Multispecific antigen-binding molecules that bind to CD38 and 4-1BB, and uses thereof

Abstract

CD38 is expressed on malignant cells. 4-1BB is a co-stimulatory molecule required for T cell activation and survival. For example, a novel multispecific antigen-binding molecule (MABM) that binds to both CD38 and 4-1BB is provided herein to provide "signal 2" to enhance T cell activation in the presence of "signal 1" provided by a tumor-associated antigen (TAA)×CD3 bispecific antibody or an allogeneic response provided by antigen-presenting cells. In certain embodiments, the multispecific antigen-binding molecule of the invention can inhibit the growth of tumors expressing CD38. The multispecific antigen-binding molecule of the invention is useful for the treatment of diseases and disorders where an upregulated or induced CD38-targeted immune response is desirable and / or therapeutically beneficial. For example, the multispecific antigen-binding molecule of the invention is useful for the treatment of various cancers including multiple myeloma, lymphoma, and leukemia.

Description

Technical Field

[0001] Field of the Invention The present invention relates to multispecific antigen-binding molecules that are specific for CD38 and 4-1BB, and methods of using the same.

[0002] Sequence Listing An official copy of the sequence listing is submitted electronically herewith simultaneously with this specification via the Patent Center. The contents of the electronic sequence listing (10927WO01_Sequence_Listing_ST26.xml, size: 151,552 bytes, and creation date: May 17, 2023) are hereby incorporated by reference in their entirety.

Background Art

[0003] Background Multiple myeloma (MM) is the second most common blood cancer after non-Hodgkin lymphoma, with a prevalence in the United States of approximately 120,000 people, and approximately 30,000 new cases and 13,000 deaths each year. MM is characterized by the clonal proliferation of malignant plasma cells that secrete cytokines in an uncontrolled manner. The production of cytokines, particularly IL-6, causes local organ and tissue damage that is responsible for many of the symptoms associated with myeloma. MM patients suffer from bone pain and osteoporosis, anemia, renal dysfunction and failure, bacterial infections, and neurological disorders. MM has a median survival of 4-5 years and is rarely cured. Although the treatment of MM has advanced, new treatments have provided disproportionate benefits to younger patients. The prognosis for relapsed MM patients is poor, and new treatment approaches are urgently needed.

[0004] CD38, also known as cyclic ADP-ribose hydrolase, is a 45KDa surface glycoprotein expressed on thymocytes, some activated peripheral blood T cells and B cells, plasma cells, and dendritic cells. CD38 hydrolyzes extracellular nicotinamide adenine dinucleotide (NAD +) and functions as an extracellular enzyme involved in the metabolism of cytoplasmic nicotinamide adenine dinucleotide phosphate (NADP) (Howard, et al. Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38. Science (1993) 262:1056-9 (Non-Patent Document 1)), and produces calcium regulators such as cyclic adenosine diphosphate (ADP) ribose, ADP ribose (ADPR), and nicotinic acid adenine dinucleotide phosphate. 2+ This results in the production of mobilizing compounds. Calcium regulation leads to the activation of signaling pathways that control a wide range of physiological functions, including lymphocyte proliferation, insulin release by the pancreas, myocardial contraction, neutrophil chemotaxis, and T cell activation. CD38 enzyme activity regulates NAD + levels and improves the function of proteasome inhibitors (Cagnetta, et al. Intracellular NAD(+) depletion enhances bortezomib-induced anti-myeloma activity. Blood (2013) 122:1243-55 (Non-Patent Document 2)). Furthermore, ADPR can be metabolized by CD203a / PC-1 and CD73 to generate the immunosuppressive molecule adenosine (ADO), which can facilitate the escape of tumor cells from immune system control (Chillemi et al. Roles and modalities of ectonucleotidases in remodeling the multiple myeloma niche. Front Immunol. (2017) 8:305 (Non-Patent Document 3)). CD38 appears to contribute to the proliferative capacity of B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma, and malignant plasma cells in the bone marrow express high and uniform levels of CD38. Anti-CD38 monoclonal antibodies are thought to deplete CD38+ immunosuppressive cells such as myeloid-derived suppressor cells, regulatory T cells, and regulatory B cells, resulting in an increase in the anti-tumor activity of immune effector cells. Daratumumab, an anti-CD38 antigen-binding molecule, is approved for patients with multiple myeloma who are resistant to conventional treatments.

[0005] Activation of T cells, which involves costimulation via the TNF receptor superfamily, is crucial for survival, acquisition of effector functions, and memory differentiation. 4-1BB (Tnfrsf9), also known as CD137, is a member of the surface glycoprotein TNF receptor superfamily. Receptor expression is induced by lymphocyte activation after priming via the TCR, and its level can be enhanced by CD28 costimulation. CD8 + Exposure of T cells to a ligand or agonistic monoclonal antibody on 4-1BB costimulates 4-1BB, contributes to clonal expansion, survival, and development of T cells, induces proliferation in peripheral monocytes, activates NF-kappaB, enhances TCR / CD3-induced T cell apoptosis, and induces activation, memory generation, and regulation of CD28 costimulation for promoting Th1 cell responses. Urelumab (BMS-663513), a fully human IgG4 monoclonal antibody, was the first anti-4-1BB therapeutic agent to enter clinical trials. Clinical development was stopped when antibody-related hepatotoxicity became apparent. Utomilumab (PF-05082566) is a humanized IgG2 monoclonal antibody that activates endogenous 4-1BB while blocking binding to endogenous 4-1BBL. Therefore, there is a need in the art for alternative approaches to treating cancer.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0007] The present invention relates, in part, to multispecific antigen-binding molecules that bind to CD38 and 4-1BB, and their use in the treatment of various diseases including cancer.

[0008] The multispecific antigen-binding molecules can be used alone or in combination with other agents for treating cancers that express CD38.

[0009] The multispecific antigen-binding molecules provided herein include two antigen-binding arms, A1 and A2. The A1 arm specifically binds to CD38. The A2 arm includes a first antigen-binding domain (R1) and a second antigen-binding domain (R2), and A2 specifically binds to 4-1BB. R1 is linked to R2 via a linker, forming a stacked antigen-binding domain on the A2 arm. The combination of the A1 arm and the stacked A2 arm is called a 1+2 format. The antigen-binding domains of A2 can be included in a Fab. See Figure 1. In some embodiments, R1 and R2 bind to different 4-1BB epitopes. In some embodiments, R1 and R2 bind to the same 4-1BB epitope. In some embodiments, the amino acid sequences of the R1 and R2 heavy chain variable regions are identical or substantially similar, i.e., there are less than 5, or less than 4, or less than 3 amino acid differences in the heavy chain variable region or heavy chain complementarity-determining regions, or 2 or 1 amino acid differences in the heavy chain variable region or heavy chain complementarity-determining regions. In some embodiments, the R1 and R2 heavy chain variable regions are different, i.e., they have different antigen-binding sequences. In some embodiments, R1 is included in a first Fab (Fab2) and R2 is included in a second Fab (Fab3). The Fab2 and Fab3 of the anti-CD38 × anti-4-1BB 1+2 construct are connected via a linker from the N-terminus of VH-2 4-1BB "in" Fab2 to the C-terminus of CH1-3 "out" Fab3.

[0010] Multispecific antigen-binding molecule comprising anti-CD38 and anti-4-1BB antigen-binding domains The present disclosure provides a multispecific antigen-binding molecule that binds to CD38 and 4-1BB. Such a multispecific antigen-binding molecule is also referred to herein as an "anti-CD38 / anti-4-1BB multispecific antigen-binding molecule". The CD38 antigen-binding arm A1 comprises one antigen-binding domain. The 4-1BB antigen-binding arm A2 comprises two antigen-binding domains, R1 and R2. R1 is referred to herein as the 4-1BB "in" binding domain, and R2 is referred to herein as the 4-1BB "out" domain. Multispecific antigen-binding molecules having stacked antigen-binding domains are referred to herein as anti-CD38 / anti-4-1BB 1+2 multispecific antigen-binding molecules, or anti-CD38×anti-4-1BB 1+2 multispecific antigen-binding molecules, and the like.

[0011] The anti-CD38 portion of the anti-CD38 / anti-4-1BB multispecific molecule is useful for targeting tumor cells (e.g., plasma cells) that express CD38, and the anti-4-1BB portion of the multispecific molecule is useful for providing co-stimulation of T cells activated by cognate MHC peptides or tumor-targeting CD3 multispecific antigen-binding molecules. Simultaneous binding of CD38 on tumor cells and 4-1BB on T cells promotes the direct killing (cytolysis) of targeted tumor cells by activated T cells. Thus, the anti-CD38 / anti-4-1BB 1+2 multispecific molecules provided herein are useful for treating diseases and disorders associated with, or caused by, CD38-expressing tumors (e.g., lymphoma, leukemia, multiple myeloma, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer).

[0012] The multispecific antigen-binding molecules provided herein include a first antigen-binding arm A1 comprising an antigen-binding domain that specifically binds to human CD38, and a second antigen-binding arm A2 comprising two antigen-binding domains R1 and R2 that specifically bind to 4-1BB. The present disclosure includes anti-CD38 / anti-4-1BB 1+2 multispecific molecules (e.g., multispecific antigen-binding molecules), and each antigen-binding domain comprises a heavy-chain variable region (HCVR) paired with a light-chain variable region (LCVR). In certain exemplary embodiments of the invention, the anti-CD38 antigen-binding domain and the anti-4-1BB antigen-binding domain each comprise distinct, different HCVRs paired with a common LCVR or a universal LCVR. For example, as exemplified in Example 4 herein, a first antigen-binding domain that specifically binds to CD38, wherein the first antigen-binding domain comprises an HCVR derived from an anti-CD38 antigen-binding molecule, a first antigen-binding domain, and a second antigen-binding domain (R1) and a third antigen-binding domain (R2) that specifically bind to 4-1BB, wherein the second antigen-binding domain (R1) and the third antigen-binding domain (R2) each comprise an HCVR derived from an anti-4-1BB antigen-binding molecule, each HCVR paired with a universal light-chain LCVR, a second antigen-binding domain (R1) and a third antigen-binding domain (R2), and A multispecific antigen-binding molecule was constructed. In such embodiments, the first, second, and third antigen-binding domains each comprise distinct anti-CD38 and anti-4-1BB HCVRs but share a common light-chain LCVR.

[0013] As used herein, (a) a first antigen-binding arm comprising three CDRs of a heavy-chain variable region (HCVR) and three CDRs of an LCVR wherein the first antigen-binding arm specifically binds to CD38, the first antigen-binding arm, and a first antigen-binding arm, and (b) A first antigen-binding region (R1) comprising three CDRs of HCVR (R1-HCVR) and three CDRs of LCVR (R1-LCVR), and a second antigen-binding region (R2) comprising three CDRs of HCVR (R2-HCVR) and three CDRs of LCVR (R2-LCVR) A second antigen-binding arm comprising wherein said second antigen-binding arm specifically binds to 4-1BB A second antigen-binding arm A bispecific antigen-binding molecule comprising is provided.

[0014] In some embodiments, R1 and R2 bind to the same epitope on 4-1BB. In some embodiments, R1 and R2 bind to different epitopes on 4-1BB.

[0015] In some embodiments, R1 and R2 are connected via a peptide linker. Exemplary peptide linkers include the peptide sequence of (GGGGS)n, where n is from 1 to 6.

[0016] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein said first antigen-binding arm comprises three CDRs of HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 40.

[0017] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein said first antigen-binding arm comprises three CDRs of LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and 48.

[0018] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein said first antigen-binding arm comprises three heavy chain complementarity determining regions (HCDR1-HCDR2-HCDR3) each comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4-6-8 and 42-44-46 respectively.

[0019] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein the first antigen-binding arm comprises three light chain complementarity determining regions (LCDR1-LCDR2-LCDR3), each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54 and comprising

[0020] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein the first antigen-binding arm comprises a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40

[0021] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein the first antigen-binding arm comprises a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48

[0022] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, wherein the first antigen-binding arm comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40 and a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48

[0023] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding arm, and the second antigen-binding arm comprises a first antigen-binding region (R1) and a second antigen-binding region (R2)

[0024] In some embodiments, R1 comprises three CDRs of a heavy chain variable region of R1 (R1-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94

[0025] In some embodiments, R1 comprises three CDRs of a light chain variable region of R1 (R1-LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48

[0026] In some embodiments, R1 is Three heavy chain complementarity determining regions (R1-HCDR1-R1-HCDR2-R1-HCDR3) each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16, 34-36-38, 64-66-68, 74-76-78, 88-90-92, and 96-98-100 comprising.

