binding to CD30 comprises a heavy chain variable region (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 531; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 532; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 533. H and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 534, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 535, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 536. L In one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 537. H CD30) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 538. L A bispecific agonist CD28 antigen-binding molecule is provided, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 537. H CD30) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 538 L CD30).

[0191] TBPG (5T4)-targeted bispecific agonist CD28 antigen-binding molecule In another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to TBPG (5T4).

[0192] In one embodiment, the antigen-binding domain capable of specifically binding to TBPG comprises a heavy chain variable region (V) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 539; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 540; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 541.H TBPG), and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 542, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 543, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 544. L In one aspect, the antigen-binding domain capable of specifically binding to TBPG comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 545. H TBPG) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 546. L A bispecific agonist CD28 antigen-binding molecule is provided, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 545. H TBPG) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 546 L TBPG).

[0193] MM-targeted bispecific agonist CD28 antigen-binding molecule The present invention also provides a novel bispecific agonist CD28 antigen-binding molecule that is particularly useful in the treatment of multiple myeloma. The molecule comprises at least one antigen-binding domain capable of specifically binding to a multiple myeloma (MM) cell surface antigen, causing cross-linking in the presence of MM cell surface antigen-expressing cells, and an Fc domain composed of a first subunit and a second subunit capable of stable association, which contain one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to Fc receptors and / or effector functions (Fc silent). Thus, nonspecific cross-linking via Fc receptors is avoided, and specific T cell activation is achieved in the presence of MM cell surface antigen-expressing cells.

[0194] Thus, provided herein is a bispecific agonist CD28 antigen-binding molecule comprising an antigen-binding domain capable of specifically binding to CD28, an antigen-binding domain capable of specifically binding to a multiple myeloma (MM) cell surface antigen, and an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or effector function. In one aspect, the bispecific agonist CD28 antigen-binding molecule described herein is characterized by monovalent binding to CD28. In a further aspect, the bispecific agonist CD28 antigen-binding molecule described herein is characterized by monovalent binding to a multiple myeloma (MM) cell surface antigen.

[0195] In one aspect, there is provided a bispecific agonist CD28 antigen-binding molecule as defined herein above, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. In a particular aspect, the Fc domain composed of a first subunit and a second subunit capable of stable association is an IgG1 Fc domain. The Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or reduce or eliminate effector function. In one aspect, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one aspect, the Fc domain is an Fc domain of the human IgG1 subclass and comprises amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0196] In one aspect there is provided a bispecific agonist CD28 antigen binding molecule as defined herein above, wherein the MM cell surface antigen is selected from the group consisting of CD38, BCMA and GPRC5D.

[0197] In another embodiment, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to CD38.

[0198] In one aspect, an antigen-binding domain capable of specifically binding to CD38 comprises a heavy chain variable region (VH) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 547; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 548; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 549. H and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 550, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 551, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 552. L In one aspect, the bispecific agonist CD28 antigen binding molecule described herein comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 553. H CD38) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 554. L The bispecific agonist CD28 antigen-binding molecule is provided, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 553. H CD38) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 554 L CD38).

[0199] In yet another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to BCMA.

[0200] In one aspect, an antigen binding domain capable of specifically binding to BCMA comprises a heavy chain variable region (V) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 555; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 556; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 557. HBCMA) and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 558, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 559, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 560. L In one aspect, the antigen-binding domain capable of specifically binding to BCMA comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 561. H BCMA) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 562. L The bispecific agonist CD28 antigen-binding molecule is provided, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 561. H BCMA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 562 L BCMA).

[0201] GPRC5D-targeted bispecific agonist CD28 antigen-binding molecule In another embodiment, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to a tumor-associated antigen is an antigen-binding domain capable of specifically binding to GPRC5D.

[0202] In one aspect, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 563; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 564; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 565. H GPRC5D) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 566, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 567, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 568. LGPRC5D).

[0203] In one aspect, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 569, SEQ ID NO: 571, SEQ ID NO: 572 and SEQ ID NO: 573. H GPRC5D) and a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 570, SEQ ID NO: 574, SEQ ID NO: 575, SEQ ID NO: 576, SEQ ID NO: 577, and SEQ ID NO: 578. L GPRC5D).

