Tumor-targeting agonistic CD28 antigen-binding molecules

Abstract

The present invention relates to tumor-targeting bispecific agonistic antigen-binding molecules characterized by monovalent binding to CD28, methods for preparing the same, pharmaceutical compositions containing the same, and methods of using the same.

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 preparing the same, pharmaceutical compositions containing the same, and their use as immunomodulators in cancer treatment. Background Art

[0002] Cancer immunotherapy is becoming an increasingly effective treatment option, producing significant and durable responses in cancers such as melanoma, non-small cell lung cancer, and renal cell carcinoma. This has been driven primarily by the success of several immune checkpoint blockers, including anti-PD-1 (e.g., Keytruda, Merck; Opdivo, BMS), anti-CTLA-4 (e.g., Yervoy, BMS), and anti-PD-L1 (e.g., Tecentriq, Roche). These agents are likely to serve as the standard of care for many types of cancer or as the basis for combination therapies, however, only a small proportion of patients (<25%) benefit from such therapies. In addition, various cancers (prostate, colorectal, pancreatic, sarcomas, non-triple-negative breast cancer, etc.) exhibit primary resistance to these immunomodulatory agents. Numerous reports indicate that the loss of pre-existing anti-tumor T cells leads to a lack of response or a poor response in some patients. In summary, despite the impressive anticancer effects of existing immunotherapies, there remains a clear medical need to treat the vast cancer patient population and to develop therapies designed to induce and enhance novel tumor-specific T cell responses.

[0003] CD28 is a founding member of the costimulatory molecule subfamily, characterized by paired V-set immunoglobulin superfamily (IgSF) domains connected to a single transmembrane domain and a cytoplasmic domain containing key signaling motifs (Carreno and Collins, 2002). Other members of this subfamily include ICOS, CTLA-4, PD1, PD1H, TIGIT, and BTLA (Chen and Flies, 2013). The expression of CD28 is limited to T cells and is ubiquitous in all untreated and most antigen-treated subgroups, including those expressing PD-1 or CTLA-4. CD28 and CTLA-4 have a high degree of homology and compete for binding to the same B7 molecules CD80 and CD86 expressed on dendritic cells, B cells, macrophages, and tumor cells (Linsley et al., 1990). CTLA-4's higher affinity for the B7 family of ligands enables CTLA-4 to outperform CD28 in ligand binding and inhibit 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). For the functional de novo initiation of naive T cells, subsequent clonal expansion, cytokine production, target cell lysis, and the formation of long-term memory, it is strictly required that CD28 be connected to the surface of professional antigen-presenting cells via CD80 or CD86. Binding of CD28 ligands also promotes the expression of inducible costimulatory molecule receptors such as OX-40, ICOS, and 4-1BB (reviewed in Acuto and Michel, 2003). After connecting to CD28, disulfide-linked homodimers, membrane-proximal YMNM motifs, and distal PYAP motifs have been shown to complex with several kinases and linker proteins (Boomer and Green, 2010). These motifs are important for inducing 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, other less characterized sites of phosphorylation and ubiquitination are found in the cytoplasmic domain of CD28. As reviewed by (Esensten et al., 2016), the CD28-initiated pathway plays a key role in promoting the proliferation and effector function of conventional T cells. CD28 ligation can also promote the anti-inflammatory function of regulatory T cells. CD28 co-stimulates T cells in part by amplifying signals from the T cell receptor, but has also been shown to mediate unique signaling events (Acuto and Michel, 2003; Boomer and Green, 2010; June et al., 1987).Signals specifically triggered by CD28 control many important aspects of T cell function, including phosphorylation and other post-translational modifications of downstream proteins (e.g., PI3K-mediated phosphorylation), transcriptional changes (e.g., Bcl-xL expression), epigenetic changes (e.g., IL-2 promoter), cytoskeletal remodeling (e.g., orientation of the microtubule-organizing center), and changes in glycolytic rate (e.g., glycolytic flux). CD28-deficient mice have reduced responses to infectious pathogens, alloantigens, graft-versus-host disease, contact hypersensitivity, and asthma (Acuto and Michel, 2003). Lack of CD28-mediated co-stimulation leads to reduced T cell proliferation in vitro and in vivo, severe inhibition of germinal center formation and immunoglobulin isotype switching, reduced helper T (Th) cell differentiation, and expression of Th2-type cytokines. CD4-dependent cytotoxic CD8+ T cell responses are also affected. Importantly, naive T cells lacking CD28 display reduced proliferative responses, especially at lower antigen concentrations. A growing body of literature supports the idea that engaging CD28 on T cells has antitumor potential. Recent evidence suggests that the anticancer effects of PD-L1 / PD-1 and CTLA-4 checkpoint inhibitors are dependent on CD28 (Kamphorst et al., 2017; Tai et al., 2007). Clinical studies investigating the efficacy of CTLA-4 and PD-1 blockade have shown promising results in patients with advanced melanoma and other cancers. Furthermore, infusions of genetically engineered T cells expressing artificial chimeric T cell receptors (comprising an extracellular antigen recognition domain fused to an intracellular TCR signaling domain (CD3z) and intracellular costimulatory molecule domains (CD28 and / or 4-1BB domains)) have demonstrated high response rates and durability in B cell cancers and other cancers.

[0004] CD28 agonistic antibodies can be divided into two categories: (i) CD28 superagonist antibodies and (ii) CD28 conventional agonist antibodies. Generally, activation of naive T cells requires engagement of the T cell antigen receptor (TCR, signal 1) and signaling of CD28 co-stimulatory molecules (signal 2). CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies that can autonomously activate T cells without explicit T cell receptor engagement (Hünig, 2012). In rodents, CD28SA activates conventional and regulatory T cells. CD28SA antibodies have therapeutic efficacy in various autoimmune, inflammatory, and transplant models. However, a phase I study of the human CD28SA antibody TGN1412 caused a life-threatening cytokine storm in 2006. Subsequent studies demonstrated that the toxicity was due to a dosing error caused by differences in the CD28 response capacity of human T cells compared to T cells from preclinical animal models. TGN1412 is currently being re-evaluated in an open-label, multicenter, dose-escalation study in patients with RA and metastatic or unresectable advanced solid malignancies. Conventional agonist antibodies to CD28, such as clone 9.3, mimic the natural ligand for CD28 and can enhance T cell activation only in the presence of a T cell receptor signal (signal 1). Published insights suggest that the binding epitope of an antibody has a significant impact on whether an agonist antibody is a superagonist or a conventional agonist (Beyersdorf et al., 2005). The superagonist TGN1412 binds to a side motif of CD28, while the conventional agonist 9.3 binds tightly to the ligand-binding epitope. Due to the differences in 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 is able to effectively form linear arrays of CD28, which presumably results in aggregated signaling components sufficient to exceed the threshold for T cell activation. On the other hand, the conventional agonist 9.3 results in a complex that is not linear in structure. An attempt to convert a conventional agonistic binding agent based on the 9.3 clone using a recombinant bispecific single chain antibody directed against melanoma-associated proteoglycans and CD28 has been previously disclosed (Otz et al., 2009). Based on the inherent tendency of bispecific single chain antibodies to form multimeric constructs, the reported bispecific single chain antibody was reported to exert "super-agonistic" activity despite the use of the conventional CD28 agonistic binding agent 9.3.

[0005] It has been found that when a limited amount of anti-CD3 bispecific antibodies (i.e., T cell bispecific antibodies (TCBs) such as CEA-TCBs) are combined with agonistic anti-CD28 molecules, better T cell activation can be achieved. Given the baseline expression of CD28 on T cells in various tumor indications (Lavin et al., 2017; Tirosh et al., 2016; Zheng et al., 2017), and the activation of CD28 signaling enhances T cell receptor signaling, it is expected that such binding of TCB molecules to tumor-targeting CD28 molecules can synergize to induce strong and long-lasting anti-tumor responses. Therefore, we describe here a novel tumor-targeting agonistic CD28 molecule that exhibits synergy with TCBs and, in the presence of TCB signals, requires CD28 binding to be monovalent for strict tumor target dependence.

[0006] Immunotherapy for solid tumors

[0007] The treatment of solid tumors is an ongoing challenge, with little progress in recent years. Typically, treatment involves a combination of surgery, chemotherapy, and / or radiation therapy. Although many new treatment modalities have been developed recently, further improvements are needed to increase survival and improve the quality of life of patients with solid tumors. Solid tumors rarely express a single tumor-specific antigen. For most solid tumors, it is more common to find a tumor-associated antigen (TAA), which is enriched in the tumor but also expressed at very low levels on normal tissues. TAAs are preferentially present on the surface of solid tumor cells or on cells in the tumor stroma. This is the case for many common TAAs that target 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. Other TAAs include HER3, EpCAM, TPBG (5T4), mesothelin, MUC1, and PSMA. Therefore, a bispecific agonistic CD28 antigen-binding molecule comprising an antigen-binding domain that specifically binds to a tumor-associated antigen will primarily target the tumor surface or tumor microenvironment and will specifically activate T cells near the tumor while avoiding systemic activation.

[0008] Rationale for targeting CD28 agonism to B-cell malignancies

[0009] Non-Hodgkin lymphoma (NHL) is one of the leading causes of cancer death in the United States and Europe. Follicular lymphoma (FL) has an indolent, slowly progressive course with a median survival of 8 to 10 years; patients with advanced clinical disease are generally incurable. Similarly, in 2% to 3% of patients each year, the FL phenotype can transform into an aggressive large cell lymphoma, which is a key event in the disease process and is associated with increased lymphoma-related mortality. Mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL) are more aggressive and have a median survival of only 6 months if left untreated. Despite significant advances in immunotherapy that have prolonged progression-free survival, the lack of a cure for many patients with both indolent and aggressive NHL subtypes remains an unmet medical need. Over the past few years, significant advances have been made, particularly with the addition of the anti-CD20 monoclonal antibody rituximab (rituximab) to intensive cytotoxic chemotherapy regimens. ), significantly prolonged survival has been observed in DLBCL. However, despite the efficacy of conventional treatment for previously untreated DLBCL, the majority of patients ultimately relapse. Similarly, advanced FL remains largely incurable with current SoCs and is characterized by repeated relapses and progressively shorter remissions. Currently, many new-generation monoclonal antibodies are in various stages of preclinical and clinical evaluation to further improve the prognosis of NHL patients and overcome rituximab resistance mechanisms. High-dose chemotherapy with autologous stem cell support or allogeneic stem cell transplantation offers only a therapeutic option for a minority (10%) of patients with relapsed / refractory (r / r) DLBCL and is associated with a significant treatment-related mortality rate. 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 / rB-NHL, but this treatment is available only in limited circumstances and can be associated with fatal neurologic events and cytokine release syndrome (CRS). Bispecific antibody constructs that redirect cytotoxic cell lysis to malignant B cells are currently in development and have shown very promising efficacy against NHL. The future of NHL holds promise for chemotherapy-free treatments, potentially based on bispecific antibodies or chimeric antigen receptor T cells (CAR T cells). Combining CD28 agonists targeting B-cell surface antigens with immunotherapy should improve survival and / or cure rates in patients with B-cell malignancies without compromising quality of life.

[0010] B cell surface antigens as targets for B cell malignancies

[0011] TAAs associated with B-cell malignancies are B-cell surface antigens. 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 of the B lymphocyte lineage. CD19 expression is maintained in B-lineage cells that undergo neoplastic transformation, making CD19 useful for diagnosing leukemias and lymphomas using monoclonal antibodies (mAbs) and flow cytometry. The CD20 antigen is also useful for diagnosing leukemias and lymphomas. Because CD19 expression is rarely lost in B-lineage leukemias and lymphomas, and because it is not expressed in pluripotent stem cells, it has become a target for various immunotherapeutic agents, including immunotoxins. CD79 is the signaling component of the B-cell receptor and consists of a covalent heterodimer containing CD79a (Igα, mb-1) and CD79b (Igβ, B29). CD79a and CD79b each comprise extracellular immunoglobulin (Ig) domain, transmembrane domain and intracellular signal transduction domain, activation motif (ITAM) domain based on immunoreceptor tyrosine, just like other signal transduction proteins (such as CD3 or activation Fc γ receptors).Therefore, CD79a and CD79b are transmembrane proteins constituting the signal transduction subunits of B cell receptor (BCR).CD79b is a 39KDa protein expressed only on B cells, and cooperates with CD79a to start the signal transduction cascade downstream of BCR, thereby causing BCR complex internalization, its translocation to endosome and antigen presentation.In B cells, antigen-induced BCR clustering triggers the tyrosine phosphorylation of the ITAM of CD79a and CD79b carried out by Src kinase.This results in raising and activating effector molecule arrays, and the effector molecule belongs to BCR signal transduction cascade, including the most famous SYK and BLNK. Further downstream, the recruitment of PLCg2, Btk, and ERK facilitates calcium flux and activates B cells, which are then poised to receive additional coactivation signals that drive their proliferation and differentiation into memory or effector cells. In this process, B cells become robust APCs and release cytokines that can influence the outcome and quality of the immune response. In addition to their role in BCR signaling, CD79 subunits are essential for the trafficking and display of membrane-bound Ig from the endoplasmic reticulum to the cell surface. The average surface expression of CD79b on NHL is similar to that of 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 would produce little or no antigenicity when administered to patients, particularly for chronic treatment.

[0012] It has been found that when a limited amount of anti-CD3 bispecific antibodies (i.e., T cell bispecific antibodies (TCBs) such as CD20 / CD3 bispecific antibodies) are combined with agonistic anti-CD28 molecules, better T cell activation can be achieved. Given the baseline expression of CD28 on T cells in various tumor indications (Lavin et al., 2017; Tirosh et al., 2016; Zheng et al., 2017), and the activation of CD28 signaling enhances T cell receptor signaling, it is expected that the combination of T cell bispecific antibodies with bispecific agonistic CD28 antigen binding molecules targeting B cell surface antigens can synergize to induce a strong and lasting anti-tumor response. Therefore, we describe herein a novel bispecific agonistic CD28 antigen binding molecule targeting B cell surface antigens that exhibits synergistic effects with TCBs and, in the presence of TCB signals, requires CD28 binding to be monovalent for strict tumor target dependence.

[0013] Immunotherapy for multiple myeloma

[0014] In the EU and the United States, multiple myeloma (MM) affects approximately 75,000 new patients each year and is one of the most common hematological malignancies, with a high medical need. Multiple myeloma is characterized by terminally differentiated plasma cells that secrete non-functional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide and proteasome inhibitors such as carfilzomib or bortezomib may still be the backbone of first-line treatment for multiple myeloma (Moreau et al., 2016). However, these drugs cannot specifically target diseased tumor cells, for example, diseased plasma cells (PC). Efforts have been made to selectively deplete plasma cells in multiple myeloma. The lack of surface proteins that specifically mark plasma cells has hindered the development of antibodies or cell therapies for multiple myeloma. To date, there have been few successful biologics, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), but it is important to note that both antigens are also expressed in other normal tissues, including hematopoietic lineages and immune effector cells, which may limit their long-term clinical utility. B-cell maturation antigen (BCMA) is a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17) that is expressed at significantly higher levels on all patient MM cells but not in normal tissues other than normal plasma cells. BCMA chimeric antigen receptor (CAR) T cells have shown significant clinical activity in patients with RRMM who have received at least three prior lines of therapy, including proteasome inhibitors and immunomodulators. Other modalities, including anti-BCMA antibody-drug conjugates, have also achieved significant clinical responses in patients who have failed at least three prior lines of therapy, including anti-CD38 antibodies, proteasome inhibitors, and immunomodulators (Cho et al., 2018). For example, one of the challenges of BCMA-targeted therapy or CD38-targeted therapy is the presence of high levels of soluble BCMA or CD38 in the serum of MM patients, which may reduce the amount of active drug in the patient's body. An alternative approach may be new targets, such as G protein-coupled receptor class C group 5 member D (GPRC5D), which is expressed differently in plasma cells in multiple myeloma compared to plasma cells of healthy donors and has no soluble form. It has been reported that GPRC5D is 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 ligand in general men, especially in cancer patients, and is almost unknown biologically.The gene encoding GPRC5D, located on chromosome 12p13.3, contains three exons and spans approximately 9.6 kb (Brauner-Osborne, H. et al., 2001). The large first exon encodes seven transmembrane domains. GPRC5D has been shown to be involved in keratin formation in animal hair follicles (Gao, Y. et al., 2016 and Inoue, S. et al., 2004). WO2018 / 017786 A2 discloses GPRC5D-specific antibodies or antigen-binding fragments.

[0015] Rationale for targeting CD28 agonism to diseased plasma cells in multiple myeloma

[0016] CD28 agonism in multiple myeloma may exert different biological functions on the corresponding MM plasma cells of immunity. Although it is expected that CD28 co-activation of T cells can drive anti-tumor responses, CD28 agonism for MM cells can mediate pro-survival signaling through the regulation of PI3K / Akt, FoxO3a and Bimm, and pro-survival signaling has been described as inducing chemotherapeutic resistance in multiple myeloma (Murray ME et al., 2014). Overexpression of CD28 on newly diagnosed multiple myeloma plasma cells is thought to be associated with poor clinical prognosis (Bahlis et al., 2007). However, although CD28 activation enhances the survival of myeloma cells, its activation inhibits the proliferation of myeloma cells. Activating CD28 in the presence of a strong immune cell-mediated response (such as T cell bispecific activation of T cells) can further enhance effective anti-tumor responses. We provide herein bispecific agonist CD28 antigen binding molecules that specifically bind to human multiple myeloma (MM) cell surface antigens. In particular, the bispecific agonist CD28 antigen-binding molecules according to the present invention targeting TAAs selected from BCMA, CD38 and GPRC5D expressed on T cells and CD28 have the potential to be used as single agents or in combination with other agents such as T cell bispecific antibodies (TCBs) targeting human MM cell surface antigens for the treatment of multiple myeloma. Summary of the Invention

[0017] The present invention describes the bispecific agonist CD28 antigen binding molecules for targeting tumors, which achieve tumor-dependent T cell activation and tumor cell killing without the need to form multimers. The bispecific CD28 antigen binding molecules of the present invention are characterized in that they are monovalently bound to CD28, and they include at least one antigen binding domain that can specifically bind to tumor-associated antigens (such as fibroblast activation protein (FAP) or carcinoembryonic antigen (CEA), CD19 or GPRC5D). In addition, they have an Fc domain consisting of a first subunit and a second subunit that can stably associate, and the amino acid substitutions reduce the binding affinity and / or effector function of the antigen binding molecules to Fc receptors. Thus, Fc receptor-mediated cross-linking is eliminated, and cross-linking is achieved by combining at least one antigen binding domain that can specifically bind to a tumor-associated antigen with its antigen, thereby achieving tumor-specific activation.