[0027] In some embodiments, R1 is Three light chain complementarity determining regions (R1-LCDR1-R1-LCDR2-R1-LCDR3) each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54 comprising.

[0028] In some aspects, R1 comprises an HCVR (R1-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94.

[0029] In some aspects, R1 comprises an LCVR (R1-LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0030] In some aspects, R1 comprises an R1-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94 and an R1-LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0031] In some aspects, R2 comprises the three CDRs of an HCVR (R2-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94.

[0032] In some aspects, R2 comprises the three CDRs of an LCVR (R2-LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0033] In some embodiments, R2 is Three heavy chain complementarity determining regions (R2-HCDR1-R2-HCDR2-R2-HCDR3) each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16, 34-36-38, 64-66-68, 74-76-78, 88-90-92, and 96-98-100 comprising.

[0034] In some embodiments, R2 is Three light chain complementarity determining regions (R2-LCDR1-R2-LCDR2-R2-LCDR3) each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54 comprising.

[0035] In some aspects, R2 comprises an HCVR (R2-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94.

[0036] In some aspects, R2 comprises an LCVR (R2-LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0037] In some aspects, R2 comprises an R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94 and an R2-LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0038] In some aspects, the bispecific antigen-binding molecule is (a) a first antigen-binding arm comprising three CDRs of an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and three CDRs of an LCVR comprising the amino acid sequence of SEQ ID NO: 48, (b) a second antigen-binding arm, (i) a first antigen-binding region (R1) comprising three CDRs of an R1-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 62, and 72 and three CDRs of an R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48, (ii) Three CDRs of R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 84, and 94, and three CDRs of R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48, a second antigen-binding region (R2), comprising a second antigen-binding arm, comprising.

[0039] In some embodiments, the bispecific antigen-binding molecule is (a) a first antigen-binding arm comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-44-46-50-52-54, (b) a second antigen-binding arm, (i) a first antigen-binding region (R1) comprising R1-HCDR1-R1-HCDR2-R1-HCDR3-R1-LCDR1-R1-LCDR2-R1-LCDR3, each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-36-38-50-52-54, 64-66-68-50-52-54, and 74-76-78-50-52-54 comprising, (ii) a second antigen-binding region (R2) comprising R2-HCDR1-R2-HCDR2-R2-HCDR3-R2-LCDR1-R2-LCDR2-R2-LCDR3, each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-66-68-50-52-54, 74-76-78-50-52-54, and 88-90-92-50-52-54 comprising, comprising a second antigen-binding arm, comprising.

[0040] In some embodiments, the bispecific antigen-binding molecule is (a) a first antigen-binding arm comprising HCVR comprising the amino acid sequence of SEQ ID NO: 40 and LCVR comprising the amino acid sequence of SEQ ID NO: 48, (b) a second antigen-binding arm, (i) An R1-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, 62, and 72, and an R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48, a first antigen-binding region (R1), (ii) An R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 62, 86, and 94, and an R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48, a second antigen-binding region (R2), comprising a second antigen-binding arm, and comprising.

[0041] In some embodiments, the bispecific antigen-binding molecule is (a) a first antigen-binding arm comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and an LCVR comprising the amino acid sequence of SEQ ID NO: 48, (b) a second antigen-binding arm, (i) a first antigen-binding region (R1) comprising an R1-HCVR comprising the amino acid sequence of SEQ ID NO: 32 and an R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48, (ii) a second antigen-binding region (R2) comprising an R2-HCVR comprising the amino acid sequence of SEQ ID NO: 86 and an R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48, comprising a second antigen-binding arm, and comprising.

[0042] In some embodiments, the bispecific antigen-binding molecule is (a) a first antigen-binding arm comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and an LCVR comprising the amino acid sequence of SEQ ID NO: 48, (b) a second antigen-binding arm, (i) a first antigen-binding region (R1) comprising an R1-HCVR comprising the amino acid sequence of SEQ ID NO: 72 and an R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48, (ii) a second antigen-binding region (R2) comprising an R2-HCVR comprising the amino acid sequence of SEQ ID NO: 94 and an R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48, comprising a second antigen-binding arm, comprises

[0043] In some embodiments, the bispecific antigen-binding molecule (a) a first antigen-binding arm comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and an LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) a second antigen-binding arm, (i) a first antigen-binding region (R1) comprising an R1-HCVR comprising the amino acid sequence of SEQ ID NO: 62 and an R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding region (R2) comprising an R2-HCVR comprising the amino acid sequence of SEQ ID NO: 62 and an R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48, comprising a second antigen-binding arm; and comprises.

[0044] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody.

[0045] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising a heavy chain constant region of the IgG1 or IgG4 isotype.

[0046] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen-binding arm and a second heavy chain comprising the R1-HCVR and R2-HCVR of the second antigen-binding arm, wherein the second heavy chain comprises the mutations H435R and Y436F (EU numbering).

[0047] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen-binding arm paired with a light chain comprising the LCVR of the first antigen-binding arm, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 58 and the light chain comprises the amino acid sequence of SEQ ID NO: 60.

[0048] In some embodiments, the bispecific antibody A second heavy chain comprising R1-HCVR and R2-HCVR of a second antigen-binding arm paired with a first light chain comprising R1-LCVR and a second light chain comprising R2-LCVR comprising wherein said second heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 70, 80, 82, and 84; wherein said first light chain comprises the amino acid sequence of SEQ ID NO: 60, and the second light chain comprises the amino acid sequence of SEQ ID NO: 60.

[0049] In some embodiments, the bispecific antibody is (a) a first antigen-binding arm that specifically binds to human CD38, comprising a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 60 and (b) a second antigen-binding arm that specifically binds to human 4-1BB, comprising a heavy chain comprising the sequence of SEQ ID NO: 56, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60 and comprising.

[0050] In some embodiments, the bispecific antibody is (a) a first antigen-binding arm that specifically binds to human CD38, comprising a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 60 and (b) a second antigen-binding arm that specifically binds to human 4-1BB, comprising a heavy chain comprising the sequence of SEQ ID NO: 70, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60 and comprising.

[0051] In some embodiments, the bispecific antibody is (a) a first antigen-binding arm that specifically binds to human CD38, comprising a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 60 and (b) a second antigen-binding arm that specifically binds to human 4-1BB, comprising a heavy chain comprising the sequence of SEQ ID NO: 80, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60 A second antigen-binding arm that specifically binds to human 4-1BB, and comprises.

[0052] In some embodiments, the bispecific antibody is (a) A first antigen-binding arm that specifically binds to human CD38, comprising a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 60 and (b) A second antigen-binding arm that specifically binds to human 4-1BB, comprising a heavy chain comprising the sequence of SEQ ID NO: 82, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60 and comprises.

[0053] In some embodiments, the bispecific antibody is (a) A first antigen-binding arm that specifically binds to human CD38, comprising a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 60 and (b) A second antigen-binding arm that specifically binds to human 4-1BB, comprising a heavy chain comprising the sequence of SEQ ID NO: 84, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60 and comprises.

[0054] As used herein, a bispecific antigen-binding molecule comprising a first antigen-binding arm that specifically binds to CD38 and a second antigen-binding arm that specifically binds to 4-1BB, wherein (a) The first antigen-binding arm comprises three CDRs of an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and three CDRs of an LCVR comprising the amino acid sequence of SEQ ID NO: 48, (b) The second antigen-binding arm (i) A first antigen-binding region (R1) comprising three CDRs of an HCVR (R1-HCVR) comprising the amino acid sequence of SEQ ID NO: 62 and three CDRs of an LCVR (R1-LCVR) comprising the amino acid sequence of SEQ ID NO: 48, (ii) The second antigen-binding region (R2) comprising three CDRs of HCVR (R2-HCVR) containing the amino acid sequence of SEQ ID NO: 62 and three CDRs of LCVR (R2-LCVR) containing the amino acid sequence of SEQ ID NO: 48, comprising a bispecific antigen-binding molecule is provided.

[0055] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody.

[0056] In some embodiments, the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen-binding arm, the first heavy chain being paired with a light chain comprising the LCVR of the first antigen-binding arm, the first heavy chain comprising the amino acid sequence of SEQ ID NO: 58, and the light chain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the bispecific antibody comprises a second heavy chain comprising R1-HCVR and R2-HCVR of the second antigen-binding arm, the second heavy chain being paired with a first light chain comprising R1-LCVR and a second light chain comprising R2-LCVR, the second heavy chain comprising the amino acid sequence of SEQ ID NO: 70, 82, or 84, the first light chain comprising the amino acid sequence of SEQ ID NO: 60, and the second light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0057] In some embodiments, the bispecific antigen-binding molecule (a) a first antigen-binding arm that specifically binds to human CD38, comprising a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 60, (b) a second antigen-binding arm that specifically binds to human 4-1BB, (i) comprising a heavy chain comprising the sequence of SEQ ID NO: 70, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60, or (ii) comprising a heavy chain comprising the sequence of SEQ ID NO: 82, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60, or (iii) comprising a heavy chain comprising the sequence of SEQ ID NO: 84, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60, a second antigen-binding arm, and comprises.

[0058] In some embodiments, the multispecific antigen-binding molecule inhibits the proliferation of CD38+ tumor cells selected from the group consisting of myeloma cells, leukemia cells, lymphoma cells, hepatocellular carcinoma cells, non-small cell lung cancer cells, melanoma cells, pancreatic ductal adenocarcinoma cells, glioma cells, or breast cancer cells.

[0059] In another aspect, there is provided a pharmaceutical composition comprising a multispecific antigen-binding molecule and a pharmaceutically acceptable carrier or diluent. In related aspects, the invention features a composition that is a combination of an anti-CD38 / anti-4-1BB 1+2 multispecific antigen-binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-CD38 / anti-4-1BB 1+2 multispecific antigen-binding molecule.

[0060] In another aspect, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding either A1 or A2. In some aspects, there are provided nucleic acid molecules comprising a nucleotide sequence encoding any one of HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, heavy chain, and / or light chain. In some embodiments, the nucleic acid molecule comprises one or more nucleotide sequences set forth in Tables 2, 4, 6, 8, or 10. The nucleic acid molecules comprising the nucleic acid sequences may be in any functional combination or arrangement thereof.

[0061] In some aspects, as used herein, an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region (HCVR) of antigen-binding arm A1 of a multispecific antigen-binding molecule, wherein A1 binds to CD38, (a) the HCVR comprises three heavy chain complementarity-determining regions (HCDR1-HCDR2-HCDR3) each comprising the amino acid sequences of SEQ ID NOs: 42, 44, and 46 or (b) the HCVR comprises the amino acid sequence of SEQ ID NO: 40, An isolated nucleic acid molecule is provided.

[0062] In some embodiments, provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain of antigen-binding arm A1 of a multispecific antigen-binding molecule, wherein A1 binds to CD38 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 58.

[0063] In some embodiments, provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region (HCVR) of antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 comprises a first antigen-binding domain (R1) that binds to 4-1BB and a second antigen-binding domain (R2) that binds to 4-1BB, (a) R1-HCVR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 34, 36, and 38, and R2-HCVR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 88, 90, and 92, or (b) R1-HCVR comprises the amino acid sequence of SEQ ID NO: 32 and R2-HCVR comprises the amino acid sequence of SEQ ID NO: 86, An isolated nucleic acid molecule is provided.

[0064] Provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain of antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 binds to 4-1BB and the heavy chain comprises the amino acid sequence of SEQ ID NO: 56.

[0065] Also provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the first heavy chain variable region (HCVR) and the second HCVR of antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 binds to 4-1BB, (a) The first HCVR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 64, 66, and 68, and the second HCVR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 64, 66, and 68, or (b) The first HCVR comprises the amino acid sequence of SEQ ID NO: 62, and the second HCVR comprises the amino acid sequence of SEQ ID NO: 62, An isolated nucleic acid molecule is also provided.

[0066] Also provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain of the antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 binds to 4-1BB and the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 82, and 84.