[0204] In one aspect, an antigen binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 569. H GPRC5D) and a light chain variable region (V) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 570. L GPRC5D). Specifically, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 569. H GPRC5D) and a light chain variable region (V L GPRC5D).

[0205] In another aspect, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 579; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 580; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 581. HGPRC5D) and a light chain variable region (V) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 582, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 583, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 584. L GPRC5D).

[0206] In one aspect, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 585, SEQ ID NO: 586, SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589 and SEQ ID NO: 590. H GPRC5D) and a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 594 and SEQ ID NO: 595. L GPRC5D).

[0207] In another embodiment, the antigen-binding domain capable of specifically binding to GPRC5D is (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 569 H GPRC5D) and a light chain variable region (V L GPRC5D), or (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 573 H GPRC5D) and a light chain variable region (V L GPRC5D), or (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 569 H GPRC5D) and a light chain variable region (V L GPRC5D), or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 586 H GPRC5D) and a light chain variable region (V L GPRC5D), or (e) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 587 H GPRC5D) and a light chain variable region (V L GPRC5D), A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0208] Bispecific agonist CD28 antigen-binding molecules, monovalent for binding to CD28 and monovalent for binding to tumor-associated antigens (1+1 format) In another aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0209] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to CEA; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0210] In one particular aspect, a bispecific agonist CD28 antigen binding molecule (molecule M) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 65, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 87, and a second light chain comprising the amino acid sequence of SEQ ID NO: 88.

[0211] In another aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to the FAP; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor;

[0023] The bispecific agonist CD28 antigen binding molecule described herein is provided, comprising:

[0212] In one particular aspect, a bispecific agonist CD28 antigen binding molecule (molecule C) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 65, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a second light chain comprising the amino acid sequence of SEQ ID NO: 68.

[0213] In a further aspect, (a) a first Fab fragment capable of specifically binding to CD28; (b) a second Fab fragment capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; Including, Provided herein is a bispecific agonist CD28 antigen-binding molecule, in which a first Fab fragment capable of specifically binding to CD28 is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab fragment capable of specifically binding to a tumor-associated antigen, which is then fused at its C-terminus to the N-terminus of one of the Fc domain subunits.

[0214] In one particular aspect, a bispecific agonist CD28 antigen binding molecule (molecule H) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 77, a second light chain comprising the amino acid sequence of SEQ ID NO: 78, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 79.

[0215] In a further aspect, (a) a Fab fragment capable of specifically binding to CD28; (b) a VH domain and a VL domain capable of specifically binding to a tumor-associated antigen; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; Including, Provided herein is a bispecific agonist CD28 antigen-binding molecule, in which a Fab fragment capable of specifically binding to CD28 is fused at its C-terminus to the N-terminus of a first Fc domain subunit, one of a VH domain and a VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of the first Fc domain subunit via a peptide linker, and the other of the VH domain and VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of a second Fc domain subunit via a peptide linker.

[0216] In one aspect, the peptide linker comprises an amino acid sequence selected from SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 151 and SEQ ID NO: 152. More particularly, the peptide linker comprises SEQ ID NO: 152.

[0217] In one particular aspect, a bispecific agonist CD28 antigen binding molecule (molecule I) is provided, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 62, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 72, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 80.

[0218] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) one cross-Fab fragment capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0219] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 HCD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) one cross-Fab fragment capable of specifically binding to CEA, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 186 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 187 L CEA), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 200 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 201 L CEA), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 513 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 514 L CEA), a cross-Fab fragment comprising (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0220] In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11A) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 352, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 351, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 353, and a second light chain comprising the amino acid sequence of SEQ ID NO: 354. In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11B) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 352, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 351, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356. In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11C) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 352, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 351, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 357, and a second light chain comprising the amino acid sequence of SEQ ID NO: 358. In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11D) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 352, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 351, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 359, and a second light chain comprising the amino acid sequence of SEQ ID NO: 354. In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11I) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 370, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 369, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 353, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356. In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11J) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 370, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 369, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 359, and a second light chain comprising the amino acid sequence of SEQ ID NO: 354. In one aspect, a bispecific agonist CD28 antigen binding molecule (molecule 11K) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 370, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 369, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 357, and a second light chain comprising the amino acid sequence of SEQ ID NO: 358.In one aspect, a bispecific agonist CD28 antigen binding molecule is provided (molecule 11L), comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 370, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 369, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 359, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356. In one aspect, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 376, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 375, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 357, and a second light chain comprising the amino acid sequence of SEQ ID NO: 358 (molecule 11R). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 376, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 375, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356 (molecule 11S). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 376, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 375, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 354 (molecule 11T).