[0018] Therefore, the present invention provides a bispecific agonist CD28 antigen-binding molecule characterized by monovalent binding to CD28, comprising:

[0019] (a) an antigen-binding domain capable of specifically binding to CD28,

[0020] (b) at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen, and

[0021] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0022] On the one hand, a bispecific agonist CD28 antigen binding molecule as defined below is provided, wherein the Fc domain is IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. In a specific aspect, the Fc domain consisting of a first subunit and a second subunit capable of stably associating is an IgG1 Fc domain. On the one hand, the Fc domain comprises amino acid substitutions L234A and L235A (numbered according to the EU index of Kabat). On the one hand, the Fc domain belongs to the human IgG1 subclass and comprises amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0023] In one aspect, a bispecific agonist CD28 antigen binding molecule as previously defined herein is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: (i) a heavy chain variable region (V HCD28), which comprises a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 36, a CDR-H2 of SEQ ID NO: 37, and a CDR-H3 of SEQ ID NO: 38; and a light chain variable region (V L CD28), which comprises a light chain complementary determining region CDR-L1 of SEQ ID NO: 39, a CDR-L2 of SEQ ID NO: 40, and a CDR-L3 of SEQ ID NO: 41; or (ii) a heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25.

[0024] In one 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 (V H CD28), which comprises CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37, and CDR-H3 of SEQ ID NO: 38, and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO:39, CDR-L2 of SEQ ID NO:40, and CDR-L3 of SEQ ID NO:41.

[0025] On the other hand, the antigen-binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen-binding molecule comprises: a heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22, and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25.

[0026] Furthermore, there is provided a bispecific agonist CD28 antigen binding molecule as previously defined herein, wherein the antigen binding domain capable of specific binding to CD28 comprises: a heavy chain variable region (V HCD28), which comprises 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; and a light chain variable region (V L CD28), which comprises 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.

[0027] In a further aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28) 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; and a light chain variable region (V L CD28), 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.

[0028] In another aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to CD28 comprises:

[0029] (a) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0030] (b) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 27, or

[0031] (c) Heavy chain variable region (V HCD28), which comprises the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 61, or

[0032] (d) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0033] (e) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0034] (f) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 59, or

[0035] (g) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 27, or

[0036] (h) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 43, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 27, or

[0037] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0038] (j) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 59, or

[0039] (k) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:27.

[0040] In one particular aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:54.

[0041] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 46; and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:53.

[0042] In a further aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 42; and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:27.

[0043] 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).

[0044] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CEA comprises:

[0045] (i) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 188, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 189, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 190; and a light chain variable region (V LCEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 191, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 192, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 193; or

[0046] (ii) (ii) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 182; and a light chain variable region (V L CEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 183, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 184, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 185; or

[0047] (iii) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 127, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 128, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 129; and a light chain variable region (V L CEA), comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 130, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 131, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 132, or

[0048] (iv) (iv) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 507, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 508, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 509; and a light chain variable region (V L CEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 510, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 511, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 512.

[0049] On the one hand, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (VH CEA), which comprises 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; and a light chain variable region (V L CEA), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 186; and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 187.

[0050] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CEA comprises:

[0051] (a) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 194, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 195, or

[0052] (b) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 196, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 197, or

[0053] (c) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 198, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 199, or

[0054] (d) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 200, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 201, or

[0055] (e) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 202, and a light chain variable region (V LCEA), which comprises the amino acid sequence of SEQ ID NO: 203, or

[0056] (f) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 204, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 205, or

[0057] (g) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 206, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 207, or

[0058] (h) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 208, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 209, or

[0059] (i) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 210, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 211, or

[0060] (j) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 212, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 213.

[0061] In particular, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 200; and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 201.

[0062] 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 fibroblast activation protein (FAP). In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to FAP comprises: (a) a heavy chain variable region (VH FAP), 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, and a light chain variable region (V L FAP), which comprises (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, or (b) a heavy chain variable region (V H FAP), 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, and a light chain variable region (V L 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. In particular, the antigen-binding domain capable of specifically binding to FAP comprises: a heavy chain variable region (V H FAP), 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; and a light chain variable region (V L 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to FAP comprises: (a) a heavy chain variable region (V H FAP), which comprises 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: 18, and a light chain variable region (V LFAP), which comprises 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: 19, or (b) a heavy chain variable region (V H FAP), which comprises 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: 10, and a light chain variable region (V L FAP), which comprises 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: 11. In particular, the antigen-binding domain capable of specifically binding to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 18; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 19.

[0063] 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 an epithelial cell adhesion molecule (EpCAM). In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to EpCAM comprises: a heavy chain variable region (V H EpCAM), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 515, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 516, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 517; and a light chain variable region (V L EpCAM), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to EpCAM comprises: (a) a heavy chain variable region (V H EpCAM), which comprises 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; and a light chain variable region (V LEpCAM), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to EpCAM comprises: a heavy chain variable region (V H EpCAM), which comprises the amino acid sequence of SEQ ID NO: 521; and a light chain variable region (V L EpCAM), which comprises the amino acid sequence of SEQ ID NO:522.

[0064] 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, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to HER3 comprises: a heavy chain variable region (V H HER3), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 523, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 524, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 525; and a light chain variable region (V L HER3), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to HER3 comprises: (a) a heavy chain variable region (V H HER3), which comprises 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; and a light chain variable region (V L HER3), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to HER3 comprises: a heavy chain variable region (V H HER3), comprising the amino acid sequence of SEQ ID NO: 529; and a light chain variable region (V L HER3), which comprises the amino acid sequence of SEQ ID NO:530.

[0065] 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 CD30. In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CD30 comprises: a heavy chain variable region (V H CD30), 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; and a light chain variable region (V L CD30), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to CD30 comprises: (a) a heavy chain variable region (V H CD30), which comprises 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 a light chain variable region (V L CD30), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to CD30 comprises: a heavy chain variable region (V H CD30), comprising the amino acid sequence of SEQ ID NO: 537; and a light chain variable region (V L CD30), which comprises the amino acid sequence of SEQ ID NO: 538.

[0066] In addition, 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. In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to TBPG comprises: a heavy chain variable region (V HTBPG), 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; and a light chain variable region (V L TBPG), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to TBPG comprises: (a) a heavy chain variable region (V H TBPG), which comprises 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; and a light chain variable region (V L TBPG), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to TBPG comprises: a heavy chain variable region (V H TBPG), comprising the amino acid sequence of SEQ ID NO: 545; and a light chain variable region (V L TBPG), which comprises the amino acid sequence of SEQ ID NO:546.

[0067] In a further aspect, the present invention provides a bispecific agonist CD28 antigen binding molecule characterized by monovalent binding to CD28, comprising: (a) an 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 stably associating, comprising 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 multiple myeloma (MM) cell surface antigen is selected from the group consisting of CD38, BCMA, and GPRC5D.

[0068] Therefore, 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, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises: (a) a heavy chain variable region (V H GPRC5D), 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, and a light chain variable region (V L GPRC5D), which comprises (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, or (b) a heavy chain variable region (V H GPRC5D), 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, and a light chain variable region (V L GPRC5D), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), which comprises 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; and a light chain variable region (V L GPRC5D), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 569; and a light chain variable region (VL GPRC5D), which comprises the amino acid sequence of SEQ ID NO:570.

[0069] 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, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CD38 comprises: a heavy chain variable region (V H CD38), 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; and a light chain variable region (V L CD38), comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 550, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 551 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 552. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to CD38 comprises: a heavy chain variable region (V H CD38), which comprises 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; and a light chain variable region (V L CD38), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to CD38 comprises: a heavy chain variable region (V H CD38), comprising the amino acid sequence of SEQ ID NO: 553; and a light chain variable region (V L CD38), which comprises the amino acid sequence of SEQ ID NO:554.

[0070] 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, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to BCMA comprises: a heavy chain variable region (V HBCMA), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 555, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 556, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 557; and a light chain variable region (V L BCMA), comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 558, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 559 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 560. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to BCMA comprises: a heavy chain variable region (V H BCMA), which comprises 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; and a light chain variable region (V L BCMA), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to FAP comprises: a heavy chain variable region (V H BCMA), comprising the amino acid sequence of SEQ ID NO: 561; and a light chain variable region (V L BCMA), which comprises the amino acid sequence of SEQ ID NO: 562.

[0071] In a further aspect, the present invention provides a bispecific agonist CD28 antigen binding molecule characterized by monovalent binding to CD28, comprising: (a) an 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 stably associating, comprising 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 B cell surface antigen is selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37.

[0072] Therefore, 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, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CD19 comprises: (a) a heavy chain variable region (V H CD19), comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 406, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 407, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 408, and a light chain variable region (V L CD19), comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 409, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 410 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 411, or (b) a heavy chain variable region (V H CD19), comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 414, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 415, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 416, and a light chain variable region (V L CD19), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to CD19 comprises: (a) a heavy chain variable region (V H CD19), which comprises an amino acid sequence that is at least about 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 412, and a light chain variable region (V L CD19), which comprises an amino acid sequence that is at least about 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 413, or (b) a heavy chain variable region (V H CD19), which comprises an amino acid sequence that is at least about 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 420, and a light chain variable region (V LCD19), which comprises an amino acid sequence that is at least about 95%, 98% or 100% identical to the amino acid sequence of SEQ ID NO: 421. In particular, the antigen binding domain capable of specifically binding to CD19 comprises: a heavy chain variable region (V H CD19), comprising the amino acid sequence of SEQ ID NO: 412; and a light chain variable region (V L CD19), which comprises the amino acid sequence of SEQ ID NO:413.

[0073] 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, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CD79b comprises: a heavy chain variable region (V H CD79b), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 422, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 423, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 424; and a light chain variable region (V L CD79b), comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 425, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 426 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 427. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to CD79b comprises: a heavy chain variable region (V H CD79b), which comprises 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; and a light chain variable region (V L CD79b), which comprises 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. In particular, the antigen binding domain capable of specifically binding to CD79b comprises: a heavy chain variable region (V H CD79b), comprising the amino acid sequence of SEQ ID NO: 428; and a light chain variable region (V L CD79b), which comprises the amino acid sequence of SEQ ID NO:429.

[0074] In a further aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as previously defined herein, wherein the antigen binding domain capable of specific binding to CD28 is a Fab fragment or a crossFab fragment.

[0075] In another aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0076] (a) a Fab fragment that can specifically bind to CD28,

[0077] (b) a crossFab fragment that specifically binds to a tumor-associated antigen; and

[0078] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0079] In a further aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0080] (a) a first Fab fragment capable of specifically binding to CD28;

[0081] (b) a second Fab fragment capable of specifically binding to a tumor-associated antigen; and

[0082] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function,

[0083] The 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 the second Fab fragment capable of specifically binding to a tumor-associated antigen, and the Fab heavy chain of the second Fab fragment is then fused at its C-terminus to the N-terminus of one of the Fc domain subunits.

[0084] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as disclosed herein, comprising:

[0085] (a) a first Fab fragment capable of specifically binding to CD28;

[0086] (b) a second Fab fragment and a third Fab fragment capable of specifically binding to a tumor-associated antigen; and

[0087] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function,

[0088] The 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 the second Fab fragment capable of specifically binding to a tumor-associated antigen, the Fab heavy chain of the second Fab fragment is then fused at its C-terminus to the N-terminus of the first subunit of the Fc domain, and the 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 subunit of the Fc domain.

[0089] In a further aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0090] (a) a Fab fragment capable of specifically binding to CD28;

[0091] (B) a VH domain and a VL domain capable of specifically binding to a tumor-associated antigen; and

[0092] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function,

[0093] The Fab fragment capable of specifically binding to CD28 is fused at its C-terminus to the N-terminus of the first subunit of the Fc domain, and one of the VH domain and the VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of the first subunit of the Fc domain via a peptide linker, and the other of the VH domain and the VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of the second subunit of the Fc domain via a peptide linker.

[0094] 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 vectors, comprising isolated polynucleotides of the present invention; and a host cell is provided, comprising isolated polynucleotides or expression vectors of the present invention. In some aspects, the host cell is a eukaryotic cell, particularly a mammalian cell. On the other hand, a method for producing a bispecific agonist CD28 antigen binding molecule as described herein is provided, comprising culturing the host cell of the present invention under conditions suitable for expressing the bispecific agonist CD28 antigen binding molecule. Optionally, the method further 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.

[0095] The present invention further provides a pharmaceutical composition comprising the bispecific agonist CD28 antigen binding molecule of the present invention and at least one pharmaceutically acceptable excipient. In one aspect, the pharmaceutical composition is used to treat cancer.

[0096] The present invention also encompasses methods of using bispecific agonist CD28 antigen binding molecules and pharmaceutical compositions of the present invention. On the one hand, the present invention provides bispecific agonist CD28 antigen binding molecules or pharmaceutical compositions according to the present invention, which are used as medicines. On the one hand, bispecific agonist CD28 antigen binding molecules as described herein are provided, which are used for (a) enhancing cell activation or (b) enhancing T cell effector function. On the one hand, bispecific agonist CD28 antigen binding molecules or pharmaceutical compositions according to the present invention are provided, which are used to treat diseases. In a specific aspect, the disease is cancer. On the other hand, bispecific agonist CD28 antigen binding molecules or pharmaceutical compositions according to the present invention are provided, which are used to treat cancer, wherein bispecific agonist CD28 antigen binding molecules are co-administered with chemotherapeutics, radiotherapy and / or other agents for cancer immunotherapy. In a further aspect, bispecific agonist CD28 antigen binding molecules or pharmaceutical compositions are provided, which are used to treat cancer, wherein bispecific agonist CD28 antigen binding molecules are co-administered with T cell activation anti-CD3 bispecific antibodies. In yet another aspect, a bispecific agonist CD28 antigen binding molecule or a pharmaceutical composition is provided for use in treating cancer, wherein the bispecific agonist CD28 antigen binding molecule is administered in combination with an anti-PD-L1 antibody or an anti-PD-1 antibody.

[0097] Also provided are uses of bispecific agonist CD28 antigen binding molecules according to the present invention in the preparation of a medicament for treating a disease; and methods for treating individual diseases, comprising administering to the individual a pharmaceutically effective amount of a bispecific agonist CD28 antigen binding molecule according to the present invention or a composition comprising a bispecific agonist CD28 antigen binding molecule according to the present invention. In a specific aspect, the disease is cancer. On the one hand, there is provided a method for (a) enhancing cell activation or (b) enhancing T cell effector function in an individual, comprising administering to the individual a pharmaceutically acceptable bispecific agonist CD28 antigen binding molecule according to the present invention or a composition comprising a bispecific agonist CD28 antigen binding molecule according to the present invention. On the other hand, there is provided uses of bispecific agonist CD28 antigen binding molecules according to the present invention in the preparation of a medicament for treating a disease, wherein treatment comprises administering in combination with chemotherapeutic agents, radiotherapy, and / or other agents for cancer immunotherapy. In a further aspect, a method for treating a disease in an individual is provided, comprising administering to the individual a pharmaceutically effective amount of a bispecific agonist CD28 antigen binding molecule according to the present invention or a composition comprising a bispecific agonist CD28 antigen binding molecule according to the present invention, wherein the method comprises administering in combination with a chemotherapeutic agent, radiotherapy, and / or other agent for cancer immunotherapy. In a further aspect, a method for treating a disease in an individual is provided, comprising administering to the individual a pharmaceutically effective amount of a bispecific agonist CD28 antigen binding molecule according to the present invention or a composition comprising a bispecific agonist CD28 antigen binding molecule according to the present invention, wherein the method comprises administering in combination with a T cell activating anti-CD3 bispecific antibody. On the other hand, a method for treating a disease in an individual is provided, comprising administering to the individual a pharmaceutically effective amount of a bispecific agonist CD28 antigen binding molecule according to the present invention or a composition comprising a bispecific agonist CD28 antigen binding molecule according to the present invention, wherein the method comprises administering in combination with an anti-PD-L1 antibody or an anti-PD-1 antibody. Also provided is a method of 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 present invention or a composition comprising the bispecific agonist CD28 antigen binding molecule according to the present invention in a pharmaceutical form, so as to inhibit tumor cell growth. In any of the above aspects, the individual is preferably a mammal, particularly a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] exist Figures 1A to 1L , a schematic diagram of the molecule is shown. Figure 1A The CD28 agonistic antibody CD28 (SA) is shown in its huIgG4 isotype (TGN1412).

[0099] Figure 1B Shown is a CD28 (SA) agonistic antibody ("Fc silent") that is of the hu IgGl PGLALA isotype.

[0100] Figure 1C 、 1D , 1E and 1F show bispecific FAP-CD28 antigen binding molecules in 1+1 format, 1+2 format, 2+2 format and 1+4 format, respectively.

[0101] Figure 1G 、 1H A and 1J show bispecific CEA-CD28 antigen binding molecules in 1+2 format, 2+2 format, and 1+1 format, respectively.