[0067] Also provided herein is an isolated nucleic acid molecule comprising nucleic acid sequences encoding the first heavy chain variable region (HCVR) and the second HCVR of the antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 binds to 4-1BB, (a) The first HCVR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 74, 76, and 78, and the second HCVR comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NOs: 96, 98, and 100, or (b) The first HCVR comprises the amino acid sequence of SEQ ID NO: 72, and the second HCVR comprises the amino acid sequence of SEQ ID NO: 94, An isolated nucleic acid molecule is also provided.

[0068] Also provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain of the antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 binds to 4-1BB and the heavy chain comprises the amino acid sequence of SEQ ID NO: 80.

[0069] Also provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a light chain variable region (LCVR) of a multispecific antigen-binding molecule, wherein (a) the LCVR comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 50, 52, and 54, or (b) the LCVR comprises the amino acid sequence of SEQ ID NO: 48, the isolated nucleic acid molecule is provided.

[0070] Also provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a light chain of a multispecific antigen-binding molecule, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 60.

[0071] Also provided herein is an expression vector or a set of expression vectors comprising one or more nucleic acid molecules of any one of the nucleic acids described above. Also provided is a host cell comprising one or more expression vectors provided herein. In some embodiments, the host cell is a mammalian cell or a prokaryotic cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or an Escherichia coli (E. coli) cell. Further provided is a composition comprising one or more nucleic acid molecules described herein.

[0072] In some embodiments, methods for producing a multispecific antigen-binding molecule are provided. As provided herein, the methods include growing a host cell under conditions that permit production of the multispecific antigen-binding molecule, wherein the host cell comprises a nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region (HCVR) of antigen-binding arm A1 of the multispecific antigen-binding molecule, a nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region (HCVR) of antigen-binding arm A2 of the multispecific antigen-binding molecule, and / or a nucleic acid molecule comprising a nucleic acid sequence encoding a common light chain variable region (LCVR). In some aspects, each nucleic acid molecule is in the same expression vector. In some aspects, one or more of the nucleic acid molecules are in different expression vectors. In some aspects, the host cell comprises a nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain of antigen-binding arm A1 of the multispecific antigen-binding molecule, a nucleic acid molecule encoding the heavy chain of antigen-binding arm A2 of the multispecific antigen-binding molecule, and / or a nucleic acid molecule comprising a nucleic acid sequence encoding a common light chain. In some aspects, each nucleic acid molecule is in the same expression vector. In some aspects, one or more of the nucleic acid molecules are in different expression vectors.

[0073] Also provided herein is a method of inhibiting the growth of a plasmacytoma tumor in a subject, the method comprising administering to the subject any one or more of the multispecific antigen-binding molecules described herein or the pharmaceutical compositions provided herein. In some aspects, the plasmacytoma tumor is multiple myeloma.

[0074] Also provided herein is the use of a multispecific antigen-binding molecule described herein or a pharmaceutical composition provided herein in the manufacture of a medicament for inhibiting the growth of a plasmacytoma tumor in a subject. The multispecific antigen-binding molecule or pharmaceutical composition can be administered to a subject. In some aspects, the plasmacytoma tumor is multiple myeloma.

[0075] Disclosed herein is a method for inhibiting tumor growth in a subject, the method comprising administering to the subject any one or more of the multispecific antigen-binding molecules described herein or the pharmaceutical compositions provided herein. In some embodiments, the tumor is selected from the group consisting of multiple myeloma, lymphoma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer characterized in part by having CD38+ cells.

[0076] Also disclosed herein is the use of a multispecific antigen-binding molecule described herein or a pharmaceutical composition provided herein in the manufacture of a medicament for inhibiting tumor growth in a subject. The multispecific antigen-binding molecule or pharmaceutical composition can be administered to a subject. In some embodiments, the tumor is selected from the group consisting of multiple myeloma, lymphoma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer characterized in part by having CD38+ cells.

[0077] Disclosed herein is a method of treating a patient suffering from multiple myeloma or another BCMA-expressing B-cell malignancy, the method comprising administering to the patient a multispecific antigen-binding molecule provided herein or a pharmaceutical composition provided herein. In some embodiments, the BCMA-expressing B-cell malignancy is selected from the group consisting of Waldenström macroglobulinemia, Burkitt lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma.

[0078] Also disclosed herein is the use of a multispecific antigen-binding molecule described herein or a pharmaceutical composition provided herein in the manufacture of a medicament for treating a patient suffering from multiple myeloma or another BCMA-expressing B-cell malignancy. The multispecific antigen-binding molecule or pharmaceutical composition can be administered to a subject.

[0079] Provided herein are methods of treating patients afflicted with CD38+ tumors and / or BCMA-expressing tumors. The methods include administering to a subject a multispecific antigen-binding molecule described herein, or a pharmaceutical composition provided herein, in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody, e.g., semiprimab (bsAb2810).

[0080] Also provided herein is the use of a multispecific antigen-binding molecule described herein, or a pharmaceutical composition provided herein, in the manufacture of a medicament for treating patients afflicted with CD38+ tumors and / or BCMA-expressing tumors.

[0081] In some embodiments, the methods or uses provided herein further comprise administering a second therapeutic agent or treatment regimen. In some embodiments, the second therapeutic agent is an antibody that binds to a plasmacytoma tumor. In some embodiments, the second therapeutic agent is an anti-BCMA / anti-CD3 bispecific antigen-binding molecule. In some embodiments, the second therapeutic agent is an anti-CD20 / anti-CD3 bispecific antigen-binding molecule. In some embodiments, the second therapeutic agent is an anti-CD28 / anti-4-1BB bispecific antigen-binding molecule. In some embodiments, the second therapeutic agent or treatment regimen comprises a chemotherapeutic agent, a DNA alkylating agent, an immunomodulatory agent, a proteasome inhibitor, a histone deacetylase inhibitor, radiation therapy, stem cell transplantation, an oncolytic virus, a cancer vaccine, an immunocytokine, CAR-T cells, different bispecific antibodies that interact with different tumor cell surface antigens and T cell or immune cell antigens, an antibody-drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 checkpoint inhibitor, a CD22 inhibitor, a BCMA inhibitor, a CD28 agonist, a CD20 inhibitor, or combinations thereof.

[0082] Furthermore, provided is the use of the multispecific antigen-binding molecules provided herein, or the pharmaceutical compositions provided herein, in the treatment of diseases or disorders associated with the expression of CD38, CD20, and / or BCMA. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the multispecific antigen-binding molecule or pharmaceutical composition is for use in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. The multispecific antigen-binding molecule, or a pharmaceutical composition comprising the multispecific antigen-binding molecule, is administered intravenously, intramuscularly, or subcutaneously.

[0083] Anti-CD38 / anti-4-1BB 1+2 multispecific antigen-binding molecules having modified glycosylation patterns are provided herein. In some applications, for example, for increasing the antibody-dependent cell cytotoxicity (ADCC) function of the antigen-binding molecule, modifications that remove undesirable glycosylation sites, i.e., antigen-binding molecules lacking fucose moieties present on the oligosaccharide chains, may be useful (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be made to modify the complement-dependent cytotoxicity (CDC) activity.

[0084] In yet another aspect, provided herein is a method of treatment for targeting / killing tumor cells expressing CD38 using the anti-CD38 / anti-4-1BB multispecific antigen-binding molecule of the invention, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of the anti-CD38 / anti-4-1BB multispecific antigen-binding molecule provided herein.

[0085] The disclosure also includes the use of the anti-CD38 / anti-4-1BB multispecific antigen-binding molecule provided herein in the manufacture of a medicament for the treatment of a disease or disorder associated with or resulting from CD38 expression.

[0086] Other embodiments will become apparent from consideration of the forms of the invention that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0087]

Figure 1

Figure 2A

Figure 2B

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

[0088] Detailed Description Before describing the present invention, it should be understood that since the specific methods and experimental conditions to be described may vary, the present invention is not limited to such methods and conditions. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are used for the purpose of describing only specific embodiments and are not intended to be limiting.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0090] As used herein, the term "about" when used in relation to a specific recited numerical value or range of values means that the value can vary by up to 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, as well as all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0091] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but exemplary methods and materials are described herein. All patents, applications, and non-patent publications mentioned herein are hereby incorporated by reference in their entirety.

[0092] Definition As used herein, the expression "4-1BB" refers to the receptor of 4-1BBL. Cross-linking of 4-1BB by 4-1BBL enhances T cell activation. Human 4-1BB (or CD137) having UniProt accession number Q07011 contains the amino acid sequence set forth in SEQ ID NO: 101, and amino acid residues 1 to 23 are the signal peptide. Residues 24 to 186 constitute the extracellular domain of the receptor.

[0093] Human 4-1BB (immunogen) amino acid sequence TIFF2025519057000002.tif25148 (SEQ ID NO: 101)

[0094] All references in this specification to proteins, polypeptides, and protein fragments are intended to refer to the human version of each such protein, polypeptide, or protein fragment, unless explicitly specified as being from non-human species. Thus, the expression "4-1BB" means human 4-1BB, unless otherwise specified as being from non-human species such as, for example, "mouse 4-1BB", "monkey 4-1BB", etc.

[0095] As used herein, an "antigen-binding molecule that binds to 4-1BB", or an "anti-4-1BB antigen-binding molecule" includes an antigen-binding molecule that specifically recognizes 4-1BB expressed on the surface of a cell. The anti-4-1BB antigen-binding molecules provided herein include the VRs and CDRs disclosed herein. In certain embodiments, the antigen-binding molecule is an antibody. In certain embodiments, the antigen-binding molecule is a bispecific antibody.

[0096] As used herein, the expression "CD38" also refers to a glycoprotein known as cyclic ADP-ribose hydrolase and expressed on malignant cells. CD38 plays a central role in the regulation of intracellular calcium levels. The protein has an N-terminal cytoplasmic tail, a single transmembrane domain, and a C-terminal extracellular region with four N-glycosylation sites. As used herein, the term "CD38" refers to the human CD38 protein unless otherwise specified as being derived from a non-human species (e.g., "mouse CD38", "monkey CD38", etc.). The human CD38 protein has the amino acid sequence shown in SEQ ID NO: 102 (human CD38 extracellular domain (V43-I300).mFc) and / or has the amino acid sequence described in NCBI accession number NP_001766.2 or NM_001775.3.

[0097] Human CD38 extracellular domain (V43-I300).mFc (immunogen) amino acids TIFF2025519057000003.tif43148 (SEQ ID NO: 102) mFc sequence is underlined

[0098] As used herein, an "antigen-binding molecule that binds to CD38" or an "anti-CD38 antigen-binding molecule" includes an antigen-binding molecule that specifically recognizes CD38.

[0099] The term "antigen-binding molecule" includes multispecific antigen-binding molecules, such as anti-CD38 × anti-4-1BB 1+2 multispecific antigen-binding molecules. The anti-CD38 antigen-binding molecules provided herein include the VRs and CDRs disclosed herein. In certain embodiments, the antigen-binding molecule is an antibody. In certain embodiments, the antigen-binding molecule is a bispecific antibody.

[0100] As used herein, the term "antigen-binding molecule" means any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD38 or 4-1BB). The term "antigen-binding molecule" encompasses immunoglobulin molecules, which include four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). The term "antigen-binding molecule" includes immunoglobulin molecules that contain two antigen-binding arms, A1 and A2. The term "antigen-binding molecule" also includes immunoglobulin molecules consisting of four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains. Each antigen-binding arm contains a heavy chain, which in turn contains at least one heavy-chain variable region (abbreviated herein as HCVR or VH-1, VH-2, or VH-3) and a heavy-chain constant region (CH1-1, CH1-2, and CH1-3). The heavy-chain constant region also includes CH2 and CH3. Each light chain contains a light-chain variable region (abbreviated herein as LCVR, VL-1 on the A1 arm, VL-2 and VL-3 on the A2 arm) and a light-chain constant region (CL-1 on the A1 arm, and CL-2 and CL-3 on the A2 arm). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), with relatively conserved regions called framework regions (FRs) arranged in between. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments herein, the FRs of the anti-CD38 antigen-binding arm or the anti-4-1BB antigen-binding arm (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on the parallel analysis of two or more CDRs.