[0221] In one particular embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 376, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 375, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356 (molecule 11S). In another embodiment, a bispecific agonist CD28 antigen binding molecule (molecule 11B) is provided, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 352, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 351, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356.

[0222] In another aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to a tumor-associated antigen; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0223] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to CEA; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0224] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a cross-Fab fragment comprising (b) one Fab fragment capable of specifically binding to CEA, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 186 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 187 L CEA), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 200 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 201 L CEA), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 513 H CEA) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 514 L CEA), a cross-Fab fragment comprising (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0225] In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 361, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 360, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 362, and a second light chain comprising the amino acid sequence of SEQ ID NO: 363 (molecule 11E). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 361, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 360, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 364, and a second light chain comprising the amino acid sequence of SEQ ID NO: 365 (molecule 11F). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 361, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 360, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 366, and a second light chain comprising the amino acid sequence of SEQ ID NO: 367 (molecule 11G). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 361, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 360, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 368, and a second light chain comprising the amino acid sequence of SEQ ID NO: 363 (molecule 11H). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 372, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 371, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 368, and a second light chain comprising the amino acid sequence of SEQ ID NO: 363 (molecule 11M). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 372, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 371, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 366, and a second light chain comprising the amino acid sequence of SEQ ID NO: 367 (molecule 11N). In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 372, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 371, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 364, and a second light chain comprising the amino acid sequence of SEQ ID NO: 365 (molecule 11O).

[0226] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to EpCAM; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0227] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 521 H EpCAM) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 522 L one cross-Fab fragment capable of specifically binding to EpCAM, comprising EpCAM; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0228] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to EpCAM; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0229] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a cross-Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 521 H EpCAM) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 522 L one Fab fragment capable of specifically binding to EpCAM, comprising EpCAM; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0230] In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 367, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 366, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 390, and a second light chain comprising the amino acid sequence of SEQ ID NO: 391 (molecule 14A).

[0231] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to HER3; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0232] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 529 H HER3) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 530 L one cross-Fab fragment capable of specifically binding to HER3, comprising HER3; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0233] In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 357, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 392, and a second light chain comprising the amino acid sequence of SEQ ID NO: 393 (molecule 14B).

[0234] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to HER3; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0235] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a cross-Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 529 H HER3) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 530 L HER3), and one Fab fragment capable of specifically binding to HER3; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0236] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to CD30; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0237] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 537 H CD30) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 538 L one cross-Fab fragment capable of specifically binding to CD30, comprising a cross-Fab fragment capable of specifically binding to CD30; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0238] In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 357, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 394, and a second light chain comprising the amino acid sequence of SEQ ID NO: 395 (molecule 14C).

[0239] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to CD30; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0240] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a cross-Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 537 H CD30) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 538 L one Fab fragment capable of specifically binding to CD30, comprising: (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0241] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to TPBG; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0242] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 545 H TPBG) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 546 L one cross-Fab fragment capable of specifically binding to TPBG, comprising TPBG; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0243] In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 357, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 396, and a second light chain comprising the amino acid sequence of SEQ ID NO: 397 (molecule 14D).

[0244] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to TPBG; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0245] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a cross-Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 545 H TPBG) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 546 L one Fab fragment capable of specifically binding to TPBG, comprising TPBG; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0246] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to CD38; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0247] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 553 H CD38) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 554 L one cross-Fab fragment capable of specifically binding to CD38, comprising CD38; (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0248] In one embodiment, the bispecific agonist CD28 antigen binding molecule comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 357, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 358, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 400, and a second light chain comprising the amino acid sequence of SEQ ID NO: 401 (molecule 16C).

[0249] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28; and (b) one Fab fragment capable of specifically binding to CD38; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0250] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one cross-Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L CD28), a cross-Fab fragment comprising (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 553 H CD38) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 554 L one Fab fragment capable of specifically binding to CD38, comprising: (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0251] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28; and (b) one cross-Fab fragment capable of specifically binding to BCMA; and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor; A bispecific agonist CD28 antigen binding molecule is provided, comprising:

[0252] In one aspect, a bispecific agonist CD28 antigen binding molecule described herein, comprising: (a) one Fab fragment capable of specifically binding to CD28, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 L CD28), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 46 H CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 53 L CD28), or (iii) a heavy chain var...