[0102] Figure 1I Schematic representation of a CD28 agonistic antibody variant that is a monovalent hu IgG1 PGLALA isotype ("Fc silent").

[0103] Figure 1K A bispecific FAP-CD28 antigen binding molecule is shown in a 1+1 format, wherein the FAP antigen binding domain is represented as a VH domain and a VL domain each fused to one C-terminus of an Fc domain subunit.

[0104] Figure 1L A 2+1 format of a bispecific FAP-CD28 antigen binding molecule is shown, wherein the CD28 antigen binding domain, represented as crossFab, is fused at its C-terminus to the N-terminus of one of the heavy chains of a "bivalent" FAP antibody.

[0105] Figure 1M Another bispecific FAP-CD28 antigen binding molecule in a 1+1 format is shown, in which the CD28 antigen binding domain, represented as crossFab, is fused at its C-terminus to the N-terminus of the FAP-binding Fab fragment.

[0106] Figure 1N Shown is a trispecific FAP-CEA-CD28 antigen binding molecule in a 1+1+1 format, wherein the CD28 antigen binding domain is represented as a Fab, fused at the C-terminus of both its 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 wherein the anti-CEA CrossFab fragment is part of the huIgG1 PG-LALA Fc hole chain.

[0107] Figure 2A 、 2B, 2C, 2D and 2E relate to the binding of CD28 agonistic antibodies and FAP-CD28 antigen binding molecules to human CD28 or human FAP on cells. Binding of CD28 (SA) in the form of IgG4 isotype and hu IgG1 PGLALA isotype to human CD28 is shown ( Figure 2A ) and different FAP-CD28 molecules and human CD28 on cells ( Figure 2B ) and human FAP ( Figure 2C ). The median fluorescence intensity of the binding of different CD28 agonistic antibodies or anti-DP47 targeting molecules to CHO cells expressing human CD28 (parental cell line CHO-k1ATCC#CCL-61, modified to stably overexpress human CD28) or 3T3 cells expressing human FAP (NIH / 3T3 cell line (ATCC CRL-1658)) was assessed by flow cytometry. Three parallel technical samples and SEM are shown. A comparison of FAP(4B9)-CD28(SA) antigen binding molecules (molecules D, E and F in Example 1) is shown in Figure 2. Figure 2D (binds to human CD28) and Figure 2E (binding to human FAP).

[0108] Figure 2F and 2G The monovalent binding of FAP-CD28 antigen binding molecules of different formats to CD28 and FAP is shown. Curves are shown 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. The binding of FAP-CD28 antibodies or anti-DP47 antibodies (negative control) to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, modified to stably overexpress human CD28) evaluated by flow cytometry are shown. Figure 2F ) or 3T3 cells expressing human FAP (NIH / 3T3 cell line (ATCC CRL-1658)) ( Figure 2G ) The median fluorescence intensity of the combination of three replicate samples and SEM are shown. Figure 2H and 2I Binding of FAP-CD28 2+1 (P1AE5231, molecule G) to CD28 and FAP is shown, respectively.

[0109] The alignment of the variable domains of CD28(SA) and its variants is shown in Figures 3A to 3DTo remove cysteine 50 and reduce the affinity of the resulting anti-CD28 binders to varying degrees, an alignment of the CD28(SA) VH domain and its variants is shown in Figure 3A and 3B Of note, in VH variants i and j, the CDRs of CD28(SA) were transplanted from the IGHV1-2 framework to the IGHV3-23 framework ( Figure 3B ). In order to reduce the affinity of the anti-CD28 binding agent obtained to varying degrees, Figure 3C and 3D An alignment of the CD28(SA) VL domain and its variants is shown in . In variant t, the CDRs are grafted into the framework sequence of the trastuzumab (Herceptin) VL sequence.

[0110] exist Figures 4A to 4C In Figure 2, binding of affinity-reduced CD28 agonistic antibody variants in a monospecific, monovalent IgG format from supernatants to human CD28 on cells is shown. The median fluorescence intensity of binding to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC #CCL-61, modified to stably overexpress human CD28) was assessed by flow cytometry and compared to a negative control (anti-DP47) and original TGN1412. Binding curves for variants 1 to 10 are shown in Figure 2. Figure 4A The binding curves of variants 11 to 22 are shown in Figure 4B The binding curves of variants 23 to 31 are shown in Figure 4C In Figure 2, two technical parallel samples and SD are shown.

[0111] exist Figure 4D and 4E Binding of FAP-targeting bispecific CD28 agonist antibody variants in the huIgG1 PG-LALA 1+1 format and selected affinity-reduced CD28 agonist antibody variants to human CD28 on cells is shown. Binding curves for bispecific 1+1 constructs with variants 8, 11, 12, 15, 16, and 17 are shown in Figure 4D The binding curves of the bispecific 1+1 constructs with variants 19, 23, 25, 27 and 29 are shown in Figure 4E In. Selected binders were selected based on affinity to generate a 1+1 bispecific format targeting FAP. Figure 4F and 4GIn the figure, binding of the same FAP-targeting bispecific CD28 agonist antibody variant to human FAP in the huIgG1 PG-LALA 1+1 format is shown. Median fluorescence intensity of binding to CHO cells expressing human CD28 (parental cell line e CHO-k1 ATCC # CCL-61, modified to stably overexpress human CD28) or 3T3 cells expressing human FAP (NIH / 3T3 cell line (ATCC CRL-1658)) assessed by flow cytometry is provided, compared to a negative control (anti-DP47) and TGN1412 (molecule A). Three replicates and SEM are shown.

[0112] exist Figure 4H 、 4I In vitro potency of selected FAP-targeting bispecific CD28 agonistic antibody variants in huIgG1 PG-LALA 1+1 format is shown in 4J. PBMC T cells were incubated with MV3 melanoma cells expressing MCSP and FAP for 5 days in the presence of limiting concentrations of MCSP-TCB (5 pM, P1AD2189) and increasing concentrations of FAP-CD28 constructs with the indicated CD28 variant binders. Figure 4H Figure 2 shows CFSE dilution as a measure of T cell proliferation of CD8 T cells assessed by flow cytometry. Error bars show SEM, and the figure shows representative results of three technical replicates from two donors. Figure 4I The K values of the CD28 binder variants are shown in D Correlation of titer (nM) with area under the curve (a) expressed as a percentage of the parental TGN1412 clone (CD28 (SA)). Figure 4J Target cell killing at 90 h is shown in FIG.

[0113] Figures 5A to 5D The establishment and mode of action of high density (HD) pre-culture involving CD28 (SA). PBMC T cells were pre-cultured at high density (HD) for 2 days or freshly isolated from PBMC and stimulated with increasing concentrations of CD28 (SA). The expression of CD28 (SA) (molecule A, P1AE1975) as an indicator of T cell proliferation after 5 days of stimulation is shown. Figure 5A ) and cytokine secretion indicators after 2 days of stimulation ( Figure 5B ) of CFSE dilution. Figure 5C Shown are the percentages of FcγRIIb expression in PBMC monocytes and B cells before and after 2 days of preculture of HD PBMCs assessed by flow cytometry. Figure 5D: HD-pre-cultured PBMCs were co-cultured with CD28(SA) for 5 days in the presence or absence of FcγRIIb blocking antibody or isotype control, and the percentage of CD4 T cells at CFSE dilution was assessed by flow cytometry. Graphs are representative of at least 6 donors ( Figure 5A 、 5B ) and 2 donors ( Figure 5C 、 5D ), each donor was evaluated in an independent experiment. These figures show three technical replicates. Error bars represent mean ± SEM. Statistical analysis was performed by Student's t-test. ***: p < 0.001. The superagonistic effect of CD28(SA)IgG4 is dependent on cross-linking with FcγRIIb.

[0114] exist Figure 6A and 6B Figure 2 shows T cell proliferation after 5 days of stimulation with either native Fc-wildtype IgG4 CD28(SA) (P1AE1975) or CD28(SA) with the P329G-LALA mutation (P1AD9289), i.e., CFSE dilution of CD4 T cells. T cells were precultured at high density for 2 days. The graph is representative of at least 3 independent experiments. Three parallel technical samples are shown. Fc silencing abolishes the hyperagonism of TGN1412. Adding a tumor-targeting moiety to Fc-silenced TGN1412 restores the hyperagonism, which then becomes dependent on the presence of the tumor target.

[0115] exist Figure 7A 、 7B , 7C and 7D, a comparison of FAP-targeted CD28 agonists in different formats (2+2 and 1+2) with superagonist (CD28(SA)) binders and conventional agonist binders (9.3, CD28(CA)) is shown. FAP-targeted CD28 agonists with conventional CD28 agonist binders did not function as superagonists. PBMC T cells were co-cultured with 3T3-huFAP cells (in the presence of FAP) in the presence of increasing concentrations of superagonist binders (SA, Figure 7A ) or conventional agonist binders (9.3, Figure 7B ) in the presence of FAP-CD28 format for 5 days. T cell proliferation was shown. PBMC T cells were then co-cultured with 3T3 WT cells (without FAP) in the presence of increasing concentrations of a superagonist binding agent (SA, Figure 7C ) or conventional agonist binders (9.3, Figure 7D) were co-cultured for 5 days in the FAP-CD28 format. CFSE dilution is shown as a measure of T cell proliferation of CD8 T cells, assessed by flow cytometry on day 5 after stimulation. The graph shows cumulative data from 3 donors in 3 independent experiments. Error bars show SEM. Cytokines were also measured from the supernatant after 2 days of co-culture in the same experimental setup. Values are provided in Figure 7E middle.

[0116] The ability of various forms of FAP-CD28 with either a superagonistic CD28 (SA) binder or a conventional agonistic binder (CD28 (CA)) to induce killing of FAP-expressing RFP-MV3 melanoma cells was assessed by live cell imaging over the course of 90 hours using IncuCyte technology. All molecules, including FAP-TCB (P1AD4645), were used at 10 nM. Figure 8A 、 8B A and 8C show representative results of three technical parallel samples from three donors, respectively. Figure 8D Cumulative results expressed as the area under the curve (AUC) at t = 90 h for three donors from three independent experiments are shown. Boxes indicate the 25th to 75th percentiles, and whiskers indicate the minimum to maximum values. Statistical analysis was performed by paired one-way ANOVA. ***: p < 0.001, ns: not significant.

[0117] Figure 9A and 9B Comparison of different formats of CD28 agonists with superagonist binders and conventional agonist binders targeting CEA is shown. CEA-CD28 induced killing of CEA-expressing RFP cells by various formats with superagonist CD28 (SA) binders or conventional agonist binders (CD28 (CA)) was assessed by live cell imaging over the course of 90 hours using IncuCyte technology. + Capacity of MKN45 gastric cancer cells. All molecules, including CEACAM5-TCB (P1AD5299), were used at 10 nM. Figure 9A Representative results of three technical parallel samples from one donor are shown. Figure 9B Statistical analysis of three replicate technical samples from one donor in one experiment at t = 90 h is shown, expressed as the area under the curve (AUC). Boxes show the 25th to 75th percentiles, and whiskers show the minimum to maximum values. Statistical analysis was performed by paired one-way ANOVA. ***: p < 0.001. The results demonstrate that CEA-targeted CD28 agonists with conventional CD28 agonist binders do not behave in a hyperagonistic manner.

[0118] exist Figure 10A 、 10BIn A and 10C, it is shown that a targeted CD28 agonist with a monovalent superagonist binder is not functionally superagonist. PBMC T cells were co-cultured with 3T3-huFAP cells for 5 days in the presence of increasing concentrations of FAP-CD28 with a bivalent CD28 binder (P1AD9011, filled circles) or in the presence of FAP-CD28 with a monovalent state for CD28 binding (P1AD4492, filled circles). Figure 10A The CFSE dilution of CD8 T cells is shown in Figure 2. In addition, the activation marker CD69 ( Figure 10B ) and CD25( Figure 10C ) to assess T cell activation. Five days after stimulation, mean fluorescence intensity (MFI) of CD69 and CD25 staining is shown. Three replicates from a single donor are shown, and error bars represent mean average (SEM). The results suggest that TGN1412-like hyperagonism requires multivalent CD28 binding.

[0119] Figure 11A and 11B We show that monovalent and bivalent CD28 binding of agonistic CD28 antigen-binding molecules targeting FAP can support TCB-mediated effector functions with comparable potency if combined with a T cell bispecific antibody (TCB), but maintaining tumor target dependence of the CD28 agonist in the presence of the TCB requires CD28 binder monovalency. Figure 11A In the presence of FAP, PBMC T cells were incubated with MV3 melanoma cells expressing MCSP and FAP in the presence of limiting concentrations of MCSP-TCB (5 pM, P1AD2189) and increasing concentrations (range 0 to 10 nM) of FAP-CD28 (SA) with bivalent or monovalent binding to CD28, respectively, for 90 h. Target cell killing at 90 h was assessed by hepatocyte imaging using IncuCyte technology. Figure 11B In the present study, PBMC T cells were co-cultured with FAP-negative MKN45 gastric cancer cells expressing CEA (in the absence of FAP) in the presence of limiting concentrations of CEACAM5-TCB (10 pM, P1AD5299) and increasing concentrations of FAP-CD28 (ranging from 0 to 10 nM) with bivalent or monovalent binding to CD28, respectively, for 90 h. Target cell killing at 90 h as assessed by IncuCyte is shown. Data show MKN45 target cell killing over time from three parallel technical samples of one donor from one experiment, with error bars representing SEM.

[0120] exist Figure 12A and 12BIn the present study, we showed that FAP-CD28(SA), which has bivalent binding to CD28, lost its FAP dependency when combined with a T cell bispecific. Figure 12A In the absence of TCB. PBMC T cells were co-cultured with CEA-expressing MKN45 and 3T3-huFAP ("FAP present", solid circles) or with 3T3-WT ("FAP absent", open circles) in the presence of increasing concentrations of FAP-CD28(SA)2+1. Figure 12B As shown. In the presence of limiting concentrations of CEACAM5-TCB (10pM, P1AD5299) and increasing concentrations of FAP-CD28 2+1SA, PBMCT cells were co-cultured with MKN45 and 3T3-huFAP expressing CEA ("FAP exists", solid circles) or co-cultured with 3T3-WT ("FAP does not exist", hollow circles). CD8 T cell proliferation 5 days after stimulation is shown. The data represent 2 independent experiments using 2 donors. Results from one donor are shown, and data points represent three parallel technical samples, with error bars representing SEM.

[0121] Figure 13A 、 13B 13C and 13C show the function of FAP-CD28 (SA) antigen binding molecules with different formats of monovalent binding to CD28. Molecule C is the classic 1+1 format of FAP-CD28 (SA) (P1AD4492), molecule H is the 1+1 "head to tail" (H2T) format of FAP-CD28 (SA) (P1AE2021), molecule I is the FAP-CD28 (SA) 1+1 format (P1AE2236) fused to the C-terminus with the FAP binder, 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 MV3 melanoma cells expressing MCSP and FAP in the presence of a limited concentration of MCSP-TCB (5pM, P1AD2189) and a given format of FAP-CD28 at increasing concentrations (range 0 to 10nM). Shown are CD8 ( Figure 13A ) and CD4 T cells ( Figure 13B ) of the CFSE dilution as a measure of T cell proliferation. Figure 13CFigure : Shows MV3 cell killing over 84 hours in the presence of 5 pM MCSP-TCB alone, compared to a combination of 5 pM MCSP-TCB and increasing concentrations of various FAP-CD28 formats. Killing was assessed by live cell imaging using the IncuCyte system. All molecules were able to support TCB-mediated effector function. Graph shows 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 additive effect was significant compared to TCB alone: *p ≤ 0.05, **p ≤ 0.01; ***p ≤ 0.001. Error bars represent SEM.

[0122] CEA-CD28 1+1 format combined with TCB kills target cells Figure 14 As shown. PBMC T cells were co-cultured with CEA-expressing MKN45 gastric cancer cells for 90 hours in the presence of a limiting concentration of CEACAM5-TCB (10 pM, P1AD5299) in combination with 2 nM of CEA-CD28 (P1AE3127) or non-targeted CD28 (P1AD8944). Data show killing of MKN45 target cells over time from one donor in one experiment. Killing was assessed by live cell imaging using the IncuCyte system. The results demonstrate that only this combination leads to target cell killing; at the given concentrations, neither molecule alone induces killing. CEA-CD28 acts synergistically with CEACAM5-TCB.

[0123] exist Figure 15 The results show that CEA-CD28 enhances CEA-TCB and CEACAM5-TCB and reduces the CEA expression threshold for TCB-induced T cell activation. PBMC T cells were incubated with increasing concentrations of 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 (medium expression, approximately 60,000 CEA binding sites / cell), and (iii) HT-29 (low expression, approximately 6,000 CEA binding sites / cell). T cell proliferation, an indicator of T cell activation, was assessed by flow cytometry.

[0124] Figure 16The binding of the selected CD28 binding agent variants with reduced affinity for the bispecific monovalent 1+1 format targeting CEA to cellular CD28 is shown. The median fluorescence intensity of the binding of CEA-CD28 antibodies or anti-DP47 antibodies (negative control) to CHO cells expressing human CD28 (parental cell line CHO-k1 ATCC#CCL-61, modified to stably overexpress human CD28) was assessed by flow cytometry. Three parallel technical samples and SEM are shown.

[0125] Figure 17A 、 17B The function of the selected affinity-reduced CD28 binder variants in a bispecific monovalent 1+1 format targeting CEA was demonstrated in Figure 17C. PBMC T cells were co-cultured with CEA-expressing MKN45 gastric cancer cells in the presence of a limiting concentration of CEACAM5-TCB (10 pM, P1AD5299) and 2 nM of a combination of CEA-CD28 1+1 molecules with CD28 binder variants. After 5 days of co-culture, CD8 T cell proliferation was assessed by flow cytometry using CFSE dilution ( Figure 17A ) and CD4 T cell proliferation ( Figure 17B Target cell killing was assessed after 90 h of incubation ( Figure 17C ). Data show MKN45 target cell killing over time from one donor in one experiment. All molecules were able to support CEACAM5-TCB-mediated effector function.