[0101] As used herein, terms such as "antigen-binding portion" of an antigen-binding molecule, "antigen-binding fragment" of an antigen-binding molecule, etc. include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antigen-binding molecule can be derived from a complete antigen-binding molecule using any suitable standard techniques, such as protein digestion or recombinant genetic engineering techniques, which involve manipulation and expression of DNA encoding the variable domain and optionally the constant domain of the antigen-binding molecule. Such DNA is known and / or can be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage antigen-binding molecule libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical or molecular biological techniques to, for example, arrange one or more variable domains and / or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0102] The anti-CD38×anti-4-1BB 1+2 antigen-binding molecule will typically comprise at least three variable domains. The variable domains can be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to or in-frame with one or more framework sequences. In having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and can include VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding molecule can include a monomeric VH or VL domain.

[0103] In certain embodiments, the antigen-binding molecule can include at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the multispecific antigen-binding molecules of the present invention include (i)V H 1-C H 1-1, (ii)V H 2-C H 1-2、 (iii)V H 3-C H 1-3、 (iv)V H 1-C H 1-C H 2、 (v)V H 1-C H 1-C H 2-C H 3、 (vi)V H 2-C H 1-2-C H 2、 (vii)V H 2-C H 1-2-C H 2-C H 3、 (viii)V H 3-C H 1-3、 (ix)V H 3-C H 1-3-V H 2-C H 1-2、 (x)V H 3-C H 1-3-V H 2-C H 1-2-C H 2、 (xi)V H 3-C H 1-3-V H 2-C H 1-2-C H 2-C H 3、 (xii)V H -C L 、 (xiii)V L -1-C L 1、 (xiv)V L -2-C L 2、 (xv)V L -3-C L 3、 (xvi)V L -3-C L3-C H 1-3、 (xvii)V L -2-C L -2-C H 1-2、 (xviii)V L 1-C L 1-C H 1-C H 2-C H 3、 (xix)V L -2-C L 2-C H 1-2、 (xx)V L -2-C L 2-C H 1-2-C H 2、 (xxi)V L -2-C L 2-C H 1-2-C H 2-C H 3、 (xxii)V L -3-C L 3-C H 1-3-V H 2-C H 1-2-C H 2、 (xxiii)V L -3-C L 3-C H 1-3-V H 2-C H 1-2-C H 2-C H 3、and (xxiv)V L -C L Examples include. In any configuration of the variable domain and the constant domain, including any of the exemplary configurations listed above, the variable domain and the constant domain may be directly connected to each other, or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a mobile or semi-mobile connection between adjacent variable domains and / or constant domains in a single polypeptide molecule.

[0104] Exemplarily, FIG. 1 shows an A1 antigen-binding arm containing Fab1 specific for CD38, and two Fabs, Fab2 and Fab3, each containing an A2 antigen-binding arm specific for 4-1BB. The Fab1, Fab2, and Fab3 light chains may be universal light chains. In this example, V L -1 is bound to C H -1 bound to C L -1 bound to C L -1. In the A2 arm, V H -3 is bound to C L -3 bound to C L -3. Similarly, V H -2 is bound to C L -2 bound to C H -2. The Fab2 and Fab3 of the anti-CD38 × anti-4-1BB 1+2 construct are connected via a linker from the N-terminus of V H -2 4-1BB "in" Fab2 to the C-terminus of C

[0105] The antigen-binding molecules of the present invention can function via complement-dependent cytotoxicity (CDC) or antigen-binding molecule-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antigen-binding molecules of the present disclosure in the presence of complement. "Antigen-binding molecule-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize the bound antigen-binding molecules on the target cells, thereby resulting in the lysis of the target cells. CDC and ADCC can be measured using assays well-known and available in the art. (See, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of the antigen-binding molecule is important in the ability of the antigen-binding molecule to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antigen-binding molecule can be selected based on whether it is desirable for the antigen-binding molecule to mediate cytotoxicity.

[0106] In certain embodiments of the present invention, the anti-CD38×anti-4-1BB 1+2 multispecific antigen-binding molecules provided herein are human antigen-binding molecules. As used herein, the term "human antigen-binding molecule" is intended to include antigen-binding molecules having variable and constant regions derived from human germline immunoglobulin sequences. The human antigen-binding molecules of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences, for example, in the CDRs, particularly in CDR3 (e.g., mutations are introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antigen-binding molecule" is not intended to include antigen-binding molecules in which CDR sequences from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

[0107] In some embodiments, the multispecific antigen-binding molecules provided herein may be recombinant human antigen-binding molecules. As used herein, the term "recombinant human antigen-binding molecule" refers to any human antigen-binding molecule prepared, expressed, created, or isolated by recombinant means, such as an antigen-binding molecule expressed using a recombinant expression vector transfected into a host cell (described in detail below), an antigen-binding molecule isolated from a recombinant, combinatorial human antigen-binding molecule library (described in detail below), an antigen-binding molecule isolated from an animal (e.g., a mouse) into which human immunoglobulin genes have been introduced (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or an antigen-binding molecule prepared, expressed, created, or isolated by any other means involving splicing from a human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antigen-binding molecules have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such genetically recombinant human antigen-binding molecules are subjected to in vitro mutagenesis (or, if transgenic animals with human Ig sequences are used, somatic mutagenesis in vivo), such that the amino acid sequences of the V H region and the V L region are related to, and derived from, the human germline V H sequence and the V L sequence, but may not naturally occur in the human antigen-binding molecule germline repertoire in vivo.

[0108] Human antigen-binding molecules can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, with the dimer held together by inter-chain heavy-chain disulfide bonds. In the second form, the dimer is not linked via inter-chain disulfide bonds, and the molecule of approximately 75-80 kDa is composed of covalently linked light and heavy chains (hapten-binding molecule). These forms were very difficult to separate even after affinity purification.

[0109] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences related to the hinge-region isotype of the antigen-binding molecule. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to levels typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention encompasses antigen-binding molecules having one or more mutations in the hinge, C H 2 region or C H 3 region, and those mutations can be desirable, for example, in production to improve the yield of the desired antigen-binding molecule form.

[0110] The multispecific antigen-binding molecules of the present invention may be isolated antigen-binding molecules. As used herein, "isolated multispecific antigen-binding molecule" means an antigen-binding molecule that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antigen-binding molecule that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antigen-binding molecule naturally occurs or is naturally produced, is an "isolated antigen-binding molecule" for the purposes of the present invention. An isolated antigen-binding molecule also includes an antigen-binding molecule in situ within a recombinant cell. An isolated antigen-binding molecule is an antigen-binding molecule that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antigen-binding molecule may be substantially free of other cellular and / or chemical substances.

[0111] The anti-CD38 × anti-4-1BB 1+2 antigen-binding molecules disclosed herein may include one or more amino acid substitutions, insertions, and / or deletions in the frameworks and / or CDRs of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antigen-binding molecules are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antigen-binding molecule sequence databases. The present disclosure includes multispecific antigen-binding molecules or antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frameworks and / or CDRs are mutated relative to the sequences provided herein. In some embodiments, the mutations are made to reflect the corresponding residues of the germline sequence from which the antigen-binding molecule was derived, or the corresponding residues of another human germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate many antigen-binding molecules that include one or more mutations, such as individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework and / or CDR residues within the V H and / or V L domains are mutated back to the residues found in the original germline sequence from which the antigen-binding molecule is derived. In other embodiments, only certain residues, e.g., only the mutated residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding molecule was originally derived).

[0112] Furthermore, the multispecific antigen-binding molecules of the invention may comprise any combination of two or more mutations within the framework and / or CDR regions. For example, certain individual residues may be mutated relative to the sequences provided herein, or to the corresponding residues of a particular germline sequence, while, on the other hand, certain other residues that are different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antigen-binding molecules containing one or more mutations can be readily tested for one or more desired properties such as improved binding specificity, increased binding affinity, improved or enhanced (where appropriate) biological properties of an antagonist or agonist, and reduced immunogenicity. Antigen-binding molecules and those obtained in this general manner are encompassed by the present invention.

[0113] Provided herein are anti-CD38 antibody × anti-4-1BB antigen-binding molecules comprising a variant of any of the HCVR, LCVR, and / or CDR disclosed herein having one or more substitutions. In some embodiments, the substitutions are conservative amino acid substitutions. For example, the present disclosure provides an anti-CD38 × anti-4-1BB antigen-binding molecule having an HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences described in Tables 1, 3, 5, or 7 herein.

[0114] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antigen-binding molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antigen-binding molecules can bind to different regions on an antigen and can have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain situations, an epitope can include a sugar, phosphoryl group, or sulfonyl group moiety on an antigen.

[0115] The term "substantial identity" or "substantially identical", when referring to a nucleic acid or a fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as described below, has nucleotide sequence identity of at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0116] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using default gap weights. As used herein, amino acid substitutions that do not substantially alter the functional properties of the multispecific antigen-binding molecule are contemplated. In some embodiments, the non-identical residue positions differ only by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference.A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 logarithmic likelihood matrix.

[0117] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used with default parameters for determining sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant proteins, including programs such as Gap and Bestfit. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, which is a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the best overlapping regions between the query sequence and the search sequence and the percent sequence identity (Pearson (2000) supra). Another preferred algorithm when comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0118] Binding characteristics of the antigen-binding molecule As used herein, in the context where an antigen-binding molecule, immunoglobulin, antigen-binding molecule binding fragment, or Fc-containing protein binds to any of a given antigen, such as a cell surface protein or a fragment thereof, the term "binding" typically refers to an interaction or association between at least two entities or molecular structures, such as an antigen-binding molecule - antigen interaction.

[0119] For example, when determined by surface plasmon resonance (SPR) technology in a BIAcore device using the antigen as a ligand and an antigen-binding molecule, Ig, antigen-binding molecule binding fragment, or Fc-containing protein as the analyte (or anti-ligand), for example, the binding affinity is typically about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, corresponding to a K D value. Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlates well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223 - 31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1 - 2):68 - 77).

[0120] Thus, the multispecific antigen-binding molecules or antigen-binding proteins herein bind to a given antigen or cell surface molecule (receptor) having an affinity corresponding to a K D value that is at least 10-fold lower than its affinity for binding to non-specific antigens (e.g., BSA, casein). According to the present disclosure, an affinity of an antigen-binding molecule corresponding to a K D value that is 10-fold or less than that for a non-specific antigen may be considered undetectable binding, but such an antigen-binding molecule can pair with a second antigen-binding arm for the production of the multispecific antigen-binding molecules of the present invention.

[0121] "K DThe term "(M)" refers to the dissociation equilibrium constant of a specific antigen-binding molecule-antigen interaction, or the dissociation equilibrium constant of an antigen-binding molecule or antigen-binding molecule binding fragment that binds to an antigen. K D has an inverse relationship with the binding affinity, and thus, K D the smaller the value, the higher the affinity, that is, the stronger. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and thus, a smaller K D value; conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, and thus, a larger K D value. In some situations, when the binding affinity (or K D ) of a specific molecule (e.g., an antigen-binding molecule) for an interaction partner molecule (e.g., antigen X) is higher compared to the binding affinity of that molecule (e.g., an antigen-binding molecule) for another interaction partner molecule (e.g., antigen Y), the larger K D value (lower, or weaker, affinity) can be expressed as a binding ratio determined by dividing by the smaller K D value (higher, or stronger, affinity), and can be expressed, for example, in some cases, as 5-fold or 10-fold higher binding affinity.

[0122] The term "k d " (sec-1 or 1 / s) refers to the dissociation rate constant of a specific antigen-binding molecule-antigen interaction, or the dissociation rate constant of an antigen-binding molecule or antigen-binding molecule binding fragment. This value is also referred to as the k off value.

[0123] The term "k a " (M-1×sec-1 or 1 / M / s) refers to the association rate constant of a specific antigen-binding molecule-antigen interaction, or the association rate constant of an antigen-binding molecule or antigen-binding molecule binding fragment.

[0124] The "K AThe term "(M - 1 or 1 / M)" refers to the association equilibrium constant of a particular antigen - binding molecule - antigen interaction, or the association equilibrium constant of an antigen - binding molecule or an antigen - binding molecule - binding fragment. The association equilibrium constant is k a divided by k d to obtain.