Claims

1. A bispecific agonist CD28 antigen-binding molecule characterized by monovalent binding to CD28, (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 47 Ｈ CD28) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 54 Ｌ one Fab fragment capable of specifically binding to CD28, comprising a fragment of the Fab fragment capable of specifically binding to CD28; (b) (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 412 Ｈ CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 413 Ｌ CD19); or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 420 Ｈ CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 421 Ｌ CD19) one cross-Fab fragment capable of specifically binding to CD19, comprising: (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, the first subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; Including, the Fc domain is of the human IgG1 subclass and contains the amino acid mutations L234A, L235A and P329G (numbering according to the Kabat EU index); Bispecific agonist CD28 antigen binding molecules.

2. The cross-Fab fragment capable of specifically binding to CD19 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

412. Ｈ CD19) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 413 Ｌ 2. The bispecific agonist CD28 antigen binding molecule of claim 1, comprising a CD28 antigen binding domain and a CD19 antigen binding domain.

3. 2. The bispecific agonist CD28 antigen binding molecule of claim 1, comprising a first light chain comprising the amino acid sequence of SEQ ID NO: 122, a first heavy chain comprising the amino acid sequence of SEQ ID NO: 114, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 430, and a second light chain comprising the amino acid sequence of SEQ ID NO:

431.

4. One or more isolated polynucleotides encoding the bispecific agonist CD28 antigen binding molecule of any one of claims 1 to 3.

5. 5. One or more vectors, in particular expression vectors, comprising the polynucleotide(s) according to claim 4.

6. 6. A host cell comprising the polynucleotide(s) of claim 4 or the vector(s) of claim 5.

7. 10. A method for producing a bispecific agonist CD28 antigen-binding molecule, comprising: a) culturing the host cell of claim 6 under conditions suitable for expression of the bispecific agonist CD28 antigen-binding molecule; and b) optionally recovering the bispecific agonist CD28 antigen-binding molecule.

8. A pharmaceutical composition comprising the bispecific agonist CD28 antigen binding molecule of any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient.

9. 9. The pharmaceutical composition of claim 8 for use in the treatment of cancer.

10. A medicament comprising the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3, or the pharmaceutical composition of claim 8.

11. A medicament for use in enhancing (a) T cell activation or (b) T cell effector function, comprising the bispecific agonist CD28 antigen binding molecule of any one of claims 1 to 3.

12. A medicament for use in the treatment of cancer, comprising the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3.

13. 4. A medicament for use in the treatment of a B-cell proliferative disorder selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL), comprising the bispecific agonist CD28 antigen binding molecule of any one of claims 1 to 3.

14. A medicament for use in the treatment of cancer, comprising the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with a chemotherapeutic agent, radiation therapy, and / or other agent for use in cancer immunotherapy.

15. A medicament for use in the treatment of cancer, comprising the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with a T-cell-activating anti-CD3 bispecific antibody.

16. A medicament for use in the treatment of cancer, comprising the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with an anti-CD20 / anti-CD3 bispecific antibody.

17. A medicament for use in the treatment of cancer, comprising the bispecific agonist CD28 antigen binding molecule of any one of claims 1 to 3, wherein the bispecific agonist CD28 antigen binding molecule is administered in combination with an anti-CD20 / anti-CD3 bispecific antibody comprising the polypeptide sequence of SEQ ID NO: 455, the polypeptide sequence of SEQ ID NO: 456, the polypeptide sequence of SEQ ID NO: 457, and the polypeptide sequence of SEQ ID NO:

458.

18. A pharmaceutical for use in the treatment of cancer, comprising the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3, wherein the bispecific agonist CD28 antigen-binding molecule is administered in combination with an anti-PD-L1 antibody or an anti-PD-1 antibody.

19. A medicament for inhibiting the growth of tumor cells in an individual, comprising an effective amount of the bispecific agonist CD28 antigen-binding molecule of any one of claims 1 to 3 or the pharmaceutical composition of claim 8.

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