[0126] Figure 18 Binding of humanized CEA (A5B7) huIgGl P329G LALA variant to MKN-45 is shown compared to binding of the parent murine A5B7 antibody. Antibodies were detected with fluorescently labeled secondary antibodies and fluorescence was measured by flow cytometry.

[0127] Figures 19A to 19C Schematic representation of recombinant proteins displaying different domains of the CEACAM5 protein used as antigen in phage display activities. Figure 19A The construct NABA-avi-His is shown, consisting of four Ig-like domains, N, A1, B and A2. Figure 19B The construct N(A2B2)A-avi-His is shown, and Figure 19C The construct NA(B2)A-avi-His is shown.

[0128] Figure 20A and 20B The VH and VL sequences of the humanized CEA antibody A5H1EL1D are shown, respectively, with randomized positions marked with Xs.

[0129] A schematic diagram of the phage vector for affinity maturation library is shown in Figure 21A (CDRH1 / H2 affinity maturation library), Figure 21B (CDRL1 / H2 affinity maturation library) and Figure 21C (CDRH3 / CDRL3 amplified library).

[0130] Figure 22A and 22B The VH amino acid sequences of affinity matured humanized CEA (A5H1EL1D) antibody variants are shown ( Figure 22A ) and VL amino acid sequence ( Figure 22B ) comparison chart.

[0131] exist Figures 23A to 23D , a schematic diagram of a bispecific CEA / CD28 antigen binding molecule as described in Example 11 is shown.

[0132] Figure 23A A bispecific CEA-CD28 antigen-binding molecule in a 1+1 format is shown, wherein the CEA antigen-binding domain is represented as a crossFab (VH / VL exchange) and to support correct pairing of the light chain, there are charged modifications in the Fab fragment with the CD28 antigen-binding domain. The Fc domain has a knob-in-hole modification and a P329G LALA mutation to eliminate binding to Fcγ receptors. Figure 23B In the figure, the CD28 antigen-binding domain is represented as a crossFab (VH / VL exchange), and the Fab fragment with the CEA antigen-binding domain contains a charged modification.

[0133] Figure 23C A bispecific CEA-CD28 antigen binding molecule in a 2+1 format is shown, wherein the CD28 antigen binding domain is represented as crossFab and two Fab fragments with the CEA antigen binding domain are fused to each other via the heavy chain (head to tail).

[0134] Figure 23D A bispecific CEA-CD28 antigen binding molecule in a 2+1 format is shown, wherein the CD28 antigen binding domain is represented as a crossFab, fused at its C-terminus to the N-terminus of one of the heavy chains of a "bivalent" CEA antibody ("classical" format).

[0135] exist Figure 24Figure 2 shows that the affinity-matured anti-CEA clone P002.139 exhibits improved binding to CEACAM5 on CEA-expressing MV3 cells. Figure 2 shows binding of CEA-CD28 bispecific antibodies carrying the affinity-matured anti-CEA clone P002.139 or the parental A5H1EL1D clone. Binding of the CEA-CD28 bispecific antibody or anti-DP47 antibody (negative control) to MV3 cells genetically engineered to express human CEACAM5 was assessed by flow cytometry. Two technical replicates with SEM are shown. The graph represents three independent experiments.

[0136] exist Figure 25A and 25B Figure 3 shows that affinity-matured anti-CEA clone P002.139 exhibits improved functionality in an IL-2 reporter assay. Luminescence readings are shown after 6 hours of incubation of MKN45 cells, IL-2 reporter cells, with 5 nM CEA-TCB and CEA-CD28 carrying affinity-matured clone P002.139 or parental clone A5H1EL1D. Figure 25A Dose response is shown. The dashed line represents the luminescence achieved by CEA-TCB alone. Figure 25B In the Figure 25A Area under the curve values calculated from the data shown. Two technical replicates with SEM are provided. The graph is representative of three independent experiments.

[0137] Figure 26 Shown is the study design for an efficacy study using a bispecific CEA-CD28 antibody (comparison of different CEA clones) in combination with a CEA TCB in MKN45 xenografts in humanized mice. The design and the different treatment groups are shown.

[0138] Figures 27A to 27E Results of an efficacy study using a combination of CEA-CD28 and CEATCB in MKN45 xenografts in humanized mice are shown. Mean tumor volumes ( Figure 27A ) or tumor growth in a single mouse, as plotted on the y-axis ( Figures 27B to 27E ). Figure 27B Tumor growth of each individual mouse in the vehicle group is shown. Figure 27C showed tumor growth in mice treated with CEA TCB alone, Figure 27D Tumor growth in mice treated with CEA TCB and CEA(T84.66)-CD28(SA_variant 15) is shown, and Figure 27EShown are tumor growth in mice treated with CEA TCB and CEA(A5H1EL1D)-CD28(SA_variant 15). As can be seen, TCB-mediated tumor regression was increased in the presence of both bispecific CEA-CD28 antibodies.

[0139] Figure 28 Shown is the study design for an efficacy study using a bispecific CEA-CD28 antibody (comparison of different CD28 clones) in combination with a CEACAM5 TCB in BXPC3 xenografts in humanized mice. Design and different treatment groups are shown.

[0140] Figure 29 Tumor growth kinetics for all treatment groups are shown (mean, + SEM) and the corresponding TGI values for each treatment arm are shown in Table 33 (Example 13.2).

[0141] In vitro Immuno-PD data show Figures 30A to 30D middle. Figure 30A Representative dot plots of stained tumor single cell suspensions for each treatment arm are shown (CD3 vs. CD45 and CD4 vs. CD8). Summary of CD3, CD8, and CD4 T cell infiltration are shown in Table 1. Figure 30B (CD3), Figure 30C (CD8) and Figure 30D (CD4) is shown.

[0142] Figure 31 Shown is the study design for an efficacy study using a bispecific CEA-CD28 antibody (CEA(A5H1EL1D)-CD28(SA_variant 8)) in combination with a CEA TCB in MKN45 xenografts in humanized mice. The design and different treatment groups are shown.

[0143] Figure 32 Tumor growth kinetics for all treatment groups are shown (mean, + SEM) and the corresponding TGI values for each treatment arm are shown in Table 35 (Example 13.3).

[0144] In vitro Immuno-PD data show Figure 33A and 33B middle. Figure 33A Representative dot plots of stained tumor single cell suspensions for each treatment arm are shown. Figure 33B Shown in.

[0145] exist Figures 34A to 34D , a schematic diagram of a bispecific CD28 antigen binding molecule as described in Example 14 is shown.

[0146] Figure 34AA 1+1 bispecific EpCAM-CD28 antigen-binding molecule is shown, in which the CD28 antigen-binding domain is represented as a crossFab (VH / VL exchange) and, to support proper pairing of the light chain, there are charge modifications in the Fab fragment with the EpCAM antigen-binding domain. The Fc domain has a knob-in-hole modification and a P329G LALA mutation to abrogate binding to Fcγ receptors.

[0147] exist Figure 34B In the figure, the Fab with CD28 antigen binding domain contains charged modifications, while the Fab with HER3 antigen binding domain is denoted as crossFab (VH / VL swap).

[0148] Figure 34C A bispecific CD30-CD28 antigen binding molecule in a 1+1 format is shown, wherein the Fab molecule with the CD28 antigen binding domain comprises a charged modification, while the Fab with the CD30 antigen binding domain is represented as a crossFab (VH / VL swap).

[0149] Figure 34D A bispecific TPBG-CD28 antigen binding molecule in a 1+1 format is shown, wherein the Fab molecule with the CD28 antigen binding domain contains a charged modification, while the Fab with the TPBG (5T4) antigen binding domain is represented as a crossFab (VH / VL swap).

[0150] Figures 35A to 35C Functional characterization of EpCAM-CD28 bispecific antigen binding molecules. Figure 35A In Figure 1, binding of EpCAM-CD28 (molecule 14A) to human CD28 on CD28-expressing CHO-k1 cells was assessed by flow cytometry. Binding to EpCAM on HT29 cells was assessed by flow cytometry and is shown in Figure 1. Figure 35B Anti-DP47 was used as a negative control for nonspecific binding of the antibody compound to the cells. The dots represent the mean of two parallel technical samples. Figure 35C Figure 2 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, is shown following 6 hours of incubation with HT-29 cells in the presence of a suboptimal concentration of anti-CD3 IgG (10 nM) and increasing concentrations of EpCAM-CD28. Points represent the mean of two technical replicates.

[0151] Figures 36A to 36C Functional characterization of a HER3-CD28 bispecific antigen binding molecule. Figure 36A In Figure 2, binding of HER3-CD28 (P1AF0151) to human CD28 on CD28-expressing CHO-k1 cells was assessed by flow cytometry. Binding of HER3-CD28 to HER3 on T-47D cells was assessed by flow cytometry. Figure 36B Anti-DP47 was used as a negative control for nonspecific binding of the antibody compound to the cells. The dots are the means of two parallel technical samples. Figure 36C Figure 1 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 readings, is shown following 6-hour 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. Points represent the mean of two technical replicates.

[0152] exist Figures 37A to 37C , a schematic diagram of a bispecific CD28 antigen binding molecule targeting a multiple myeloma (MM) cell surface antigen as described in Example 16 is shown.

[0153] Figure 37A A 1+1 bispecific GPRC5D-CD28 antigen-binding molecule is shown, in which the CD28 antigen-binding domain is represented as a crossFab (VH / VL exchange) and, to support proper pairing of the light chain, there are charge modifications in the Fab fragment with the GPRC5D antigen-binding domain. The Fc domain has a knob-in-hole modification and a P329G LALA mutation to abrogate binding to Fcγ receptors.

[0154] exist Figure 37B In the figure, the Fab with the CD28 antigen-binding domain contains charged modifications, while the Fab with the CD38 antigen-binding domain is denoted as crossFab (VH / VL exchange).

[0155] Figure 37C A bispecific BCMA-CD28 antigen-binding molecule in a 1+1 format is shown, wherein the Fab molecule with the CD28 antigen-binding domain is represented as a crossFab (VH / VL swap), while the Fab with the BCMA antigen-binding domain contains a charged modification.

[0156] Figure 37DShown is an anti-GPRC5D / anti-CD3 bispecific antibody (GPRC5DTCB) in a 2+1 format, wherein the Fab molecule with the GPCR5D antigen-binding domain contains charged modifications, while the Fab with the CD3 antigen-binding domain is represented as a crossFab (VH / VL swap).

[0157] Figures 38A to 38F Binding of bispecific antigen binding molecules targeting CD28 and multiple myeloma (MM) cell surface antigens to cells (Example 17.1) is shown. Binding of bispecific antigen binding molecules to the following antigens expressed on the indicated cell lines: human CD28 ( Figure 38A and 38E ), human CD38 on OCI-Ly18 cells ( Figure 38B ), human BCMA (B cell mutant antigen, Figure 38C ), and human GPRC5D (such as 38D and 38F) on CHO huGPRC5D L2 cells. The relative median fluorescence values (MFI) and SD of two parallel samples are shown. The EC of binding 50 Values were calculated by GraphPad Prism and included in Table 38.

[0158] like Figures 39A to 39F As shown, T cell activation of bispecific antigen binding molecules targeting CD28 and multiple myeloma (MM) cell surface antigens was evaluated in an IL-2 reporter gene assay. IL2 reporter gene cell assays (determined by luminescence) after 5 to 22 hours of incubation are shown. IL2 reporter gene effectors and target cells expressing GPRC5D 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 CD28 bispecific antigen binding molecules targeting MM as shown were titrated as indicated. Representative dose response curves for CD38-CD28 are shown in Figure 39A (after 5 hours of incubation) and Figure 39B Representative dose-response curves of BCMA-CD28 are shown in (after 22 hours). Figure 39C (after 5 hours) and Figure 39D (after 22 hours) and representative dose-response curves of GPRC5D-CD28 are shown in Figure 39E (after 5 hours) and Figure 39F (after 22 h of incubation) are shown.

[0159] Figures 40A to 40C Shown are 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 ) were used to evaluate the increase in T cell-mediated lysis of the MM cell line NCI-H929 expressing GPRC5D. Lysis was determined after 22 hours of co-incubation of human pan-T cells and MM tumor target cells at a final E:T ratio of 1:1. Two replicate technical samples and the 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.

[0160] Figure 41A Shown is a schematic diagram of a bispecific CD19-CD28 antigen-binding molecule in a 1+1 format as described in Example 18, wherein in the Fab comprising the CD19 antigen-binding domain, the VH domain and the VL domain are exchanged with each other (VH / VLcrossfab), and wherein in the Fab comprising the CD28 antigen-binding domain, certain amino acids in the CH1 domain and the CL domain are exchanged (charge variants) to allow better pairing with the light chain. Figure 41B The corresponding molecule is shown, in which the CD19 antigen binding domain has been replaced by the CD79b antigen binding domain (anti-CD79b crossfab).

[0161] Figure 42 Determination of kinetic and thermodynamic parameters involving CD79b (pollotuzumab) in the construct CD79b (huMA79b.v28)-CD28 (v15) 1+1. Soluble recombinant CD79b-His was captured on a CM5 chip via an anti-pentameric-His antibody, and the bispecific CD79b (huMA79b.v28)-CD28 (v15) 1+1 was used as the analyte. The smooth line represents the overall fit of the data to a 1:1 interaction model.

[0162] exist Figure 43A In the Figure 5, the median fluorescence intensity (MFI) of CD19-CD28 variant 15 (P1AE9040) binding to four different B cell lines expressing different levels of CD19 is shown. Binding was assessed by flow cytometry. Two parallel technical samples with SEM are shown. Figure 43B In the figure, FACS staining (MFI) of CD19 against four different B cell lines is shown.

[0163] The binding of CD19-CD28 with different CD28 affinities to human CD19 and CD28 on cells is shown in Figure 44A and 44B The median fluorescence intensity (MFI) of the binding to CHOk1-CD28 cells is shown ( Figure 44A) and the median fluorescence intensity (MFI) of binding to CD19 on Nalm6 B cells ( Figure 44B ). Points represent two parallel technical samples with SEM. The corresponding EC 50 Values are shown in Table 42 (CHOk1-CD28) and Table 43 (Nalm6) of Example 20. Binding was assessed by flow cytometry.

[0164] exist Figures 45A to 45D Figure 2 shows 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), is shown after 6 hours of incubation with different B cell lines in the presence of suboptimal concentrations of CD20-TCB and increasing concentrations of CD19-CD28v15. Points represent two replicates with SEM. Suboptimal CD20-TCB concentrations varied depending on the target cell line: 10 nM for Nalm6 and 0.05 nM for RCK8, WSU DLCL2, and Z138.

[0165] Figure 46 Show that CD19-CD28 with various CD28 affinities enhances the T cell activation mediated by CD20-TCB.Show that in the presence of suboptimal concentration of CD20-TCB (10nM) and increasing concentration of CD19-CD28v15, after incubation for 6 hours with Nalm6B cells, the IL-2 reporter gene cell activation measured by luminescence reading (LUM).Point represents two parallel technical samples with SEM.

[0166] The activation status of T cells derived from PBMCs was assessed after co-culture with CD20-expressing target cells (Nalm6) (E:T ratio of 5:1) and CD19-CD28 in the absence or presence of CD20-TCB. The activity of CD19-CD28 in the absence or presence of TCR signaling is shown in Figure 47 Figure 2. CD69 expression by PBMC-derived CD4 T cells after 48 h of incubation with Nalm6 cells and increasing concentrations of CD19-CD28v15 in the presence or absence of 10 nM CD20-TCB. Points represent three technical replicates with SEM.

[0167] exist Figures 48A to 48DIn the figure, it is shown that CD19-CD28 alone does not induce cytokine secretion in PBMC. Cytokine release in whole PBMC after 48 hours of co-culture with CD19-CD28 molecules in the presence or absence of CD20-TCB is shown. The bars represent the mean + SEM of three parallel technical samples. The data are representative of 2 donors. Cytokine secretion was assessed by the Bio-Plex Pro human cytokine 17-plex assay. IFNγ ( Figure 48A ), IL-2( Figure 48B )、IL-10( Figure 48C ) and TNF( Figure 48D ).

[0168] exist Figure 49A and 49B In the present study, functional data related to CD79b-CD28 enhancing CD20-TCB in the presence of Z138 B cells were shown. Figure 49A In the figure, the median fluorescence intensity (MFI) of binding to CD79b on Z138 B cells is shown. Figure 49B Figure 2 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) is shown 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. Points represent two technical replicates with SEM.

[0169] Figure 50 Shown is 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.

[0170] Figures 51A to 51D Results of an efficacy study using CD19-CD28 in NALM6 xenografts in humanized mice are shown. Mean tumor volumes ( Figure 51A ) or tumor growth in a single mouse, as plotted on the y-axis ( Figures 51B to 51D ). Figure 51B Tumor growth of each individual mouse in the vehicle group is shown. Figure 51C Tumor growth in mice treated with CD19-CD28 (variant 15) is shown, and Figure 51DShown are tumor growth in mice treated with CD19-CD28 (variant 8). As can be seen, CD19-CD28 (variant 8) as a single agent induced a stronger tumor growth inhibition effect compared to CD19-CD28 (variant 15). DETAILED DESCRIPTION

[0171] definition

[0172] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which the invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa.

[0173] As used herein, the term "antigen binding molecule" in its broadest sense refers 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.