[0125] The term "EC50" or "EC 50 " refers to the half - maximal effective concentration, including the concentration of an antigen - binding molecule that induces a reaction midway between the baseline and the maximum after a specified exposure time. EC 50 basically represents the concentration of an antigen - binding molecule at which 50% of its maximum effect is observed. In certain embodiments, the EC 50 value is equal to, for example, the concentration of an antigen - binding molecule of the present invention that gives half of the maximum binding to cells expressing 4 - 1BB or a tumor - associated antigen (e.g., CD38) as determined by a FACS binding assay. Thus, as the EC 50 or half - maximal effective concentration value increases, a decrease or weakening of binding is observed.

[0126] In one embodiment, the decrease in binding can be defined as an increase in the EC 50 antigen - binding molecule concentration that enables binding to half of the maximum amount of target cells.

[0127] In another embodiment, the EC 50 value represents the concentration of an antigen - binding molecule of the present invention that induces maximum half - depletion of target cells by the cytotoxic activity of T cells. Thus, an increase in cytotoxic activity (e.g., T - cell - mediated tumor cell killing) is observed along with a decrease in the EC 50 , or half - maximal effective concentration value.

[0128] Multispecific antigen - binding molecule The antigen-binding molecule of the present invention binds to both CD38 and 4-1BB. The multispecific antigen-binding molecule may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for multiple target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

[0129] According to certain exemplary embodiments, the present invention includes a multispecific antigen-binding molecule that specifically binds to 4-1BB and CD38. Such molecules may be referred to herein, for example, as "anti-CD38 × anti-4-1BB 1+2", or "anti-CD38 / anti-4-1BB 1+2", or "anti-CD38 × 4-1BB 1+2", or "CD38 × 4-1BB 1+2" multispecific molecules, or other similar terms.

[0130] The present disclosure includes a multispecific antigen-binding molecule, wherein one arm A2 of the immunoglobulin has two antigen-binding domains that bind to human 4-1BB, a 4-1BB "in" domain and a 4-1BB "out" domain, and the other arm A1 of the immunoglobulin is specific for binding to human CD38. The 4-1BB-binding arm may contain any of the HCVR / LCVR or CDR amino acid sequences described in Table 3 (anti-4-1BB "in") or Table 5 (anti-4-1BB "out") herein in a stacked format.

[0131] In certain embodiments, the 4-1BB-binding arm binds to human 4-1BB and promotes human T cell activation. In certain embodiments, the 4-1BB-binding arm binds to human 4-1BB and induces human T cell activation. In other embodiments, the 4-1BB-binding arm binds to human 4-1BB and induces tumor-associated antigen-expressing cell killing in the context of a multispecific or multispecific antigen-binding molecule. The CD38-binding arm may contain any of the amino acid sequences of HCVR / LCVR or CDR described in Table 1 herein.

[0132] As used herein, the expression "antigen-binding molecule" means a protein, polypeptide, or molecular complex that contains, or consists of, at least one complementarity-determining region (CDR) that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen-binding molecule is an antigen-binding molecule or a fragment of an antigen-binding molecule, and those terms are defined elsewhere in this specification.

[0133] As used herein, the expression "multispecific antigen-binding molecule" means a protein, polypeptide, or molecular complex that comprises at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the multispecific antigen-binding molecule contains at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding arm A1 specifically binds to a first antigen (e.g., CD38), and the second antigen-binding arm A2 specifically binds to second and third antigens (e.g., 4-1BB) distinct from the first antigen.

[0134] The multispecific antigen-binding molecules contemplated herein may contain a human IgG heavy-chain constant region. In some cases, the human IgG heavy-chain constant region is isotype IgG1. In some cases, the human IgG heavy-chain constant region is isotype IgG4. In various embodiments, the multispecific antigen-binding molecule contains a chimeric hinge that reduces Fcγ receptor binding as compared to the wild-type hinge of the same isotype.

[0135] The first antigen-binding arm A1 and the second antigen-binding arm A2 can be directly or indirectly connected to each other to form the multispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding arm A1 and the second antigen-binding arm A2 can each be connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a multispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, the multimerization domain can be an immunoglobulin C H polypeptide containing 3 domains. Non-limiting examples of multimerization components are (C H 2 to C H 3 domains) the Fc portion of an immunoglobulin, for example, isotypes IgG1, IgG2, IgG3, and IgG4, and the Fc domain of IgG selected from any allotype within each isotype group.

[0136] The multispecific antigen-binding molecule of the present invention will typically comprise two multimerization domains, for example, two Fc domains that are each part of a separate antigen-binding molecule heavy chain. The first and second multimerization domains can be of the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains can be of different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0137] In certain embodiments, the multimerization domain is an Fc fragment, or an amino acid sequence 1 to about 200 amino acids in length that contains at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue, or a short-chain cysteine-containing peptide. Other multimerization domains include, or consist of, peptides or polypeptides that include a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0138] Any multispecific antigen-binding molecule format or technique can be used to generate the multispecific antigen-binding molecules of the present disclosure. For example, an antigen-binding molecule having a first antigen-binding specificity, or a fragment thereof, can be functionally linked (e.g., by chemical bonding, genetic fusion, or non-covalent association, or other means) to one or more other molecular entities, such as another antigen-binding molecule or an antigen-binding molecule fragment having a second binding specificity, to produce a multispecific antigen-binding molecule. Other specific exemplary multispecific formats that can be used in connection with the present invention include, without limitation, for example, scFv-based or diabody multispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (e.g., common light chains having knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Multispecific formats are included (e.g., regarding the overview of the formats described above, see Klein et al. 2012, mAbs 4:6, 1-11 and the references cited therein).

[0139] In the context of the multispecific antigen-binding molecules of the present invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid alterations (e.g., insertions, deletions or substitutions) compared to the naturally occurring version of the wild-type Fc domain. For example, the present invention provides multispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain having a modified binding interaction (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the multispecific antigen-binding molecule contains modifications in the C H 2 or C H 3 region, which modifications increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, the modifications include the modifications of 428L (e.g., M428L) and 434S (e.g., N434S), the modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), the modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), the modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), the modifications of 250Q and 428L (e.g., T250Q and M428L), and the modifications of 307 and / or 308 (e.g., 308F and / or 308P).

[0140] The present disclosure also includes multispecific antigen-binding molecules that contain a first C H 3 domain and a second Ig C H 3 domain, wherein the first and second Ig C HThe three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the multispecific antigen-binding molecule to protein A as compared to a bispecific antigen-binding molecule lacking the amino acid difference. In one embodiment, the first Ig C H The three domains bind to protein A, and the second Ig C H The three domains contain mutations that reduce or abolish protein A binding, such as, for example, the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C H The three may further include the Y96F modification (by IMGT; Y436F by EU). The second C H The three may further include the L105P modification (by IMGT; L455P by EU). See, for example, U.S. Patent No. 8,586,713. Further modifications that may be found in the second C H The three include the following: for IgG1 antigen-binding molecules, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT, D356E, L358M, N384S, K392N, V397M, and V422I in EU), for IgG2 antigen-binding molecules, N44S, K52N, and V82I (IMGT, N384S, K392N, and V422I in EU), and for IgG4 antigen-binding molecules, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU).

[0141] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be a part or all of the C H 2 region derived from human IgG1, human IgG2, or human IgG4 C H 2 sequence, and the C HIt may include some or all of the 3 arrays. The chimeric Fc domain may also include a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any of the antigen-binding molecules described herein are, from the N-terminus to the C-terminus, [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein is, from the N-terminus to the C-terminus, [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the present invention are described in U.S. Patent No. 9,359,437, the entire disclosure of which is incorporated herein by reference. Chimeric Fc domains having these general structural arrangements and variants thereof may have altered Fc receptor binding, and as a result, affect Fc effector functions.

[0142] Sequence variant The antigen-binding molecules and multispecific antigen-binding molecules of the present invention may include one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antigen-binding molecule sequence databases. Antigen-binding domains derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids within one or more frameworks and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antigen-binding molecule is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"), may be included in the antigen-binding molecules of the present invention. One of ordinary skill in the art can readily generate many antigen-binding molecules that include one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework residues and / or CDR residues within the V H domain and / or the V L domain are mutated back to the residues found in the original germline sequence from which the antigen-binding domain originally derived. In other embodiments, only certain residues, e.g., only the mutated residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain originally derived).

[0143] Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions. For example, while certain individual residues mutate to the corresponding residues of a particular germline sequence, certain other residues different from the original germline sequence are maintained or mutate to the corresponding residues of a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be readily tested for one or more desired properties such as improved binding specificity, increased binding affinity, improved or enhanced (where appropriate) biological properties of an antagonist or agonist, and decreased immunogenicity. Multispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed by the present disclosure.

[0144] pH-dependent binding The present invention includes anti-CD38×anti-4-1BB multispecific antigen-binding molecules having pH-dependent binding properties. For example, the anti-CD38 antigen-binding arm of the present invention may exhibit a decrease in binding to CD38 at acidic pH compared to neutral pH. Alternatively, the anti-CD38 antigen-binding arm of the present invention may exhibit an enhancement in binding to CD38 at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values of less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH" means a pH of from about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0145] In certain examples, "a decrease in binding to... at acidic pH compared to neutral pH" is represented by the ratio (or its inverse) of the K D value of the antigen-binding molecule that binds to the antigen at acidic pH to the K D value of the antigen-binding molecule that binds to the antigen at neutral pH. For example, the antigen-binding molecule is such that the antibody or its antigen-binding fragment has an acidic / neutral K DWhen presenting a ratio, for the purpose of the invention, it can be considered to present "reduction of binding to CD38 at acidic pH compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K D ratio for the antigen-binding molecule of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0, or more.

[0146] Antigen-binding molecules having pH-dependent binding properties can be obtained, for example, by screening a collection of antigen-binding molecules for a reduction (or enhancement of binding) in binding to a specific antigen at an acidic pH compared to neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can result in an antigen-binding molecule having pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antigen-binding molecule having reduced antigen binding at acidic pH compared to neutral pH can be obtained.

[0147] Antigen-binding molecules comprising an Fc variant According to certain embodiments of the present invention, for example, provided is an anti-CD38×anti-4-1BB bispecific antigen-binding molecule comprising an Fc domain containing one or more mutations that enhance or decrease antigen-binding molecule binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention relates to the C H 2 or C HAn antigen-binding molecule containing a mutation in 3 regions, where the mutation increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH in the range of about 5.5 to about 6.0). Such a mutation can result in an extended serum half-life of the antigen-binding molecule when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P).

[0148] For example, the present disclosure includes an anti-CD38×anti-4-1BB 1+2 multispecific antigen-binding molecule comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antigen-binding molecule variable domains disclosed herein are contemplated to be within the scope of the present invention.

[0149] Biological Characteristics of Antigen-Binding Molecules and Multispecific Antigen-Binding Molecules Provided herein are anti-CD38×anti-4-1BB multispecific antigen-binding molecules that bind to CD38 expressed on MOLP8 cells. As shown in Example 6, dose-dependent binding of CD38×4-1BB 1+2 (REGN7633, REGN7647, and REGN7650) and 1+1 (REGN7150) bispecific antibodies was observed in the presence of MOLP8 cells, and the maximum gMFI was 8.8×10 4 ~1.4×10 5 and the range was from 4.23×10 50 M to 9.27×10 -9 M. The EC -9 was in the range of 4.23×10

[0150] Provided herein are anti-CD38×anti-4-1BB multispecific antigen-binding molecules that bind to 4-1BB expressed on HEK293 cells engineered to express 4-1BB. Dose-dependent binding of CD38×4-1BB 1+2 (REGN7633, REGN7647, and REGN7650) and 1+1 (REGN7150) bispecific antibodies was observed in the presence of HEK293 / h4-1BB cells, and the maximum gMFI was 7.4x10 5 ~2.4x10 6 and the range was from 1.47×10 50 M to 9.97×10 -8 M. The EC -10 was in the range of 1.47×10

[0151] The CD38×4-1BB (1+1 and 1+2) multispecific antigen-binding molecule mimics the natural ligand of 4-1BB by crosslinking CD38+ target cells with 4-1BB receptor-positive T cells. By doing so, the construct provides "Signal 2" and enhances T cell activation in the presence of "Signal 1" provided by a tumor-associated antigen (TAA)×CD3 bispecific antibody or an allogeneic response provided by an APC. As shown in Example 7, in the presence of the target and "Signal 1" (provided by REGN1979), the multispecific antigen-binding molecule resulted in a higher maximum IL-2 response and greater potency than the corresponding isotype control in a T cell activation assay. As shown in Example 8, the multispecific antigen-binding molecule had a dose-dependent increase in IL-2 and IFNγ and higher potency even when the linker length between Fab2 and Fab3 was varied.