[0174] As used herein, the term "antigen binding domain that binds to a tumor-associated antigen" or "a portion that is capable of specifically binding to a tumor-associated antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding domain is capable of activating signal transduction through its target cell antigen. In a specific aspect, the antigen binding domain is capable of guiding an entity attached thereto (e.g., a CD28 antibody) to a target site, for example, to a specific type of tumor cell or tumor stroma carrying an antigenic determinant. Antigen binding domains that are capable of specifically binding to a target cell antigen include antibodies and fragments thereof as further defined herein. In addition, antigen binding domains that are 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., WO2002 / 020565).

[0175] About antigen binding molecules, i.e. antibodies or their fragments, the term "antigen binding domains that are combined with target cell antigens" refers to a part of a molecule that includes a part or all of the specific binding to an antigen and a region that is complementary thereto. Antigen binding domains that can be specifically antigen-bound can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Specifically, antigen binding domains that can be specifically antigen-bound include antibody light chain variable region (VL) and antibody heavy chain variable region (VH). On the other hand, "antigen binding domains that can be specifically bound to tumor-associated antigens" can also be Fab fragments or crossFab fragments. On the other hand, "antigen binding domains that can be specifically bound to tumor-associated antigens" can also be Fab fragments or crossFab fragments. As used herein, the terms "first", "second" or "third" with respect to antigen binding domains, etc., are used for convenience in distinguishing when there is more than one type of part. Unless explicitly stated, the use of these terms is not intended to give a particular order or orientation to a part.

[0176] As used herein, the term "antigen binding domain capable of specifically binding to a B cell surface antigen" or "part capable of specifically binding to a B cell surface antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant on the surface of a B cell. In one aspect, the antigen binding domain is capable of activating signal transduction through its target cell antigen. In a specific aspect, the antigen binding domain is capable of guiding an entity attached thereto (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. In addition, 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., WO2002 / 020565).

[0177] The term "antigen binding domain capable of specifically binding to a multiple myeloma (MM) cell surface antigen" or "a portion 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 aspect, the antigen binding domain is capable of activating signal transduction through its target cell antigen. In a specific aspect, the antigen binding domain is capable of directing an entity attached thereto (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. In addition, 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 sequence proteins or designed repeat sequence domains (see, e.g., WO 2002 / 020565).

[0178] The term "antibody" herein is used in the broadest sense and encompasses various 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.

[0179] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants typically exist in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0180] As used herein, the term "monospecific" antibody refers to an antibody with one or more binding sites, each binding site being bound to the same epitope of the same antigen. The term "bispecific" means that an antigen binding molecule can specifically bind to at least two unique antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which has specificity for different antigenic determinants. However, a bispecific antigen binding molecule can also comprise an additional antigen binding site combined with other antigenic determinants. In some aspects, a bispecific antigen binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two unique cells or on the same cell. Therefore, the term "bispecific" according to the present invention can also include trispecific molecules, for example, bispecific molecules comprising a CD28 antibody and two antigen binding domains directed against two different target cell antigens.

[0181] Term " valence " used in the application represents that there is a certain number of specific binding sites to a unique antigenic determinant in the antigen binding molecules that have specificity to a unique antigenic determinant.Therefore, the terms " bivalent ", " tetravalent " and " hexavalent " represent respectively that there are two binding sites, four binding sites and six binding sites that are specific to a specific antigenic determinant in the antigen binding molecules.In a particular aspect of the present invention, the bispecific antigen binding molecules according to the present invention can be monovalent to a specific antigenic determinant, meaning that they only have one binding site to the antigenic determinant, or can be divalent or tetravalent to a specific antigenic determinant, meaning that they have two binding sites or four binding sites respectively to the antigenic determinant.

[0182] The terms "full-length antibody" and "complete antibody" are used interchangeably herein to refer to antibodies with a structure substantially similar to that of a natural antibody structure. "Native antibody" refers to natural immunoglobulin molecules with different structures. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH) (also referred to as a variable heavy chain domain or a heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL) (also referred to as a variable light chain domain or a light chain variable domain), followed by a light chain constant domain (CL) (also referred to as a light chain constant region). The heavy chains of antibodies can be assigned to one of five types, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0183] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an 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, crossFab 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, for example, Plückthun in The harmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion of Fab fragments and F(ab')2 fragments comprising salvage receptor binding epitope residues and having extended in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments having two antigen-binding sites that can be bivalent or bispecific, see, for example, 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 comprising 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, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent 6,248,516 B1). Antibody fragments can be prepared by various 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.

[0184] Papain digestion of intact antibodies produces two identical antigen-binding fragments called "Fab" fragments, each of which contains a heavy chain variable domain and a light chain variable domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to an antibody fragment comprising: a light chain fragment comprising a variable light chain (VL) domain and a constant domain (CL) of the light chain; and a variable heavy chain (VH) domain and the first constant domain (CH1) of the heavy chain. The difference between a Fab' fragment and a Fab fragment is that the Fab' fragment has some residues added to the carboxyl terminus of the heavy chain CH1 domain, which include one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domain have a free thiol group. Pepsin treatment produces a F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0185] The term "crossFab fragment" or "xFab fragment" or "exchange Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. Two different chain compositions of exchange Fab molecules are possible and are included in the bispecific antibodies of the present invention: in one aspect, the variable regions of the Fab heavy and light chains are exchanged, that is, the exchange Fab molecule comprises a peptide chain consisting of a light chain variable (VL) domain and a heavy chain constant domain (CH1), and a peptide chain consisting of a heavy chain variable domain (VH) and a light chain constant domain (CL). This exchange Fab molecule is also called CrossFab (VLVH) On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule comprises 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 also called a CrossFab. (CLCH1) .

[0186] "Single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: 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; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized via a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by creating an interchain disulfide bond by inserting a cysteine residue (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0187] "Exchange single-chain Fab fragments" or "x-scFabs" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen-binding site that specifically binds to an antigen, and wherein the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by creating interchain disulfide bonds through the insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0188] A "single-chain variable fragment (scFv)" is a fragment of the heavy chain variable region (V H ) and light chain variable region (V L ) fusion proteins, connected by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and can be V H The N-terminal and V LThe antibody fragment is connected to the C-terminus of the antibody or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original antibody. ScFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96). In addition, the antibody fragment comprises a single-chain polypeptide characterized by having a VH domain, i.e., capable of assembling into a functional antigen-binding site together with the VL domain; or having the characteristics of a VL domain, i.e., capable of assembling into a functional antigen-binding site together with the VH domain, thereby providing the antigen-binding properties of a full-length antibody.

[0189] "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, for example, 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 (Evibody), lipocalin (Anticalin), protein A derived molecules such as the Z domain of protein A (Affibody), A domain (Avimer / giant antibody), serum transferrin (transbody); designed ankyrin repeat proteins (DARPin), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanoantibodies, aVH), V NAR fragment, fibronectin (AdNectin), C-type lectin domain (tetranectin); variable domain of neoantigen receptor β-lactamase (V NAR fragments), human γ-crystallin or ubiquitin protein (Affilin molecule); Kunitz-type domains of human protease inhibitors, minibodies (such as proteins from the knottin family), peptide aptamers and fibronectin (adnectin). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a protein that is mainly expressed in CD4 +A CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the antibody CDRs can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also called Evibodies (e.g., US7166697B1). Evibodies are roughly 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, bile acids, retinoids, and lipids. They have a rigid β-sheet secondary structure with many loops at the open ends of the pyramidal structure and can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US7250297B1 and US20070224633. Affimers are scaffolds derived from protein A of Staphylococcus aureus that can be engineered to bind antigens. The domain consists of a three-helix bundle of approximately 58 amino acids. Libraries have been formed by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP1641818A1. Avimers are multidomain proteins derived from the A domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide bonded structure. Diversity is formed by the natural variation exhibited by the recombinant 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 in permitted surface loops. Examples of engineered transferrin scaffolds include trans-isomers. For further details, see J.Biol.Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to cell scaffolds. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn.They can be engineered to bind to different target antigens by randomizing the residues in the first α-helix and β-turn in each repeat. Their 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 US20040132028A1. Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of the antibody heavy chain of camelids (nanoantibodies or V H H fragment). In addition, the term single domain antibody includes autologous human heavy chain variable domain (aVH) or V derived from shark NAR Fragments. Fibronectin can be engineered to bind to an antigen scaffold. Adnectins consist of a backbone of the native amino acid sequence of the 10th domain of 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered to enable the Adnectin to specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamers are combined recognition molecules composed of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins that contain three to four cysteine bridges and are 25 to 50 amino acids long. Examples of these microproteins include KalataBI and conotoxins, as well as knottins. Microproteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0190] 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 competition assay, and conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competition assay.

[0191] The term "antigen binding domain" refers to a part of an antigen binding molecule that includes a region that specifically binds to and is complementary to a part or all of an antigen. In the case where the antigen is very large, the antigen binding molecule can only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0192] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a conformational configuration consisting of a continuous stretch of amino acids or different regions of non-continuous amino acids) to which an antigen-binding portion binds, thereby forming an antigen-binding portion-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, in free matter and / or in the extracellular matrix (ECM) in serum. Unless otherwise indicated, the protein used as an antigen herein can be any natural form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a specific embodiment, the antigen is a human protein. When referring to a specific protein herein, the term encompasses "full length", unprocessed protein, and any form of protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

[0193] "Specific binding" refers to binding that is selective for an antigen and can be distinguished from unwanted or nonspecific 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 familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, for example, as measured by SPR, the degree of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen binding molecule to the antigen. In certain embodiments, the dissociation constant (Kd) of the molecule bound to the antigen is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or lower, such as 10 -8 M to 10 -13 M, for example 10-9 M to 10 -13 M).

[0194] "Affinity" or "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0195] As used herein, "tumor-associated antigen" or TAA refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor (such as a cancer cell, a cell of a tumor stroma, a malignant B lymphocyte, or a melanoma cell). In some aspects, the target cell antigen is an antigen on the surface of a tumor cell. On the one hand, 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 specific 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 specific 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 specific aspect, the tumor-associated antigen is fibroblast activation protein (FAP) or carcinoembryonic antigen (CEA). On the one hand, 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. On the one hand, the TAA is a multiple myeloma (MM) cell surface antigen selected from the group consisting of CD38, BCMA and GPRC5D.

[0196] The term "fibroblast activation protein (FAP)" is also referred to as prolyl endopeptidase FAP or Seprase (EC 3.4.21). Unless otherwise indicated, the term refers to any natural FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed FAP, as well as any form of FAP produced by processing in cells. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. In one embodiment, the antigen binding molecules of the present invention are capable of specifically binding to human, mouse and / or cynomolgus macaque 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 amino acid position 760. The amino acid sequence of the human FAP ECD with a His tag 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 amino acid position 761. SEQ ID NO: 137 shows the amino acid sequence of the mouse FAP ECD with a His tag. SEQ ID NO: 138 shows the amino acid sequence of the cynomolgus macaque FAP ECD with a His tag. Preferably, the anti-FAP binding molecules of the present invention bind to the extracellular domain of FAP.

[0197] The term "carcinoembryonic antigen (CEA)" is also referred to as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). Unless otherwise indicated, the term refers to any naturally occurring CEA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human CEA is shown in UniProt accession number 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). CEA was originally classified as a protein expressed only in fetal tissues, but has now been identified in a variety of normal adult tissues. These tissues are primarily derived from epithelia, including cells of the gastrointestinal, respiratory, and urogenital tracts, as well as cells of the colon, cervix, sweat glands, and prostate (Nap et al., Tumour Biol., 9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelial origin and their metastases contain CEA as a tumor-associated antigen. The presence of CEA itself does not indicate transformation into cancer cells, but the distribution of CEA is indicative. In normal tissues, CEA is usually expressed on the apical surface of cells ( S., Semin Cancer Biol. 9(2):67-81(1999)), making it impossible for antibodies in the bloodstream to be absorbed. Compared with normal tissues, CEA tends to be expressed on the entire surface of cancer cells ( S., Semin Cancer Biol. 9(2): 67-81(1999)). This change in expression pattern makes CEA easy to bind to antibodies in cancer cells. In addition, CEA expression in cancer cells increases. In addition, increased CEA expression promotes increased cell-cell adhesion, which may lead to metastasis (Marshall J., Semin Oncol., 30(a Suppl. 8): 30-6, 2003). CEA expression is generally very high in various tumor entities. According to published data, my own analysis of tissue samples confirmed the high incidence of CEA, with an incidence of about 95% in colorectal cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, co-expressed with HER3), and 40% in breast cancer; and low expression levels were found in small cell lung cancer and glioblastoma.

[0198] CEA is easily cleaved from the cell surface and flows from the tumor into the bloodstream directly or through lymphatic vessels. Due to this property, serum CEA levels have been used as a clinical indicator for diagnosing cancer and screening for recurrence of cancer (especially colorectal cancer) (Goldenberg DM., 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).

[0199] The term "epithelial cell adhesion molecule (EpCAM)" refers to any natural EpCAM from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed EpCAM, as well as any form of EpCAM produced by processing in cells. The term also encompasses naturally occurring variants of EpCAM, e.g., splice variants or allelic variants. In one embodiment, the antigen binding molecules of the present invention are capable of specifically binding to human, mouse and / or cynomolgus macaque EpCAM. The amino acid sequence of human EpCAM is shown in UniProt (www.uniprot.org) accession number 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 signal transducer 1 (TACSTD1), 17-1A and CD326, is a type I transmembrane glycoprotein of about 40 kDa, which is often in cancers of epithelial origin and overexpressed by cancer stem cells, and is therefore an important molecule of interest for treatment and diagnosis. The extracellular domain EpCAM can be cut to produce a soluble extracellular domain molecule EpEX and an intracellular molecule EpICD. EpICD has been shown to associate with other proteins to form a nuclear complex that upregulates the expression of genes that promote cell proliferation. EpCAM may also be involved in the transformation (EMT) of epithelial to mesenchymal cells and may contribute to the formation of large metastatic lesions.

[0200] "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. 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 lymphomagenesis through anti-apoptotic mechanisms, and its impact on cell survival. Given the restriction of CD30 to certain tumor types, a logical extension is to attempt to use it as a therapeutic target. CD30 is a 120 kD transmembrane glycoprotein receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily, having an intracellular domain, a transmembrane domain, and an extracellular domain. The amino acid sequence of human CD30 is shown in UniProt Accession No. P28908 (SEQ ID NO: 472).

[0201] 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 indicated, it refers to any natural TPBG from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human TPBG is shown in UniProt accession number Q13641 (SEQ ID NO: 473).

[0202] The term "FolR1" refers to the folate receptor alpha, which has been identified as a potential prognostic and therapeutic target for a number of cancers. Unless otherwise indicated, the term refers to any native FolR1 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human FolR1 is shown in UniProt Accession No. P15328 (SEQ ID NO: 139), murine FolR1 has the amino acid sequence of UniProt Accession No. P35846 (SEQ ID NO: 140), and cynomolgus macaque FolR1 has the amino acid sequence shown in UniProt Accession No. G7PR14 (SEQ ID NO: 141). FolR1 is an N-glycosylated protein expressed on the plasma membrane. FolR1 has a high affinity for folic acid and several reduced folic acid derivatives and mediates the delivery of the physiological folate 5-methyltetrahydrofolate to the cell interior. FOLR1 is an ideal target for FOLR1-directed cancer therapy because it is overexpressed in the vast majority of ovarian cancers, as well as many uterine, endometrial, pancreatic, renal, lung, and breast cancers, whereas FOLR1 expression in normal tissues is restricted to the proximal tubules of the kidney, alveolar pneumocytes of the lung, bladder, testis, choroid plexus, and the apical membrane of epithelial cells of the thyroid gland. Recent studies have identified that FolR1 expression is particularly high in triple-negative breast cancer (Necela et al. PloS One 2015, 10(3), e0127133).

[0203] The term "melanoma-associated chondroitin sulfate proteoglycan (MCSP)" is also referred to as chondroitin sulfate proteoglycan 4 (CSPG4), and unless otherwise indicated, refers to any naturally occurring MCSP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human MCSP is shown in UniProt accession number Q6UVK1 (version 103, SEQ ID NO: 142). MCSP is a highly glycosylated integral membrane chondroitin sulfate proteoglycan expressed on the cell membrane by an N-linked 280 kDa glycoprotein component and a 450-kDa chondroitin sulfate proteoglycan component (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 is found in more than 90% of benign nevi and melanoma lesions. 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.

[0204] The term "epidermal growth factor receptor (EGFR)" is also referred to as the proto-oncogene c-ErbB-1 or receptor tyrosine protein kinase erbB-1. Unless otherwise indicated, the term refers to any native EGFR from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human EGFR is shown in UniProt accession number 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 to some extent homologous to the human epidermal growth factor receptor. HER2 is also known in the art as c-erbB-2 and sometimes as the rat homolog neu. Amplification and / or overexpression of HER2 is associated with a variety of human malignancies and appears to be integrally involved in the progression of 25% to 30% of human breast and ovarian cancers. In addition, the degree of amplification is inversely proportional to the observed median survival time of patients (Slamon, DJ et al., Science 244:707-712 (1989)). The amino acid sequence of human HER2 is shown in UniProt accession number P04626 (version 230, SEQ ID NO:144). The term "p95HER2" used herein refers to the carboxyl terminal fragment (CTF) of the HER2 receptor protein, also referred to as "611-CTF" or "100-115kDa p95HER2". By starting the translation of HER2 mRNA at codon position 611 of the full-length HER2 molecule, p95HER2 fragments are produced in cells (Anido et al., EMBO J 25; 3234-44 (2006)). It has a molecular weight of 100 to 115 kDa and is expressed on 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.