[0152] According to certain embodiments, the multispecific antigen-binding molecules provided herein activate the 4-1BB receptor and stimulate 4-1BB activity in the presence of target cells expressing CD38, as demonstrated in an engineered reporter assay. As shown in Example 9, constructs with different linker lengths were used to achieve 4-1BB activation.

[0153] In certain embodiments, the multispecific antigen-binding molecules provided herein cause a dose-dependent increase in the release of IL-2 and IFNγ. As shown in Example 10, in the presence of allogeneic NALM-6 cells engineered to express PD-L1 or NALM-6 cells, CD38×4-1BB 1+2 antibody treatment (REGN7633, REGN7647, and REGN7650) resulted in a dose-dependent increase in the release of IL-2 and IFNγ and greater potency.

[0154] According to certain embodiments, when administered in vivo in combination with a BCMA×CD3 bispecific antibody, treatment with the multispecific antigen-binding molecule results in more potent antitumor efficacy that is superior to either treatment alone. In Example 11, 4×10 6Immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were administered human multiple myeloma cells. After receiving the tumor cells, the mice were treated with 0.4 mg / kg of a CD3-binding control bispecific Ab or BCMA×CD3 (REGN5458) bsAb, in combination with 4 mg / kg of a 4-1BB-binding control bispecific Ab (1+2 format) or CD38×4-1BB (1+2 format, REGN9686). The treatment combinations were administered a further 2 times, on days 7 and 14, for a total of 3 administrations. Combination treatment with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb demonstrated more potent combination antitumor efficacy than either therapy alone.

[0155] Epitope mapping and related techniques The epitope on CD38 and / or 4-1BB to which the antigen-binding molecule of the present invention binds can consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD38 or 4-1BB protein. Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) of CD38 or 4-1BB.

[0156] As used herein, the term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antigen-binding molecule p.m., known as a paratope. A single antigen can have more than one epitope. Thus, different antigen-binding molecules can bind to different regions on an antigen and can have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain situations, an epitope can include a sugar, phosphoryl group, or sulfonyl group moiety on the antigen.

[0157] Using various techniques known to those skilled in the art, it is possible to determine whether the antigen-binding domain of an antigen-binding molecule "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays such as those described in Antigen binding molecules, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutagenesis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which the antigen-binding domain of an antigen-binding molecule interacts is hydrogen / deuterium exchange detected by mass spectrometry. As a general term, hydrogen / deuterium exchange involves labeling the target protein with deuterium and then binding the antigen-binding molecule to this deuterium-labeled protein. Next, the protein / antigen-binding molecule complex is transferred to water, and hydrogen-deuterium exchange is allowed to occur at all residues except those protected by the antigen-binding molecule (which remain deuterium-labeled). After dissociation of the antigen-binding molecule, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal the deuterium-labeled residues corresponding to the specific amino acids with which the antigen-binding molecule interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259, Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallographic analysis of the antigen / antigen-binding molecule complex can also be used for the purpose of epitope mapping.

[0158] As used herein, provided is an anti-CD38 antigen-binding arm A1 that binds to the same epitope as any of the specific exemplary antigen-binding arms described herein (e.g., an antigen-binding molecule comprising any of the amino acid sequences set forth in Table 1 herein). Similarly, the present invention also includes an anti-CD38 antigen-binding arm A1 that competes for binding to CD38 with any of the specific exemplary antigen-binding arms described herein (e.g., an antigen-binding molecule comprising any of the amino acid sequences set forth in Table 1 herein).

[0159] As used herein, provided is an anti-4-1BB antigen-binding arm A2 comprising a first antigen-binding domain (R1) and a second antigen-binding domain (R2), wherein either R1 or R2 binds to the same epitope as any of the specific exemplary antigen-binding domains described herein (e.g., an antigen-binding arm comprising any of the amino acid sequences set forth in Table 3 or Table 5 herein). Similarly, the present invention also includes an anti-4-1BB antigen-binding molecule that competes for binding to 4-1BB with any of the specific exemplary antigen-binding domains described herein (e.g., an antigen-binding arm comprising any of the amino acid sequences set forth in Table 3 or Table 5 herein).

[0160] Similarly, as used herein, provided is a multispecific antigen-binding molecule comprising a first antigen-binding arm (Fab1) that specifically binds to human CD38 and second antigen-binding arms (Fab2 and Fab3) that specifically bind to human 4-1BB, wherein the first antigen-binding domain competes for binding to CD38 with any of the specific exemplary CD38-specific antigen-binding arms described herein, and / or the second antigen-binding arm competes for binding to 4-1BB with any of the specific exemplary 4-1BB-specific antigen-binding Fabs described herein.

[0161] Whether a particular antigen-binding molecule (e.g., a multispecific 1+2 antigen-binding molecule) or an antigen-binding fragment thereof binds to the same epitope as the reference antigen-binding molecule of the present invention or competes for binding can be readily determined by using conventional methods known in the art. For example, to determine whether a test antigen-binding molecule binds to the same epitope on CD38 (or 4-1BB) as the reference multispecific antigen-binding molecule of the present invention, the reference multispecific molecule is first bound to the CD38 protein (or 4-1BB protein). Next, the ability of the test antigen-binding molecule to bind to the CD38 (or 4-1BB) molecule is evaluated. If the test antigen-binding molecule can bind to CD38 (or 4-1BB) after saturation binding with the reference multispecific antigen-binding molecule, it can be concluded that the test antigen-binding molecule binds to a different epitope of CD38 (or 4-1BB) than the reference multispecific antigen-binding molecule. On the other hand, if the test antigen-binding molecule cannot bind to the CD38 (or 4-1BB) molecule after saturation binding with the reference multispecific antigen-binding molecule, the test antigen-binding molecule may bind to the same epitope of CD38 (or 4-1BB) as the epitope bound by the reference multispecific antigen-binding molecule. Additional conventional experiments (e.g., peptide mutagenesis and binding analysis) can be performed to confirm whether the observed lack of binding of the test antigen-binding molecule is actually due to binding to the same epitope as the reference multispecific antigen-binding molecule or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antigen-binding molecule binding assay available in the art. According to a particular embodiment of the present invention, when measured in a competitive binding assay, for example, an excess of 1, 5, 10, 20, or 100 times of one antigen-binding protein inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or 99%, the two antigen-binding proteins bind to the same (or overlapping) epitope (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, if substantially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, the two antigen-binding proteins are considered to bind to the same epitope. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antigen-binding protein reduce or eliminate the binding of the other, the two antigen-binding proteins are considered to have "overlapping epitopes".

[0162] To determine whether an antigen-binding molecule or its antigen-binding domain competes with a reference antigen-binding molecule for binding, the above binding methodology is carried out in two directions as follows: In the first direction, the reference antigen-binding molecule is bound to the CD38 protein (or 4-1BB protein) under saturation conditions, and then the binding of the test antigen-binding molecule to the CD38 (or 4-1BB) molecule is evaluated. In the second direction, the test antigen-binding molecule is bound to the CD38 protein (or 4-1BB protein) under saturation conditions, and then the binding of the reference antigen-binding molecule to the CD38 (or 4-1BB) molecule is evaluated. In both directions, if only the first (saturated) antigen-binding molecule can bind to the CD38 (or 4-1BB) molecule, it is concluded that the test antigen-binding molecule and the reference antigen-binding molecule compete for binding to CD38 (or 4-1BB). As will be understood by those skilled in the art, antigen-binding molecules that compete for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antigen-binding molecule, but may sterically block the binding of the reference antigen-binding molecule by binding to overlapping or adjacent epitopes.

[0163] Preparation of Antigen-Binding Domains and Construction of Multispecific Molecules Antigen-binding domains specific for a particular antigen can be prepared by any antigen-binding molecule generation technique known in the art. Once obtained, different antigen-binding domains provided herein that are specific for two different antigens (e.g., CD38 and 4-1BB) can be appropriately arranged relative to each other and the multispecific antigen-binding molecules of the invention can be produced using conventional methods. (Consideration of exemplary multispecific antigen-binding molecule formats that can be used to construct the multispecific antigen-binding molecules of the invention is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized, or fully human antigen-binding molecules. Methods for making such antigen-binding molecules are well known in the art. For example, one or more of the heavy and / or light chains of the multispecific antigen-binding molecules of the invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antigen-binding molecule generation technology), high-affinity chimeric antigen-binding molecules for a particular antigen (e.g., CD38 or 4-1BB) having human variable regions and murine constant regions are first isolated. The antigen-binding molecules are characterized and selected for desirable features including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the multispecific antigen-binding molecules of the invention.

[0164] Genetically engineered animals may be used to produce human multispecific antigen-binding molecules. For example, genetically modified mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences may be used, which mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human antigen-binding molecules that include two different heavy chains that associate with the same light chain and include a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US2011 / 0195454). By "fully human" is meant an antigen-binding molecule or an antigen-binding fragment thereof or an immunoglobulin domain thereof that includes an amino acid sequence encoded by DNA derived from a human sequence over the entire length of each polypeptide of the antigen-binding molecule, or antigen-binding fragment thereof, or immunoglobulin domain thereof. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other cases, the fully human protein or protein sequence includes a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, for example, as compared to any wild-type human immunoglobulin region or domain.

[0165] Biological equivalents Disclosed herein are antigen-binding molecules having amino acid sequences that are different from those of the exemplary molecules disclosed herein but that retain the ability to bind to CD38 and / or 4-1BB. Such variant molecules may include one or more additions, deletions, or substitutions of amino acids as compared to the parent sequence, but exhibit a biological activity that is essentially equivalent to the biological activity of the described multispecific antigen-binding molecules.

[0166] An antigen-binding molecule that is biologically equivalent to any of the exemplary antigen-binding molecules described herein is a pharmaceutical equivalent or pharmaceutical alternative if, under similar experimental conditions, when administered at the same molar dose, either as a single dose or multiple doses, it shows no significant difference in the rate and extent of its absorption. For some antigen-binding proteins, the extent of their absorption is equivalent but their absorption rates are not. Moreover, such differences in absorption rates are intentional and reflected in the labeling, for example, they are not essential for achieving an effective in vivo drug concentration in chronic use and are considered not medically important for the particular pharmaceutical studied, and thus can be considered biological equivalents and can be considered equivalents or pharmaceutical alternatives.

[0167] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, and potency.

[0168] In one embodiment, two antigen-binding proteins are biologically equivalent if a patient can be switched one or more times between a reference product and a biological product, and there is no expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or a decrease in efficacy, compared to continuous therapy without such switching.

[0169] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by one or more common mechanisms to the known extent of such mechanisms for one or more conditions of use.

[0170] Biological equivalence can be demonstrated by in vivo and in vitro methods. Biological equivalence measurement methods include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antigen-binding molecule or its metabolite is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of an antigen-binding molecule (or its target) is measured as a function of time, and (d) appropriately controlled clinical trials that demonstrate the safety, efficacy, bioavailability, or biological equivalence of an antigen-binding protein.

[0171] Biologically equivalent variants of the exemplary multispecific antigen-binding molecules described herein can be constructed, for example, by causing various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges during regeneration. In other contexts, biologically equivalent antigen-binding proteins can include variants of the exemplary multispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation properties of the molecule, such as mutations that eliminate or remove glycosylation.

[0172] Species selectivity and species cross-reactivity According to certain embodiments of the invention, antigen-binding molecules are provided that bind to human 4-1BB but not to 4-1BB from other species. Antigen-binding molecules that bind to human CD38 but not to CD38 from other species are also provided. The invention also includes antigen-binding molecules that bind to human 4-1BB and CD38 from one or more non-human species, and / or antigen-binding molecules that bind to human 4-1BB and 4-1BB from one or more non-human species.