[0205] "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 to IV). Subdomains I and III are rich in leucine and are primarily involved in ligand binding. Subdomains II and IV are rich in cysteine and most likely promote protein conformation and stability by forming disulfide bonds. Subdomain II also contains the dimerization loop required for dimer formation. The cytoplasmic domain contains a membrane-proximal segment, a kinase domain, and a C-terminal domain. Although there is no evidence that overexpression, constitutive activation, or mutation alone of ErbB3 are oncogenic, https: / / en.wikipedia.org / wiki / ERBB3-cite_note-pmid8632008-18 However, proteins that act as heterodimerization partners (most importantly, binding to ErbB2) are associated with promotion of growth, proliferation, chemotherapy resistance, and invasion and metastasis. ErbB3 has been linked to resistance to targeted therapeutics in various cancers. The amino acid sequence of human HER3 is shown in UniProt accession number P21860 (version 224, SEQ ID NO: 471).

[0206] As used herein, "B cell surface antigen" refers to an antigenic determinant present on the surface of a B lymphocyte, particularly a malignant B lymphocyte (in this case, the antigen is also referred to as a "malignant B cell surface antigen"). With respect to immunotherapy of hematological malignancies, several B cell surface antigens are of interest. In one aspect, the B cell surface antigen is selected from the group consisting of CD19, CD79b, CD20, CD22, and CD37.

[0207] The term "CD19" refers to the B lymphocyte antigen CD19, also referred to as the B lymphocyte surface antigen B4 or the T cell surface antigen Leu-12, and unless otherwise indicated, the term includes any natural CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human CD19 is shown in UniProt accession number P15391 (version 160, SEQ ID NO: 434). The term encompasses "full-length" unprocessed human CD19, as well as any form of human CD19 produced by processing in cells, as long as the antibodies reported herein bind to it. CD19 is a structurally different cell surface receptor expressed on the surface of human B cells, including but not limited to pre-B cells, B cells in early developmental stages (i.e., immature B cells), mature B cells and malignant B cells that are terminally differentiated into plasma cells. CD19 is expressed in most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B cell chronic lymphocytic leukemias (CLL), prolymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias and some non-acute lymphocytic leukemias. The expression of CD19 on plasma cells further suggests that it may be expressed in differentiated B cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia.

[0208] "CD79b" refers to the protein β chain associated with the B cell surface antigen receptor complex, also referred to as Ig-β or B cell specific glycoprotein B29, and unless otherwise indicated, the term includes any natural CD79b from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). The amino acid sequence of human CD79b is shown in UniProt accession number P40259 (version 180, SEQ ID NO: 435). CD79b is a 39KDa protein expressed only on B cells, which collaborates with CD79a to initiate the signal transduction cascade downstream of BCR, thereby causing BCR complex internalization, its translocation to endosomes, and antigen presentation. CD79 (composed of subunits CD79a and CD79b) is a heterodimeric signal transduction component of the B cell receptor, which is commonly expressed in mature B cell lymphomas and is placed on the cell surface by the earliest committed B cell progenitors before the expression of immunoglobulin μ. The term "CD79b" includes "full-length" unprocessed CD79b, as well as any form of CD79b produced by processing in the cell. The term also encompasses naturally occurring variants of CD79b, such as splice variants or allelic variants.

[0209] "CD20" refers to the B lymphocyte antigen CD20, also known as the B lymphocyte surface antigen B1 or the leukocyte surface antigen Leu-16, and unless otherwise indicated, the term includes any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human CD20 is shown in UniProt accession number P11836 (version 149, SEQ ID NO: 436). CD20 is a hydrophobic transmembrane protein expressed on pre-B lymphocytes and mature B lymphocytes with a molecular weight of approximately 35 kD. 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 new protein family are characterized by a common structure and similar intron / exon splicing boundaries and show 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 located within a cluster of family members, at 11q12. Alternative splicing of this gene produces two transcript variants that encode the same protein. The term "CD20" includes "full-length," unprocessed CD20, as well as any form of CD20 produced by processing in the cell. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants.

[0210] "CD22" refers to the B cell receptor CD22, also known as B lymphocyte adhesion molecule or SIGLEC2, and unless otherwise indicated, the term includes any natural CD22 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human CD22 is shown in UniProt accession number P20273 (version 209, SEQ ID NO: 437). CD22 is a molecule belonging to the lectin SIGLEC family, found on the surface of mature B cells and less found on certain immature B cells. Thus, CD22 is a B cell-restricted cell surface phosphoglycoprotein of 130 kDa to 150 kDa that can regulate B lymphocyte antigen receptor (BCR)-mediated signals, as well as the generation of BCR-independent signals. The term "CD22" includes "full-length" unprocessed CD22, as well as any form of CD22 produced by processing in cells. The term also encompasses naturally occurring variants of CD22, such as splice variants or allelic variants.

[0211] "CD37" refers to the leukocyte antigen CD37, also known as transmembrane tetraprotein-26 (Tspan-26), and unless otherwise indicated, the term includes any natural CD37 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human CD37 is shown in UniProt accession number P11049 (version 162, SEQ ID NO: 438). The expression of CD37 is limited to cells of the immune system, with the highest abundance on mature B cells, while lower expression is found on T cells and myeloid cells. Glycoprotein CD37 is a member of the transmembrane 4 superfamily and controls humoral and cellular immune responses. The term "CD37" includes "full-length" unprocessed CD37, as well as any form of CD37 produced by processing in cells. The term also encompasses naturally occurring variants of CD37, such as splice variants or allelic variants.

[0212] As used herein, "multiple myeloma (MM) cell surface antigen" refers to an antigenic determinant presented on the surface of multiple myeloma (MM) cells. For immunotherapy of multiple myeloma, several MM cell surface antigens are of interest. In one aspect, the MM cell surface antigen is selected from the group consisting of CD38, BCMA, and GPRC5D.

[0213] The term "CD38", also known as cluster of differentiation 38 or cyclic ADP ribose hydrolase, is a glycoprotein found on the surface of many immune cells (white blood cells), including CD4+ lymphocytes, CD8+ lymphocytes, B lymphocytes and natural killer cells. CD38 also plays a role in cell adhesion, signal transduction and calcium signaling. Under normal circumstances, the expression level of CD38 in myeloid cells and lymphoid cells and certain non-hematopoietic tissues is relatively low. In contrast, normal plasma cells and multiple myeloma (MM) cells have high levels of CD38 expression, which makes CD38 a target for targeting cell surface molecules in MM. As used herein, unless otherwise indicated, CD38 protein refers to any CD38 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). The amino acid sequence of human CD38 is shown in UniProt (www.uniprot.org) Accession No. P28907 (SEQ ID NO: 474).

[0214] The term "BCMA" refers to B cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRS17), and is a type III transmembrane protein without a signal peptide but containing a cysteine-rich extracellular domain. BCMA is expressed at significantly higher levels in all patient MM cells, but is not expressed in other normal tissues other than normal plasma cells. BCMA, together with two related TNFR superfamily B cell activating factor receptors (BAFF-R) and transmembrane activators and calcium regulators and cyclophilin ligand interactors (TACI), critically regulates the proliferation and survival of B cells, as well as maturation and differentiation into plasma cells. Receptors associated with these three functions support the long-term survival of B cells at different developmental stages by binding to BAFF and / or APRIL (their cognate ligands). As used herein, unless otherwise indicated, BCMA refers to any BCMA protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). The amino acid sequence of human BCMA is shown in UniProt (www.uniprot.org) Accession No. Q02223 (SEQ ID NO: 475).

[0215] The term "GPRC5D" refers to G protein-coupled C class receptor group 5 member D, which is a target identified from plasma cells of multiple myeloma using RNA sequencing. It is reported that GPRC5D is associated with poor prognosis and tumor burden in patients with multiple myeloma. Otherwise, GPRC5D refers to any GPRC5D protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The amino acid sequence of human GPRC5D is shown in UniProt (www.uniprot.org) accession number Q9NZD1 (SEQ ID NO: 476).

[0216] Unless otherwise indicated, the term "CD28" (cluster of differentiation 28, Tp44) refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques) and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells by CD28 can also provide effective signals for the production of various interleukins. CD28 is a receptor for CD80 (B7.1) protein and CD86 (B7.2) protein, and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747 (SEQ ID NO: 1).

[0217] "Agonist antibodies" refer to antibodies with agonist function against a given receptor. Typically, when an agonist ligand (factor) binds to a receptor, the tertiary structure of the receptor protein changes, and the receptor is activated (when the receptor is a membrane protein, it usually transduces cell growth signals, etc.). If the receptor is a type that forms dimers, the agonist antibody can dimerize the receptor at an appropriate distance and angle, thereby acting similarly to the ligand. Appropriate anti-receptor antibodies can mimic the receptor dimerization performed by the ligand and thus become agonist antibodies.

[0218] "CD28 agonistic antigen binding molecule" or "CD28 conventional agonistic antigen binding molecule" is an antigen binding molecule that mimics the action of CD28 natural ligands (CD80 or CD86) to enhance 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, the formation of "signal 1" is the interaction of the peptide / major histocompatibility complex (MHC) complex on the T cell receptor (TCR) molecule and the antigen presenting cell (APC), while "signal 2" is produced by the binding of co-stimulatory molecule receptors (e.g., CD28). The CD28 agonistic antigen binding molecule can co-stimulate T cells (signal 2). It can also induce T cell proliferation and cytokine secretion by binding to molecules that are specific for the TCR complex, but the CD28 agonistic antigen binding molecule cannot fully activate T cells without additional stimulation of the TCR. However, there is a subclass of CD28-specific antigen binding molecules, the so-called CD28 super-agonistic antigen binding molecules. A "CD28 superagonist antigen-binding molecule" is a CD28 antigen-binding molecule that can fully activate T cells without the need for additional TCR stimulation. A CD28 superagonist antigen-binding molecule can induce T cell proliferation and cytokine secretion without prior T cell activation (Signal 1).

[0219] The term "variable domain" or "variable region" refers to the domain of the antibody heavy chain or light chain that participates in the binding of an 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, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0220] As used herein, the term "hypervariable region" or "HVR" refers to the individual regions of an antigen-binding variable domain that are highly variable in sequence and determine antigen-binding specificity, such as "complementarity determining regions" ("CDRs"). Typically, an antigen-binding domain comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0221] (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));

[0222] (b) CDRs present 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

[0223] (c) Antigenic contact points present 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)).

[0224] Unless otherwise stated, CDR is determined according to the method described by Kabat et al. (supra). Those skilled in the art will understand that CDR names can also be determined according to the method described by Chothia (supra), McCallum (supra) or any other scientifically accepted nomenclature. Kabat et al. also defined a numbering system for the variable region sequence applicable to any antibody. Those of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise stated, the numbering of specific amino acid residue positions in the antibody variable region is based on the Kabat numbering system.

[0225] As used herein, in the context of antigen-binding molecules (e.g., antibodies), the term "affinity maturation" refers to an antigen-binding molecule derived from a reference antigen-binding molecule (e.g., by mutation) that binds to the same antigen, preferably to the same epitope, as the reference antibody; and has a higher affinity for the antigen than the affinity of the reference antigen-binding molecule. Affinity maturation typically 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.

[0226] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0227] For the purposes of this paper, an "acceptor human framework" is a framework comprising an 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 an amino acid sequence identical to that of the human immunoglobulin framework or the human consensus framework, or it 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.

[0228] 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.

[0229] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0230] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are antibodies in which the constant regions have been additionally modified or altered relative to the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0231] A "human" antibody is one that has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0232] The term "CH1 domain" refers to the portion of an antibody heavy chain polypeptide that extends approximately from EU position 118 to EU position 215 (EU numbering system according to Kabat). In one aspect, the CH1 domain has the amino acid sequence of ASTKGPSVFP LAPSSKSTSGGTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDKKV (SEQ ID NO: 477). Typically, a segment having the amino acid sequence of EPKSC (SEQ ID NO: 480) subsequently connects the CH1 domain to the hinge region.

[0233] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that engages the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, for example, from about position 216 to about position 230 (according to the EU numbering system of Kabat), or from about position 226 to about position 230 (according to the EU numbering system of Kabat). The hinge regions of other IgG subclasses can be determined by comparing the hinge region cysteine residues with the IgG1 subclass sequence. The hinge region is typically a dimer molecule consisting of two polypeptides with the same amino acid sequence. The hinge region typically comprises up to 25 amino acid residues and is flexible, thereby allowing the relevant target binding sites to move independently. The hinge region can be subdivided into three regions: an upper hinge domain, a middle hinge domain, and a lower hinge domain (see, for example, Roux et al., J. Immunol. 161 (1998) 4083).

[0234] In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 481), wherein X is S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 482), wherein X is S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 483), wherein X is S or P.

[0235] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises the IgG CH2 domain and the IgG CH3 domain.

[0236] The "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about EU position 231 to an amino acid residue at about EU position 340 (according to the EU numbering system of Kabat). In one aspect, the CH2 domain has an amino acid sequence of APELLGGPSVFLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 478). The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete native Fc region. It has been speculated that carbohydrates can provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.

[0237] A "CH3 domain" comprises an extension of the C-terminal residues of the CH2 domain in the Fc region and represents the portion of an antibody heavy chain polypeptide that extends approximately from EU position 341 to EU position 446 (EU numbering system according to Kabat). In one aspect, the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKLTVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 479). The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "knob" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains can be used to promote the heterodimerization of two different antibody heavy chains as described herein. In one embodiment, the 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) in the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0238] The "knob-into-hole" technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In one embodiment, the knob is modified to include the amino acid substitution T366W in one of the two subunits of the Fc domain, while the hole is modified to include the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another embodiment, the subunit comprising the knob-modified Fc domain additionally includes the amino acid substitution S354C, while the subunit comprising the hole-modified Fc domain additionally includes the amino acid substitution Y349C. The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0239] "A region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having modifications that produce substitutions, additions, or deletions but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular toxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially losing biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, for example, Bowie, JU et al., Science 247: 1306-10 (1990)).

[0240] The term "wild-type Fc domain" encompasses an amino acid sequence identical to that of an Fc domain found in nature. Wild-type human Fc domains include native sequence 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 and naturally occurring variants thereof. Wild-type Fc regions are represented in SEQ ID NO: 484 (IgG1, Caucasian allotype), SEQ ID NO: 485 (IgG1, African American allotype), SEQ ID NO: 486 (IgG2), SEQ ID NO: 487 (IgG3), and SEQ ID NO: 488 (IgG4).

[0241] The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the amino acid sequence of a "wild-type" (human) Fc domain due to at least one "amino acid mutation." In one aspect, the variant Fc region has at least one amino acid mutation, e.g., about one to about ten amino acid mutations, compared to a native Fc region, and in one aspect, about one to about five amino acid mutations compared to a native Fc region. In one aspect, the (variant) Fc region has at least about 95% homology to the wild-type Fc region.

[0242] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary 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, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0243] 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 specific cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and to lyse erythrocytes and various other cellular targets (e.g., tumor cells) coated with the corresponding antibody through antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: the 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.

[0244] Cross-linking of the Fc region receptors of IgG antibodies (FcγRs) triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, and regulation of immune complex clearance and antibody production. Three classes of FcγRs have been identified in humans, including:

[0245] -FcγRI (CD64) binds to monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification of at least one amino acid residue E233-G236, P238, D265, N297, A327, and P329 (numbered according to the EU index of Kabat) in the Fc region of IgG reduces binding to FcγRI. The IgG2 residues at positions 233-236 are replaced by IgG1 and IgG4, resulting in a 103-fold reduction in binding to FcγRI and the elimination of the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29 (1999) 2613–2624).

[0246] -FcγRII (CD32) binds to complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found in many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and appears to activate the killing process. FcγRIIB appears to play a role in the inhibition process and is found in B cells, macrophages, as well as mast cells and eosinophils. On B cells, it appears to act to inhibit further production of immunoglobulins and isotype conversion to, for example, the IgE class. On macrophages, FcγRIIB serves to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B type may help to inhibit the activation of these cells through the binding of IgE to its individual receptors. For example, antibodies comprising an IgG Fc region comprising a mutation in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index of Kabat) were found to have reduced binding to FcγRIIA.

[0247] -FcγRIII (CD16) binds to IgG with medium to low affinity and includes two types. FcγRIIIA is present on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB has a high expression level on neutrophils. It was found that, for example, antibodies (including IgG Fc regions with mutations of at least one amino acid residue E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (according to the EU index numbering of Kabat)) have a reduced binding force to FcγRIIIA.

[0248] Shields, RL et al. (J. Biol. Chem. 276 (2001) 6591-6604) describe the location of the binding site on human IgG1 for Fc receptors, the mutation sites mentioned above, and methods for measuring binding to FcγRI and FcγRIIA.

[0249] The term "ADCC" or "antibody-dependent cellular cytotoxicity" is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells that specifically bind to an antibody or derivative thereof comprising an Fc region, typically through the protein portion at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a decrease in the number of target cells that are lysed by the ADCC mechanism defined above at a given antibody concentration in the culture medium surrounding the target cells within a given time, and / or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells by the ADCC mechanism within a given time in the culture medium surrounding the target cells. Reduced ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell but not engineered using the same standard production, purification, formulation, and storage methods (such methods are known to those skilled in the art). For example, a reduction in ADCC mediated by an antibody comprising an amino acid substitution in the Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831). For example, the ability of an antibody to induce the initial steps of mediating ADCC is studied by measuring the binding of the antibody to cells expressing Fcγ receptors, such as cells that recombinantly express FcγRI and / or FcγRIIA or NK cells (which express FcγRIIIA in nature). Specifically, binding to FcγRs on NK cells is measured.

[0250] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, causes a signaling event that stimulates cells bearing the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0251] An "extracellular domain" is a domain of a membrane protein that extends into the extracellular space (ie, the space outside the target cell). The extracellular domain is typically the portion of the protein that initiates surface contacts, leading to signal transduction.