[0173] According to certain exemplary embodiments of the present invention, there are provided antigen-binding molecules that bind to human CD38 and / or human 4-1BB and that may or may not bind to one or more of CD38 and / or 4-1BB of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. For example, in certain exemplary embodiments disclosed herein, a multispecific antigen-binding molecule comprising a first antigen-binding arm that binds to human CD38 and cynomolgus monkey CD38 and a second antigen-binding arm comprising a first antigen-binding domain and a second antigen-binding domain, wherein the second antigen-binding arm specifically binds to human 4-1BB, or a multispecific antigen-binding molecule comprising a second antigen-binding arm comprising first and second antigen-binding domains that bind to human 4-1BB and cynomolgus monkey 4-1BB and a first antigen-binding arm that specifically binds to human CD38, is provided.

[0174] Therapeutic formulations and administration The present invention provides a pharmaceutical composition comprising a multispecific antigen-binding molecule disclosed herein. The pharmaceutical composition of the present invention is formulated with a suitable carrier, excipient, and other agents that provide improvements in transport, delivery, tolerability, and the like. Numerous suitable formulations can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids containing vesicles (cationic or anionic) (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0175] The dosage of the multispecific antigen-binding molecule administered to a patient can vary depending on the patient's age and physical build, the target disease, condition, route of administration, etc. Preferred dosages are typically calculated according to body weight or body surface area. When the multispecific antigen-binding molecule of the present invention is used for therapeutic purposes in adult patients, the multispecific antigen-binding molecule of the present invention is usually administered intravenously at a single dose of about 0.01 to about 20 mg per kg of body weight, more preferably about 0.02 to about 7 mg, about 0.03 to about 5 mg, or about 0.05 to about 3 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. An effective dosage and schedule for administering the multispecific antigen-binding molecule can be determined empirically. For example, the patient's progress can be monitored by regular evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0176] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. For example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-dependent endocytosis, etc. (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). The introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by injection or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. The administration can be systemic or local.

[0177] The pharmaceutical composition of the present invention can be delivered subcutaneously, intramuscularly, or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices can be readily applied in the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are sold pre-filled with the pharmaceutical composition held within a reservoir inside the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0178] A number of reusable pen-type and auto-injector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. By way of example only, and not limitation, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany) may be mentioned. Examples of disposable pens and / or auto-injector delivery devices having use in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ Pen (Abbott Labs, Abbott Park IL).

[0179] In certain situations, the pharmaceutical composition can be delivered by a controlled release system. In one embodiment, a pump may be used (see Langer, Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 above). In another embodiment, multimeric materials can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in the vicinity of the target of the composition. Thus, only a fraction of the systemic dose is required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0180] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by known methods. For example, an injectable preparation can be prepared by dissolving, suspending, or emulsifying the antigen-binding molecule or a salt thereof described above in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of the injectable aqueous medium include physiological saline, isotonic solutions containing glucose, and other adjuvants, which can be used in combination with appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of the oily medium include sesame oil, soybean oil, etc., which can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol. The injection solution thus prepared is preferably filled into an appropriate ampoule.

[0181] Advantageously, the pharmaceutical composition for oral or parenteral use described above is formulated into dosage forms in unit dosages suitable for adapting the dosage of the active ingredient. Such dosage forms in unit dosages include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of the aforementioned antigen-binding molecule contained is generally about 1 to about 1000 mg per dosage form in unit dosage, and particularly in the form of injection, the above antigen-binding molecule is preferably contained in an amount of about 1 to about 100 mg, and for other dosage forms, it is preferably contained in an amount of about 10 to about 250 mg. In some embodiments, the unit dosage can be the same as about 750 mg, 800 mg, 900 mg, or 1000 mg.

[0182] Therapeutic use of antigen-binding molecules The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition of a multispecific antigen-binding molecule that specifically binds to CD38 and 4-1BB. The therapeutic composition can include any of the multispecific antigen-binding molecules disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "a subject in need thereof" means a human or non-human animal (e.g., a subject expressing a tumor or suffering from any of the cancers described below) showing symptoms or signs of one or more cancers, or otherwise a subject who would benefit from inhibition or reduction of CD38 activity or depletion of CD38+ cells (e.g., multiple myeloma cells).

[0183] The multispecific antigen-binding molecules (and therapeutic compositions comprising the same) of the present invention are useful, in particular, for treating any disease or disorder in which stimulation, activation, and / or targeting of an immune response is beneficial. In particular, the anti-CD38×anti-4-1BB 1+2 multispecific antigen-binding molecules of the present invention can be used for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by the expression or activity of CD38 and / or BCMA, or the proliferation of CD38+ and / or BCMA+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved includes killing, in the presence of effector cells, cells expressing, for example, CD38, by, for example, CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. CD38-expressing cells that can be inhibited or killed using the multispecific antigen-binding molecules of the present invention include, for example, multiple myeloma cells.

[0184] The multispecific antigen-binding molecules of the present disclosure can be used for treating diseases or disorders associated with CD38 expression, including, for example, multiple myeloma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer characterized in part by having CD38+ cells.

[0185] According to certain embodiments, the anti-CD38×anti-4-1BB 1+2 antigen-binding molecule is useful for inhibiting the proliferation of plasmacytoma tumors in a subject. In some aspects, the plasmacytoma tumor is multiple myeloma.

[0186] The multispecific antigen-binding molecules of the present disclosure can be used for inhibiting the proliferation of tumors in a subject. The tumor is selected from the group consisting of multiple myeloma, lymphoma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer characterized in part by having CD38+ cells.

[0187] According to certain embodiments, the anti-CD38×anti-4-1BB 1+2 antigen-binding molecule is useful, for example, in the treatment of tumor cells expressing BCMA or CD20. The antigen-binding molecules provided herein are useful, for example, in the treatment of cancers including multiple myeloma or other B-cell or plasma cell cancers, such as Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma, in diseases or disorders associated with BCMA expression. According to certain embodiments of the invention, the anti-CD38×anti-4-1BB antigen-binding molecule is useful in the treatment of patients suffering from multiple myeloma. According to other related embodiments of the invention, provided herein is a method comprising administering the anti-CD38×anti-4-1BB multispecific antigen-binding molecule provided herein in combination with an anti-BCMA antigen-binding molecule, or an anti-BCMA×anti-CD3 multispecific antigen-binding molecule, or an anti-CD20×anti-CD3 multispecific antigen-binding molecule, or an anti-CD28×anti-4-1BB multispecific antigen-binding molecule disclosed herein to a patient suffering from cancer cells expressing BCMA or CD20. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to confirm whether a patient has multiple myeloma or another B-cell lineage cancer.

[0188] In some embodiments, the anti-CD38×anti-4-1BB multispecific antigen-binding molecules provided herein can be administered in combination with a second therapeutic agent or treatment regimen comprising a chemotherapeutic agent, a DNA alkylating agent, an immunomodulatory agent, a proteasome inhibitor, a histone deacetylase inhibitor, radiation therapy, stem cell transplantation, different bispecific antibodies that interact with different tumor cell surface antigens and T-cell or immune cell antigens, antibody-drug conjugates, bispecific antibodies conjugated to anti-tumor agents, PD-1 inhibitors (anti-PD-1 antibodies, such as, for example, semiprimab), PD-L1 inhibitors, CTLA-4 checkpoint inhibitors, or combinations thereof.

[0189] The present disclosure also includes a method for treating residual cancer in a subject. As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with anti-cancer therapy.

[0190] According to certain aspects, the invention provides a method for treating a disease or disorder associated with CD38 expression (e.g., multiple myeloma), the method comprising administering to a subject one or more of the anti-CD38×anti-4-1BB 1+2 antigen-binding molecular antibodies described herein after the subject has been determined to have multiple myeloma. For example, the present disclosure includes a method for treating multiple myeloma comprising administering an anti-CD38×anti-4-1BB multispecific antigen-binding molecule to a patient one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, or four weeks, two months, four months, six months, eight months, one year, or more after the subject has received other immunotherapy or chemotherapy.

[0191] Combination Therapies and Formulations The present disclosure provides a method comprising administering a pharmaceutical composition comprising any of the exemplary antigen-binding molecules and multispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that can be therapeutically combined with or administered in combination with the antigen-binding molecules of the present invention include, for example, anti-tumor agents (e.g., melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), chemotherapeutic agents, or others known to be effective in the treatment of the subject's plasma cell tumors). In some embodiments, the second therapeutic agent comprises a steroid. In some embodiments, the second therapeutic agent comprises a targeted therapy comprising thalidomide, lenalidomide, and bortezomib, which are therapies approved for treating newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents effective in the treatment of relapsed multiple myeloma.

[0192] In some embodiments, the second therapeutic agent is an anti-BCMA×CD3 bispecific antigen-binding molecule. Exemplary anti-BCMA×CD3 bispecific antigen-binding molecules are disclosed in U.S. 2020 / 0024356, which is incorporated herein by reference. An exemplary anti-BCMAxCD3 bispecific antigen-binding molecule is REGN5458, as disclosed in U.S. 2020 / 0024356. In some embodiments, the second therapeutic agent is an anti-CD20×CD3 bispecific antigen-binding molecule. Exemplary anti-CD20×CD3 bispecific binding molecules are disclosed in U.S. Patent No. 9,657,102, which is incorporated herein by reference. An exemplary anti-CD20×CD3 bispecific antigen-binding molecule is REGN1979 (U.S. Patent No. 9,657,102).

[0193] In certain embodiments, the second therapeutic agent is a regimen including radiation therapy or stem cell transplantation. In certain embodiments, the second therapeutic agent may be an immunomodulatory agent. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may be a monoclonal antibody, an antibody-drug conjugate, a multispecific / dbispecific / monospecific antigen-binding molecule conjugated to an anti-tumor agent, a checkpoint inhibitor, an oncolytic virus, a cancer vaccine, CAR-T cells, or a combination thereof. Other agents that may be beneficially administered in combination with the antigen-binding molecule of the present invention include small molecule cytokine inhibitors, and cytokine inhibitors including antigen-binding molecules that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising the anti-CD38×anti-4-1BB multispecific antigen-binding molecule disclosed herein) may also include monoclonal antigen-binding molecules other than those described herein that may interact with different antigens on the plasma cell surface; multispecific antigen-binding molecules in which one arm binds to an antigen on the tumor cell surface and the other arm binds to an antigen on a T cell; antibody-drug conjugates; bispecific antibodies conjugated to anti-tumor agents; checkpoint inhibitors, e.g., those targeting PD-1 or CTLA-4; or a combination thereof, and may be administered as part of a treatment regimen including one or more combinations selected therefrom. In certain embodiments, the checkpoint inhibitor may be selected from PD-1 inhibitors such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab (REGN2810, Libtayo).In certain embodiments, the checkpoint inhibitor may be selected from PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from CTLA-4 inhibitors such as ipilimumab (Yervoy). Other combinations that can be used in conjunction with the antigen-binding molecules of the present invention are described above.

[0194] The present disclosure also includes therapeutic combinations comprising any of the antigen-binding molecules referred to herein and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antigen-binding molecule fragment (e.g., Fab fragment; F(ab’)2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules such as diabodies, triabodies, tetra-bodies, minibodies, and minimal recognition units). The antigen-binding molecules of the present invention may also be administered and / or co-formulated in combination with antiviral agents, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen-binding molecules of the present invention may also be administered as part of a treatment regimen that includes radiotherapy and / or conventional chemotherapy.

[0195] The additional therapeutic active ingredient may be administered immediately before, simultaneously with, or immediately after administration of the antigen-binding molecules of the present invention; (for the purposes of the present disclosure, such an administration regimen is considered an administration of the antigen-binding molecules "in combination with" the additional therapeutic active ingredient).

[0196] The present invention includes pharmaceutical compositions in which the antigen-binding molecules of the present invention are co-formulated with one or more of the additional therapeutic active ingredients described elsewhere herein.

[0197] Dosing regimen According to certain embodiments of the invention, multiple doses of an antigen-binding molecule (e.g., a bispecific antigen-binding molecule that specifically binds an anti-CD38 antibody and 4-1BB) may be administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering multiple doses of the bispecific antigen-binding molecule of the invention to a subject. As used herein, "administered sequentially" means that each dose of the antigen-binding molecule is administered to the subject at different times, e.g., on different days at a predetermined interval (e.g., time, day, week, or month). The invention includes methods comprising sequentially administering a single initial dose of the antigen-binding molecule, then one or more secondary doses of the antigen-binding molecule, and optionally, thereafter, one or more tertiary doses of the antigen-binding molecule, to a patient.