[0252] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G4S) n 、(SG4) n or G4 (SG4) nPeptide linkers, wherein "n" is typically a number between 1 and 5, typically between 2 and 4, and in particular 2, i.e. a peptide 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 including the sequences GSPGSSSSGS (SEQ ID NO: 150), (G4S)3 (SEQ ID NO: 151), (G4S)4 (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 GGNGSGSG (SEQ ID NO: 160). Specific peptide linkers of 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).

[0253] The term "amino acid" as used in this application 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).

[0254] By "fused" or "linked" is meant that the components (eg, a polypeptide and the extracellular domain of the TNF ligand family member) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0255] " amino acid sequence identity percentage (%) " relative to a reference polypeptide (protein) sequence is defined as after comparing the amino acid residue in the candidate sequence with the amino acid residue in the reference polypeptide sequence and introducing a gap (if necessary) to realize maximum sequence identity percentage, and when any conservative substitution is not considered a part for the sequence identity, the percentage of the amino acid residue in the candidate sequence identical with the amino acid residue in the reference polypeptide sequence. Comparison for determining amino acid sequence identity percentage can be achieved in various ways within the scope 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 the appropriate parameters for aligning sequences, including any algorithm required for maximum alignment over the full length of the compared sequence. However, for purposes herein, the values of amino acid sequence identity % are generated using sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should 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 change. In the case of amino acid sequence comparisons performed using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as the % amino acid sequence identity that a given amino acid sequence A has or comprises with a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where 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 be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0256] In certain embodiments, amino acid sequence variants of the CD28 antigen binding molecules provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the CD28 antigen binding molecules. The 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 and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding. The sites of interest for substitution mutagenesis include HVR and framework (FR). Conservative substitutions are provided under the heading "preferred substitutions" in Table B and are further described below with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the molecule of interest, and the product can be screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0257] Table A

[0258]

[0259] Amino acids can be grouped according to common side chain properties:

[0260] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0261] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0262] (3) Acidic: Asp, Glu;

[0263] (4) Basic: His, Lys, Arg;

[0264] (5) Residues that affect chain orientation: Gly, Pro;

[0265] (6) Aromatic: Trp, Tyr, Phe.

[0266] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0267] The term "amino acid sequence variant" includes substantial variants, wherein there is an amino acid substitution in one or more hypervariable region residues of a parent antigen binding molecule (such as humanized or human antibody). Typically, relative to the parent antigen binding molecule, one or more resulting variants selected for further study will have changes (such as, improvements) and / or will substantially retain certain biological properties of the parent antigen binding molecule in terms of certain biological properties (such as, affinity increases, immunogenicity decreases). Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated using, for example, affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated and variant antigen binding molecules are displayed on phage and screened for specific biological activity (such as binding affinity). In certain embodiments, substitution, insertion or deletion may occur in one or more HVRs, as long as such changes do not substantially reduce the antigen binding ability of the antigen binding molecules. For example, conservative changes (such as, as provided herein, conservative substitutions) that do not substantially reduce binding affinity can be carried out in HVR. The method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys and Glu) are 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. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and the antigen are identified using the crystal structure of the antigen-antigen binding molecule complex. Such contact residues and adjacent residues as candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they possess desired properties.

[0268] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of insertions include CD28 antigen-binding molecules fused to the N-terminus or C-terminus of a polypeptide that increases the serum half-life of the CD28 antigen-binding molecule.

[0269] In certain embodiments, the CD28 antigen binding molecules provided herein are altered to increase or decrease the degree of antibody glycosylation. One or more glycosylation sites can be generated or removed by changing the amino acid sequence, thereby conveniently obtaining glycosylation variants of the molecule. When the agonist ICOS binding molecule comprises an Fc domain, the carbohydrate attached thereto can be changed. Natural antibodies produced by mammalian cells typically comprise biantennary oligosaccharides with side chains, which are typically attached to Asn297 of the CH2 domain of the Fc region via N-links. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In certain embodiments, the oligosaccharides in the agonist ICOS binding molecule can be modified to produce variants with certain improved properties. In one aspect, variants of agonist ICOS binding molecules are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function, see, for example, U.S. Patent Publication No. US 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Other variants of the CD28 antigen-binding molecules of the present invention include variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants having 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.).

[0270] In certain embodiments, it is desirable to produce a cysteine engineered variant of the CD28 antigen binding molecule of the present invention, such as a "thioMAb", in which one or more residues of the molecule are replaced by cysteine residues. In a specific embodiment, the substituted residue is present at an accessible site of the molecule. By replacing those residues with cysteine, the reactive thiol group is thus located at an accessible site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate. In certain embodiments, any one or more of the following residues can 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 can be formed as described, for example, in U.S. Patent No. 7,521,541.

[0271] In some aspects, the CD28 antigen binding molecules provided herein can be further modified so that they contain other non-protein moieties known in the art and easily obtained. Suitable parts for antibody derivatization 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), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol and their mixtures. Due to its stability in water, polyethylene glycol propionaldehyde can have advantages in manufacturing. Polymer can have any molecular weight and can have side chains or not. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers for derivatization can be determined based on the following considerations, including but not limited to whether the specific characteristics or functions to be improved in the antibody, bispecific antibody derivatives will be used for therapy under limited conditions, etc. On the other hand, there is provided a conjugate of an antibody and a non-proteinaceous portion that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous portion is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). Radiation can have any wavelength, and includes but is not limited to no harm to ordinary cells, but the wavelength at which the non-proteinaceous portion is heated to a temperature at which cells near the antibody-non-proteinaceous portion are killed. On the other hand, the immunoconjugates of the CD28 antigen binding molecules provided herein can be obtained." immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0272] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA or plasmid DNA (pDNA). Polynucleotides may comprise conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those present in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments. Each nucleotide is composed of a base, particularly 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. Typically, nucleic acid molecules are described by base sequences, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically expressed as from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, 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 can be linear or circular. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present 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 can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817 or EP 2 101 823 B1).

[0273] With respect to "isolated" nucleic acid molecules or polynucleotides, it is meant a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Other embodiments of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially purified) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules that are contained in cells that typically contain polynucleotide molecules, but that are present in extrachromosomal or chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention also include such molecules produced by synthesis. In addition, polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0274] In reference to a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with additional nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, or may be interspersed individually among the residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be routinely determined using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

[0275] The term "expression cassette" refers to a polynucleotide generated by recombination or synthesis, which has a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector also includes, in addition to other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0276] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene into a target cell operably associated therewith and to direct the expression of the gene. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are integrated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the transcription of a large amount of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cell's transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding the bispecific antigen binding molecule of the present invention or a fragment thereof.

[0277] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived from the primary transformed cells, regardless of passage number. Progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. This article includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, for example, cultured mammalian cells, such as CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells included in transgenic animals, transgenic plants, or cultured plants or animal tissues.

[0278] An "effective amount" of an agent is that amount required to produce a physiological change in the cells or tissues to which it is administered.

[0279] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0280] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, the individual or subject is a human.

[0281] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0282] "Pharmaceutically acceptable excipients" refer to ingredients in a pharmaceutical composition other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

[0283] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0284] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the invention are used to delay the development of a disease or to slow the progression of a disease.

[0285] As noted herein, the term "combination therapy" or "co-administration" encompasses both combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case administration of the antibodies as reported herein may be prior to, concurrently with, and / or after administration of the additional therapeutic agent or agents (preferably one or more antibodies).

[0286] A "B-cell proliferative disorder" refers to 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, and particularly wherein the increased number of B cells is a cause or hallmark of the disease.

[0287] The term "hematological cancer" refers to or describes a physiological condition characterized by uncontrolled cell growth / proliferation in mammals. Therefore, the term cancer used herein refers to a proliferative disease, 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 a B cell proliferative disorder. On the one hand, 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).

[0288] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Thus, 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" includes lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar 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, and ovarian cancer. In one embodiment, the present invention relates to a cancer comprising: a thyroid cancer, a parathyroid cancer, an adrenal cancer, a soft tissue sarcoma, a urethra cancer, a penile cancer, a prostate cancer, a bladder cancer, a kidney cancer or a ureter cancer, a renal cell carcinoma, a renal pelvis cancer, a mesothelioma, a hepatocellular carcinoma, a bile duct cancer, a central nervous system (CNS) tumor, a spinal axis tumor, a brain stem glioma, a glioblastoma multiforme, an astrocytoma, a neurilemmoma, an ependymoma, a medulloblastoma, a meningioma, a squamous cell carcinoma, a pituitary adenoma, and an 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 aspect, the cancer is a solid tumor. In another aspect, the cancer is a hematological cancer, particularly a leukemia, most particularly acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

[0289] Bispecific agonistic CD28 antigen binding molecules of the present invention

[0290] 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, an extended dosage range that can be given to patients, and thus potentially enhanced efficacy. Novel bispecific agonist CD28 antigen binding molecules comprise an Fc domain consisting of a first subunit and a second subunit that can stably associate, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function (Fc silencing) of the antigen binding molecules to Fc receptors, thereby avoiding nonspecific cross-linking via Fc receptors. Alternatively, they comprise at least one antigen binding domain that can specifically bind to tumor-associated antigens such as fibroblast activation protein (FAP) or carcinoembryonic antigen (CEA), which causes cross-linking at the tumor site. Therefore, tumor-specific T cell activation is achieved.

[0291] Provided herein is a bispecific agonist CD28 antigen binding molecule that monovalently binds to CD28, comprising:

[0292] (a) an antigen-binding domain capable of specifically binding to CD28;

[0293] (b) at least one antigen-binding domain capable of specifically binding to a tumor-associated antigen, and

[0294] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0295] On the one hand, a bispecific agonist CD28 antigen binding molecule as defined above is provided, wherein the Fc domain is IgG, particularly IgG1 Fc domain or IgG4 Fc domain. In a specific aspect, the Fc domain consisting of the first subunit and the second subunit capable of stably associating 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 the Fc receptor and / or reduce or eliminate effector function. On the one hand, the Fc domain comprises amino acid substitutions L234A and L235A (numbered according to the EU index of Kabat). On the one hand, the Fc domain belongs to the human IgG1 subclass and comprises amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index). On the one hand, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding molecule comprises the Fc domain consisting of the first subunit and the second subunit capable of stably associating, 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.

[0296] In one aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as previously defined herein, wherein the antigen binding domain capable of specific binding to CD28 comprises:

[0297] (i) Heavy chain variable region (V H CD28), which comprises a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 20, a CDR-H2 of SEQ ID NO: 21, and a CDR-H3 of SEQ ID NO: 22; and a light chain variable region (V L CD28), which comprises a light chain complementary determining region CDR-L1 of SEQ ID NO: 23, a CDR-L2 of SEQ ID NO: 24, and a CDR-L3 of SEQ ID NO: 25; or

[0298] (ii) (ii) Heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37, and CDR-H3 of SEQ ID NO: 38; and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO:39, CDR-L2 of SEQ ID NO:40, and CDR-L3 of SEQ ID NO:41.

[0299] In one 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 (VH CD28), which comprises CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37, and CDR-H3 of SEQ ID NO: 38, and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO:39, CDR-L2 of SEQ ID NO:40, and CDR-L3 of SEQ ID NO:41.

[0300] On the other hand, the antigen-binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen-binding molecule comprises: a heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25.

[0301] Furthermore, there is provided a bispecific agonist CD28 antigen binding molecule as previously defined herein, wherein the antigen binding domain capable of specific binding to CD28 comprises: a heavy chain variable region (V H CD28), which comprises an amino acid sequence that is about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26; and a light chain variable region (V L CD28), which comprises 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.

[0302] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises:

[0303] (a) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47; and the light chain variable region (V L CD28), the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54, or

[0304] (b) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47; and the light chain variable region (VL CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 27, or

[0305] (c) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 61, or

[0306] (d) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 53, or

[0307] (e) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46; and the light chain variable region (V L CD28), the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54, or

[0308] (f) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, or

[0309] (g) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 27, or

[0310] (h) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 27, or

[0311] (i) Heavy chain variable region (V HCD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 53, or

[0312] (j) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42; and the light chain variable region (V L CD28), the light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, or

[0313] (k) Heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42; and the light chain variable region (V L The light chain variable region comprises the CDRs of CD28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 27.

[0314] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 47; and the light chain variable region (V L CD28), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54. On the other hand, the antigen-binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen-binding molecule comprises: a heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 489, CDR-H2 of SEQ ID NO: 490, and CDR-H3 of SEQ ID NO: 491; and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO: 492, CDR-L2 of SEQ ID NO: 493, and CDR-L3 of SEQ ID NO: 494.

[0315] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46; and the light chain variable region (V LCD28), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 53. On the other hand, the antigen binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen binding molecule comprises: a heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 495, CDR-H2 of SEQ ID NO: 496, and CDR-H3 of SEQ ID NO: 497; and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO: 498, CDR-L2 of SEQ ID NO: 499, and CDR-L3 of SEQ ID NO: 500.

[0316] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42; and the light chain variable region (V L CD28), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 27. On the other hand, the antigen-binding domain capable of specifically binding to CD28 of the bispecific agonist CD28 antigen-binding molecule comprises: a heavy chain variable region (V H CD28), which comprises CDR-H1 of SEQ ID NO: 501, CDR-H2 of SEQ ID NO: 502, and CDR-H3 of SEQ ID NO: 503; and a light chain variable region (V L CD28), which comprises CDR-L1 of SEQ ID NO:504, CDR-L2 of SEQ ID NO:505 and CDR-L3 of SEQ ID NO:506.

[0317] In a further aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28) 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; and a light chain variable region (V LCD28), 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.

[0318] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises:

[0319] (a) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0320] (b) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 27, or

[0321] (c) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 61, or

[0322] (d) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0323] (e) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0324] (f) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V LCD28), which comprises the amino acid sequence of SEQ ID NO: 59, or

[0325] (g) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 27, or

[0326] (h) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 43, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 27, or

[0327] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0328] (j) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 59, or

[0329] (k) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:27.

[0330] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein an antigen binding domain capable of specifically binding to CD28 binds to CD28 with reduced affinity compared to an antigen binding domain comprising: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 26; and a light chain variable region (V L CD28) comprising the amino acid sequence of SEQ ID NO: 27. Affinity was measured by flow cytometry as binding to CHO cells expressing CD28. In one aspect, an antigen binding domain capable of specifically binding to CD28 binds to CD28 with reduced affinity compared to an antigen binding domain comprising: a heavy chain variable region (V HCD28), which comprises the amino acid sequence of SEQ ID NO: 26, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27; comprising: a heavy chain variable region (V H CD28), the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47, and the light chain variable region (V L CD28), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 54. In one aspect, compared to an antigen binding domain comprising: a heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 26, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27, wherein the antigen-binding domain capable of specifically binding to CD28 with reduced affinity comprises: a heavy chain variable region (V H CD28), which comprises 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, and a light chain variable region (V L CD28), which comprises 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.

[0331] In one particular aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:54.

[0332] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 46; and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:53.

[0333] In a further specific aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CD28 comprises: a heavy chain variable region (V H CD28), comprising the amino acid sequence of SEQ ID NO: 42; and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO:27.

[0334] Bispecific agonistic CD28 antigen-binding molecule targeting CEA

[0335] 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).

[0336] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CEA comprises:

[0337] (i) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 188, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 189, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 190; and a light chain variable region (V L CEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 191, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 192, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 193; or

[0338] (ii) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 182; and a light chain variable region (V L CEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 183, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 184, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 185; or

[0339] (iii) Heavy chain variable region (V HCEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 127, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 128, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 129; and a light chain variable region (V L CEA), comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 130, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 131, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 132, or

[0340] (iv) Heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 507, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 508, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 509; and a light chain variable region (V L CEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 510, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 511, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 512.

[0341] In one particular aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), comprising: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 188, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 189, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 190; and a light chain variable region (V L CEA), comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 191, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 192, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 193.

[0342] In particular, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises 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; and a light chain variable region (V LCEA), which comprises 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. In one aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 133; and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 134.

[0343] On the other hand, the antigen-binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises 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: 186; and a light chain variable region (V L CEA), which comprises 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. In one aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 186; and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 187.

[0344] On the other hand, the antigen-binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises 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: 513; and a light chain variable region (V L CEA), which comprises 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. In one aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 513; and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 514.

[0345] In another aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to CEA comprises:

[0346] (a) Heavy chain variable region (VH CEA), which comprises the amino acid sequence of SEQ ID NO: 194, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 195, or

[0347] (b) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 196, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 197, or

[0348] (c) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 198, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 199, or

[0349] (d) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 200, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 201, or

[0350] (e) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 202, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 203, or

[0351] (f) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 204, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 205, or

[0352] (g) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 206, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 207, or

[0353] (h) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 208, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 209, or

[0354] (i) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 210, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 211, or

[0355] (j) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 212, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 213.

[0356] In particular, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 200; and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 201.

[0357] Bispecific agonistic CD28 antigen-binding molecules targeting FAP

[0358] 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 fibroblast activation protein (FAP).

[0359] In one aspect, a bispecific agonistic CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to FAP comprises:

[0360] (a) Heavy chain variable region (V H FAP), 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; and a light chain variable region (V L 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; or

[0361] (b) Heavy chain variable region (V HFAP), 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; and a light chain variable region (V L 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.

[0362] In particular, the antigen binding domain capable of specifically binding to FAP comprises: a heavy chain variable region (V H FAP), 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; and a light chain variable region (V L 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to FAP comprises: (a) a heavy chain variable region (V H FAP), which comprises 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: 18, and a light chain variable region (V L FAP), which comprises 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: 19, or (b) a heavy chain variable region (V H FAP), which comprises 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: 10, and a light chain variable region (V L FAP), which comprises 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: 11. In particular, the antigen-binding domain capable of specifically binding to FAP comprises: a heavy chain variable region (V HFAP), which comprises the amino acid sequence of SEQ ID NO: 18; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 19.

[0363] Bispecific agonistic CD28 antigen-binding molecule targeting EpCAM

[0364] 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 an epithelial cell adhesion molecule (EpCAM).

[0365] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to EpCAM comprises: a heavy chain variable region (V H EpCAM), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 515, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 516, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 517; and a light chain variable region (V L EpCAM), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to EpCAM comprises: a heavy chain variable region (V H EpCAM), which comprises 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; and a light chain variable region (V L EpCAM), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to EpCAM comprises: a heavy chain variable region (V H EpCAM), comprising the amino acid sequence of SEQ ID NO: 521; and a light chain variable region (V L EpCAM), which comprises the amino acid sequence of SEQ ID NO:522.

[0366] Bispecific agonistic CD28 antigen-binding molecule targeting HER3

[0367] 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.

[0368] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to HER3 comprises: a heavy chain variable region (V H HER3), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 523, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 524, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 525; and a light chain variable region (V L HER3), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to HER3 comprises: a heavy chain variable region (V H HER3), which comprises 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; and a light chain variable region (V L HER3), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to HER3 comprises: a heavy chain variable region (V H HER3), comprising the amino acid sequence of SEQ ID NO: 529; and a light chain variable region (V L HER3), which comprises the amino acid sequence of SEQ ID NO:530.

[0369] Bispecific agonistic CD28 antigen-binding molecule targeting CD30

[0370] 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 CD30.

[0371] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CD30 comprises: a heavy chain variable region (V H CD30), 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; and a light chain variable region (V L CD30), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to CD30 comprises: a heavy chain variable region (V H CD30), which comprises 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 a light chain variable region (V L CD30), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to CD30 comprises: a heavy chain variable region (V H CD30), comprising the amino acid sequence of SEQ ID NO: 537; and a light chain variable region (V L CD30), which comprises the amino acid sequence of SEQ ID NO: 538.

[0372] Bispecific agonistic CD28 antigen-binding molecule targeting TBPG (5T4)

[0373] On the other hand, 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).

[0374] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to TBPG comprises: a heavy chain variable region (V HTBPG), 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; and a light chain variable region (V L TBPG), 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. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to TBPG comprises: a heavy chain variable region (V H TBPG), which comprises 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; and a light chain variable region (V L TBPG), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to TBPG comprises: a heavy chain variable region (V H TBPG), comprising the amino acid sequence of SEQ ID NO: 545; and a light chain variable region (V L TBPG), which comprises the amino acid sequence of SEQ ID NO:546.

[0375] Bispecific agonistic CD28 antigen-binding molecules targeting MM

[0376] The present invention also provides novel bispecific agonist CD28 antigen binding molecules, which are particularly useful in the treatment of multiple myeloma. The molecule includes at least one antigen binding domain that can specifically bind to multiple myeloma (MM) cell surface antigens, and the antigen binding domain causes crosslinking in the presence of cells expressing MM cell surface antigens;With the Fc domains consisting of the first subunit and the second subunit that can stably associate, it includes one or more amino acid substitutions, and the amino acid substitutions can reduce the binding affinity and / or effector function (Fc silence) of the antigen binding molecules to Fc receptors. Therefore, nonspecific crosslinking via Fc receptors is avoided, and specific T cell activation is achieved in the presence of cells expressing MM cell surface antigens.

[0377] Therefore, a bispecific agonist CD28 antigen binding molecule is provided herein, 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 consisting of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen binding molecule to an Fc receptor. On the one hand, 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.

[0378] On the one hand, a bispecific agonist CD28 antigen binding molecule as defined above is provided, wherein the Fc domain is IgG, particularly IgG1 Fc domain or IgG4 Fc domain. In a specific aspect, the Fc domain consisting of the first subunit and the second subunit capable of stably associating 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 the Fc receptor and / or reduce or eliminate effector function. On the one hand, the Fc domain comprises amino acid substitutions L234A and L235A (numbered according to the EU index of Kabat). On the one hand, the Fc domain belongs to the human IgG1 subclass and comprises amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0379] In one aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as defined above, wherein the MM cell surface antigen is selected from the group consisting of CD38, BCMA and GPRC5D.

[0380] 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 CD38.

[0381] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to CD38 comprises: a heavy chain variable region (V H CD38), 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; and a light chain variable region (V LCD38), comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 550, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 551 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 552. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to CD38 comprises: a heavy chain variable region (V H CD38), which comprises 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; and a light chain variable region (V L CD38), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to CD38 comprises: a heavy chain variable region (V H CD38), comprising the amino acid sequence of SEQ ID NO: 553; and a light chain variable region (V L CD38), which comprises the amino acid sequence of SEQ ID NO:554.

[0382] 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 BCMA.

[0383] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to BCMA comprises: a heavy chain variable region (V H BCMA), comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 555, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 556, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 557; and a light chain variable region (V L BCMA), comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 558, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 559 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 560. In one aspect, a bispecific agonist CD28 antigen-binding molecule as described herein is provided, wherein the antigen-binding domain capable of specifically binding to BCMA comprises: a heavy chain variable region (V HBCMA), which comprises 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; and a light chain variable region (V L BCMA), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to FAP comprises: a heavy chain variable region (V H BCMA), comprising the amino acid sequence of SEQ ID NO: 561; and a light chain variable region (V L BCMA), which comprises the amino acid sequence of SEQ ID NO: 562.

[0384] Bispecific agonistic CD28 antigen-binding molecule targeting GPRC5D

[0385] 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 GPRC5D.

[0386] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), 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; and a light chain variable region (V L GPRC5D), 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.

[0387] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), which comprises 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, and a light chain variable region (V LGPRC5D), 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.

[0388] In one aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), which comprises 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; and a light chain variable region (V L GPRC5D), which comprises 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. In particular, the antigen-binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 569; and a light chain variable region (V L GPRC5D), which comprises the amino acid sequence of SEQ ID NO:570.

[0389] In another aspect, a bispecific agonist CD28 antigen binding molecule as described herein is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V H GPRC5D), 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; and a light chain variable region (V L GPRC5D), 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.

[0390] In one aspect, a bispecific agonist CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises: a heavy chain variable region (V HGPRC5D), which comprises 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, and a light chain variable region (V L GPRC5D), 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.

[0391] In another aspect, a bispecific agonistic CD28 antigen binding molecule is provided, wherein the antigen binding domain capable of specifically binding to GPRC5D comprises:

[0392] (a) Heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 569, and a light chain variable region (V L GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 570, or

[0393] (b) Heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 573, and a light chain variable region (V L GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 576, or

[0394] (c) Heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 569, and a light chain variable region (V L GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 572, or

[0395] (d) Heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 586, and a light chain variable region (V L GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 593, or

[0396] (e) Heavy chain variable region (V H GPRC5D), which comprises the amino acid sequence of SEQ ID NO: 587, and a light chain variable region (V L GPRC5D), which comprises the amino acid sequence of SEQ ID NO:592.

[0397] Bispecific agonistic CD28 antigen binding molecules that are monovalent for binding to CD28 and monovalent for binding to tumor-associated antigens (1+1 format)

[0398] In another aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0399] (a) a Fab fragment that can specifically bind to CD28,

[0400] (b) a crossFab fragment that specifically binds to a tumor-associated antigen, and

[0401] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0402] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0403] (a) a Fab fragment that can specifically bind to CD28,

[0404] (b) a crossFab fragment that specifically binds to CEA, and

[0405] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0406] In one specific aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule M).

[0407] In another aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0408] (a) a Fab fragment that can specifically bind to CD28,

[0409] (b) a crossFab fragment that specifically binds to FAP, and

[0410] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0411] In one specific aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule C).

[0412] In a further aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0413] (a) a first Fab fragment capable of specifically binding to CD28;

[0414] (b) a second Fab fragment capable of specifically binding to a tumor-associated antigen; and

[0415] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function,

[0416] The 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 the second Fab fragment capable of specifically binding to a tumor-associated antigen, and the Fab heavy chain of the second Fab fragment is then fused at its C-terminus to the N-terminus of one of the Fc domain subunits.

[0417] In one specific aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule H).

[0418] In a further aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0419] (a) a Fab fragment capable of specifically binding to CD28;

[0420] (B) a VH domain and a VL domain capable of specifically binding to a tumor-associated antigen; and

[0421] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function,

[0422] The Fab fragment capable of specifically binding to CD28 is fused at its C-terminus to the N-terminus of the first subunit of the Fc domain, and one of the VH domain and the VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of the first subunit of the Fc domain via a peptide linker, and the other of the VH domain and the VL domain capable of specifically binding to a tumor-associated antigen is fused to the C-terminus of the second subunit of the Fc domain via a peptide linker.

[0423] In one aspect, the peptide linker comprises an amino acid sequence selected from the group consisting of 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.

[0424] In one specific aspect, a bispecific agonist CD28 antigen binding molecule 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 (Molecule 1).

[0425] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0426] (a) a Fab fragment capable of specifically binding to CD28, comprising:

[0427] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 54,

[0428] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0429] (iii) Heavy chain variable region (VH CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0430] (b) a crossFab fragment that specifically binds to a tumor-associated antigen, and

[0431] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0432] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0433] (a) a Fab fragment capable of specifically binding to CD28, comprising:

[0434] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0435] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0436] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0437] (b) a crossFab fragment capable of specifically binding to CEA, comprising:

[0438] (i) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 186, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 187, or

[0439] (ii) Heavy chain variable region (V HCEA), which comprises the amino acid sequence of SEQ ID NO: 200, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 201, or

[0440] (iii) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 513, and a light chain variable region (V L CEA), comprising the amino acid sequence of SEQ ID NO: 514,

[0441] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0442] In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11A). In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11B). In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11C). In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11D). In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11I). In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11J).In one aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11K). In one aspect, a bispecific agonist CD28 antigen binding molecule 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: 356 (molecule 11L). 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 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: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 356 (molecule 11S). 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: 355, and a second light chain comprising the amino acid sequence of SEQ ID NO: 354 (molecule 11T).

[0443] In one specific aspect, a 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 specific aspect, a bispecific agonist CD28 antigen binding molecule 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 (molecule 11B).

[0444] In another aspect, there is provided a bispecific agonistic CD28 antigen binding molecule as described herein, comprising:

[0445] (a) A crossFab fragment that specifically binds to CD28;

[0446] (b) a Fab fragment capable of specifically binding to a tumor-associated antigen; and

[0447] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0448] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0449] (a) A crossFab fragment that specifically binds to CD28;

[0450] (a) a Fab fragment capable of specifically binding to CEA; and

[0451] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0452] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0453] (a) a crossFab fragment capable of specifically binding to CD28, comprising:

[0454] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0455] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0456] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0457] (b) a Fab fragment capable of specifically binding to CEA, comprising:

[0458] (i) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 186, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 187, or

[0459] (ii) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 200, and a light chain variable region (V L CEA), which comprises the amino acid sequence of SEQ ID NO: 201, or

[0460] (iii) Heavy chain variable region (V H CEA), which comprises the amino acid sequence of SEQ ID NO: 513, and a light chain variable region (V L CEA), comprising the amino acid sequence of SEQ ID NO: 514,

[0461] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0462] In one aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 110).

[0463] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0464] (a) a Fab fragment that can specifically bind to CD28,

[0465] (b) a crossFab fragment that specifically binds to EpCAM, and

[0466] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0467] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0468] (a) a Fab fragment capable of specifically binding to CD28, comprising:

[0469] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0470] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0471] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0472] (b) a crossFab fragment capable of specifically binding to EpCAM, comprising: a heavy chain variable region (V HEpCAM), which comprises the amino acid sequence of SEQ ID NO: 521; and a light chain variable region (V L EpCAM), comprising the amino acid sequence of SEQ ID NO: 522,

[0473] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0474] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0475] (a) A crossFab fragment that specifically binds to CD28;

[0476] (b) a Fab fragment that specifically binds to EpCAM, and

[0477] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0478] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0479] (a) a crossFab fragment capable of specifically binding to CD28, comprising:

[0480] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0481] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0482] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0483] (b) a Fab fragment capable of specifically binding to EpCAM, comprising: a heavy chain variable region (V H EpCAM), which comprises the amino acid sequence of SEQ ID NO: 521; and a light chain variable region (V L EpCAM), comprising the amino acid sequence of SEQ ID NO: 522,

[0484] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0485] In one aspect, 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).

[0486] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0487] (a) a Fab fragment that can specifically bind to CD28,

[0488] (b) a crossFab fragment that specifically binds to HER3, and

[0489] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0490] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0491] (a) a Fab fragment capable of specifically binding to CD28, comprising:

[0492] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0493] (ii) Heavy chain variable region (V HCD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0494] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0495] (b) a crossFab fragment capable of specifically binding to HER3, comprising: a heavy chain variable region (V H HER3), comprising the amino acid sequence of SEQ ID NO: 529; and a light chain variable region (V L HER3), comprising the amino acid sequence of SEQ ID NO: 530,

[0496] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0497] In one aspect, 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).

[0498] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0499] (a) A crossFab fragment that specifically binds to CD28;

[0500] (b) a Fab fragment capable of specifically binding to HER3, and

[0501] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0502] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0503] (a) a crossFab fragment capable of specifically binding to CD28, comprising:

[0504] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0505] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0506] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0507] (b) a Fab fragment capable of specifically binding to HER3, comprising: a heavy chain variable region (V H HER3), comprising the amino acid sequence of SEQ ID NO: 529; and a light chain variable region (V L HER3), comprising the amino acid sequence of SEQ ID NO: 530,

[0508] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0509] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0510] (a) a Fab fragment that can specifically bind to CD28,

[0511] (b) a crossFab fragment that specifically binds to CD30, and

[0512] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0513] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0514] (a) a Fab fragment capable of specifically binding to CD28, comprising:

[0515] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0516] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0517] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0518] (b) a crossFab fragment capable of specifically binding to CD30, comprising: a heavy chain variable region (V H CD30), comprising the amino acid sequence of SEQ ID NO: 537; and a light chain variable region (V L CD30), comprising the amino acid sequence of SEQ ID NO: 538,

[0519] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0520] In one aspect, 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).

[0521] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0522] (a) A crossFab fragment that specifically binds to CD28;

[0523] (b) a Fab fragment capable of specifically binding to CD30, and

[0524] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0525] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0526] (a) a crossFab fragment capable of specifically binding to CD28, comprising:

[0527] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0528] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0529] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0530] (b) a Fab fragment capable of specifically binding to CD30, comprising: a heavy chain variable region (V H CD30), comprising the amino acid sequence of SEQ ID NO: 537; and a light chain variable region (V L CD30), comprising the amino acid sequence of SEQ ID NO: 538,

[0531] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0532] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0533] (a) a Fab fragment that can specifically bind to CD28,

[0534] (b) a crossFab fragment that specifically binds to TPBG, and

[0535] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0536] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0537] (a) a Fab fragment capable of specifically binding to CD28, comprising:

[0538] (i) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 47, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 54, or

[0539] (ii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (V L CD28), which comprises the amino acid sequence of SEQ ID NO: 53, or

[0540] (iii) Heavy chain variable region (V H CD28), which comprises the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (V L CD28), comprising the amino acid sequence of SEQ ID NO: 27,

[0541] (b) a crossFab fragment capable of specifically binding to TPBG, comprising: a heavy chain variable region (V H TPBG), which comprises the amino acid sequence of SEQ ID NO: 545; and a light chain variable region (V L TPBG), comprising the amino acid sequence of SEQ ID NO: 546,

[0542] and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, which comprises one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0543] In one aspect, 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).

[0544] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0545] (a) A crossFab fragment that specifically binds to CD28;

[0546] (a) a Fab fragment capable of specifically binding to TPBG; and

[0547] (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, comprising one or more amino acid substitutions that reduce the binding affinity of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0548] In one aspect, there is provided a bispecific agonist CD28 antigen binding molecule as described herein, comprising:

[0549] (a) a crossFab fragment capable...

Claims

1. A bispecific agonist CD28 antibody characterized by monovalent binding to CD28, comprising: (a) a Fab fragment capable of specifically binding to CD28, comprising: Heavy chain variable region (V H CD28), which has an amino acid sequence as shown in SEQ ID NO: 47, and a light chain variable region (V L CD28), which is represented by the amino acid sequence of SEQ ID NO: 54, (b) a crossFab fragment capable of specifically binding to CD19, comprising: Heavy chain variable region (V H CD19), which has an amino acid sequence as shown in SEQ ID NO: 412, and a light chain variable region (V L CD19), which has an amino acid sequence as shown in SEQ ID NO: 413, and (c) Fc domain of human IgG1 subclass, wherein amino acid L at position 234 is substituted by A, amino acid L at position 235 is substituted by A, and amino acid P at position 329 is substituted by G, numbering according to the Kabat EU index.

2. The bispecific agonist CD28 antibody 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.

3. One or more isolated polynucleotides encoding the bispecific agonist CD28 antibody according to claim 1 or 2.

4. One or more vectors comprising the polynucleotide according to claim 3.

5. The one or more vectors according to claim 4, wherein the vector is an expression vector. A host cell comprising the polynucleotide according to claim 3 or the vector according to claim 4 or 5.

7. A method for producing a bispecific agonistic CD28 antibody, comprising the following steps: a) culturing the host cell of claim 6 under conditions suitable for expression of the bispecific agonistic CD28 antibody, and b) optionally recovering the bispecific agonistic CD28 antibody.

8. A bispecific agonistic CD28 antibody prepared by the method according to claim 7.

9. A pharmaceutical composition comprising the bispecific agonistic CD28 antibody according to any one of claims 1, 2 and 8 and at least one pharmaceutically acceptable excipient.

10. A combination product comprising the bispecific agonistic CD28 antibody according to any one of claims 1, 2 and 8 and a T cell activating anti-CD3 antibody.

11. The combination product of claim 10, wherein the T cell activating anti-CD3 antibody is an anti-CD20 / anti-CD3 bispecific antibody.

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