[0198] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the antigen-binding molecule of the invention. Thus, an "initial dose" is a dose administered at the start of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of antigen-binding molecule, but generally may differ from each other with respect to dosing frequency. However, in certain embodiments, the amounts of antigen-binding molecule contained in the initial dose, secondary dose, and / or tertiary dose differ from each other (e.g., adjusted as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of a treatment regimen, and subsequent doses are administered on a less frequent basis (e.g., a "maintenance dose").

[0199] In an exemplary embodiment of the present invention, each secondary dose and / or tertiary dose is administered 1 to 26 weeks (e.g., 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, 13.5 weeks, 14 weeks, 14.5 weeks, 15 weeks, 15.5 weeks, 16 weeks, 16.5 weeks, 17 weeks, 17.5 weeks, 18 weeks, 18.5 weeks, 19 weeks, 19.5 weeks, 20 weeks, 20.5 weeks, 21 weeks, 21.5 weeks, 22 weeks, 22.5 weeks, 23 weeks, 23.5 weeks, 24 weeks, 24.5 weeks, 25 weeks, 25.5 weeks, 26 weeks, 26.5 weeks, or more) after the immediately preceding dose. As used herein, the phrase "immediately preceding administration" means the order of multiple administrations, and the administration of the very next dose in that order means the administration of the antigen-binding molecule that is administered to the patient without the intervening administrations.

[0200] The method according to this aspect of the invention may comprise administering to a patient any number of secondary doses and / or tertiary doses of an antigen-binding molecule (e.g., a multispecific 1+2 antigen-binding molecule that specifically binds to CD38 and 4-1BB). For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0201] In embodiments that include multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the previous administration. Similarly, in embodiments that include multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the previous administration. Alternatively, the frequency at which the secondary dose and / or tertiary dose is administered to the patient may change during the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment according to the needs of the individual patient after clinical examination.

[0202] Diagnostic uses of antigen-binding molecules The anti-CD38 antigen-binding molecules of the present disclosure can also be used, for example, for diagnostic purposes to detect and / or measure CD38 or CD38-expressing cells in a sample. For example, an anti-CD38 antigen-binding molecule, or a fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression of CD38 (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for CD38 can include, for example, contacting a sample obtained from a patient with an anti-CD38 antigen-binding molecule disclosed herein, where the anti-CD38 antigen-binding molecule is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CD38 antigen-binding molecule can be used for diagnostic purposes in combination with a secondary antigen-binding molecule that is itself detectably labeled. Detectable labels or reporter molecules can be, for example 3 H, 14 C, 32 P, 35 S, or 125 radioisotopes such as I, fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-CD38 antigen-binding molecules herein is for the non-invasive identification and tracking of tumor cells in a subject, for 89 such as Zr-desferrioxamine labeling, 89Comprising a Zr-labeled antibody (e.g., for positron emission tomography (PET) imaging). (See, e.g., Tavare, R. et al. Cancer Res. 2016 Jan 1;76(1):73-82; and Azad, B. B. et al. Oncotarget. 2016 Mar 15;7(11):12344-58.) Specific exemplary assays that can be used to detect or measure CD38 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0203] Samples that can be used in the CD38 diagnostic assay according to the present invention include any tissue or body fluid sample obtainable from a patient that contains a detectable amount of CD38 protein or a fragment thereof, under normal or pathological conditions. Generally, the CD38 level in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal CD38 levels or activity) is measured to first establish a baseline or standard CD38 level. This CD38 baseline level can then be compared to the CD38 level measured in a sample obtained from an individual suspected of having a CD38-related disease (e.g., a tumor containing CD38-expressing cells) or condition.

[0204] Device The present invention also provides a container (e.g., a vial or chromatography column) or an injection device (e.g., a syringe, a pre-filled syringe, or an autoinjector) comprising the bispecific antigen-binding molecule described herein (e.g., a pharmaceutical formulation thereof). The container or injection device can be packaged within a kit.

[0205] An injection device is a device for introducing a substance into the body of a subject (e.g., a human) via a parenteral route, such as intraocular, intravitreal, intramuscular, subcutaneous, or intravenous. For example, the injection device may include, for example, a cylinder or barrel for holding a fluid to be injected (e.g., containing an antigen-binding molecule, or a fragment or pharmaceutical formulation thereof), a syringe (e.g., a pre-filled syringe such as an auto-injector) including a needle for penetrating the skin, blood vessel, or other tissue for injection of the fluid, and a plunger for pushing the liquid from the cylinder through the needle hole into the subject's body.

[0206] The pharmaceutical compositions provided herein can be delivered subcutaneously or intravenously with standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices can be readily applied in the delivery of the pharmaceutical compositions of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are sold pre-filled with the pharmaceutical composition held within a reservoir inside the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0207] A number of reusable pen-type and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. By way of example only, and not limitation, there may be mentioned AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices having use in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, by way of example only, and not limitation, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.).

[0208] Provided herein are methods for administering the multispecific antigen-binding molecules of the present disclosure, including, for example, introducing the molecule into a subject's body, such as by injection, using an injection device.

[0209] Expression method In the present specification, a recombinant method for producing a multispecific antigen-binding molecule of the present invention, or an immunoglobulin chain thereof, comprising: (i) introducing into a host cell one or more polynucleotides encoding a light and / or heavy immunoglobulin chain of such a multispecific antigen-binding molecule, wherein the one or more polynucleotides are comprised within one or more vectors and / or integrated into the host cell chromosome and / or operably linked to a promoter; (ii) culturing the host cell (e.g., mammalian, fungal, Chinese hamster ovary (CHO), Pichia or Pichia pastoris) under conditions favorable for polynucleotide expression; (iii) optionally, isolating the multispecific antigen-binding molecule, or immunoglobulin chain, from the host cell and / or the medium in which the host cell is grown; is provided. The products of such methods also form part of the present disclosure, together with their pharmaceutical compositions.

[0210] In some embodiments, step (i) comprises cloning the individual A1 heavy chain, A2 heavy chain, and universal light chain into separate expression vectors. For example, the CD38-binding heavy chain variable region (HCVR) (VH-1) can be cloned into a heavy chain expression plasmid (CH1-1_CH2_CH3). The 4-1BB-binding heavy chain variable region (HCVR) (VH-3) fused to a CH1 domain (CH1-3) having linkers of various lengths (linkers), followed by another 4-1BB-binding heavy chain variable region (HCVR) (VH-2) containing the mutations H435R and Y436F, can be cloned into a heavy chain expression plasmid (CH1-2_CH2_CH3(*)) (EU numbering) (US8,586,713). Together with a plasmid containing the universal light chain, the expression plasmids can be transfected into a host cell such as CHO cells. The host cell can then produce the multispecific antigen-binding molecule described herein.

[0211] In embodiments, methods for making multispecific antigen-binding molecules include, for example, methods of purifying the molecules by column chromatography, precipitation, and / or filtration. The products of such methods also form part of the present disclosure together with their pharmaceutical compositions.

[0212] Host cells containing a polynucleotide encoding a multispecific antigen-binding molecule of the present disclosure and / or an immunoglobulin chain of such a molecule (e.g., in a vector) are also part of the invention. Examples of host cells include mammalian cells such as Chinese hamster ovary (CHO) cells and fungal cells such as Pichia cells (e.g., P. pastoris).

Examples

[0213] The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0214] The antigen-binding molecules used as controls for Examples 6-10 include the following. · A CD20×CD3 bispecific antibody (REGN1979) (US10,550,193) used as Signal 1 together with the corresponding isotype control (REGN7540). Both REGN1979 and REGN7540 contain an hIgG4 P-PVA isotype (US9,359,437). · A second CD20×CD3 bispecific antibody (REGN2281) used as Signal 1. REGN2281 contains the same variable regions as REGN1979, which contains an hIgG4 isotype with an S108P substitution (hIgG4 P ) · BCMA×CD3 (REGN5458) (US11,384,153), which is also used as Signal 1. · Comparator 1: 4-1BB bivalent agonist (REGN4249) (US7,288,638) containing the variable region of the antibody "10C7". · Anti-PD-1 antibody, semaprimab (REGN2810, LIBTAYO®) (US9,987,500). · Isotype control for 4-1BB bivalent agonist (hIgG4P) (REGN1945), and REGN1945 was also used as the isotype control for semaprimab. · Bispecific 4-1BB×non-TAA isotype control (REGN13168).

[0215] Proper T cell activation requires two signals, "Signal 1" and "Signal 2". "Signal 1" is induced by binding the T cell receptor (TCR) on the T cell to the peptide-bound major histocompatibility complex (MHC) molecule on the antigen-presenting cell (APC). "Signal 2" is provided by binding a costimulatory receptor to the T cell. One such costimulatory receptor is the 4-1BB receptor, which is an inducible type I membrane protein and a member of the tumor necrosis factor receptor (TNFR) superfamily. The expression of the 4-1BB receptor is induced on the surface of T cells after antigen or mitogen-induced activation. Activation of 4-1BB occurs through binding to 4-1BBL present on the APC. Thus, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0216] Description of cell lines The characteristics of the cell lines used in some of the following examples are provided below.

[0217] NALM6 (ACL14036) The NALM6 clone is an acute lymphoblastic leukemia (ALL) cell line [NALM6 clone G5 (ATCC, #CRL-3273)] isolated from a 19-year-old male. NALM6 cells are maintained in RPMI 1640 + 10% FBS + P / S / G at 37°C in 5% CO2. Staining for expression confirmation

[0218] NALM6 / hPD-L1 (ACL16389) NALM6 cells genetically engineered to stably express human PD-L1 (amino acids M1 - T290 of accession number NP_054862.1). The cells are maintained in RPMI 1640 + 10% FBS + P / S / G + 1 μg / ml Puro at 37°C in 5% CO2. Viral transfection and transduction Staining for expression confirmation

[0219] HEK293 parent (ACL2397) A human embryonic kidney cell line [HEK-293 (ATCC, #CRL-1573)] isolated from a fetus. It is referred to as the HEK293 parental cell line. HEK293 cells are maintained in DMEM + 10% FBS + P / S / G.

[0220] HEK293 / hCD20 (ACL14268) A cell line generated by stably transducing HEK293 (HZ) cells with human CD20 (Uniprot accession number: P11836, amino acids M1 - P297). The engineered strain is maintained in 5% CO2 in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 100 μg / mL hygromycin. Viral transfection / production Cell line transduction Staining for expression confirmation

[0221] HEK293 / hCD20 / hCD38 (ACL14270) A cell line prepared by stably transducing HEK293(HZ) cells with human CD20 (Uniprot accession number: P11836, amino acids M1 - P297) and human CD38 (Uniprot accession number P28907, amino acids M1 - I300). The engineered strain is maintained in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 500 μg / mL G418 + 100 μg / mL hygromycin under 5% CO2. Viral transfection / production Cell line transduction Staining for expression confirmation

[0222] HEK293 / h4-1BB(ACL7888) A cell line prepared by stably transducing HEK293(HZ) cells with human TNFRSF9 (4-1BB) (accession number: NM_001561, amino acids M1 - L255). The engineered strain is maintained in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 500 μg / mL G418 under 5% CO2. Viral transfection / production Cell line transduction Staining for expression confirmation

[0223] HEK293 / NFkB-Luc(ACL5021) A cell line prepared by transfecting a firefly luciferase-IRES-GFP gene driven by five copies of the NF-κB response element located upstream of the minimal TATA promoter. Selection of positive cell lines was performed by flow cytometry, and single clone D9 was isolated. Plasmid generation Staining for expression confirmation

[0224] HEK293 / NFkB-Luc / h4-1BB(ACL11090) A cell line generated by stably transducing HEK293 / NFkB-Luc cells with human TNFRSF9 (4-1BB) (Accession number: NM_001561, amino acids M1-L255). The engineered strain is maintained in 5% CO2 in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 500 μg / mL G418. Viral transfection / production Cell line transduction Staining for confirmation of expression

[0225] MOLP8 (ACL14198) A human multiple myeloma cell line established from the peripheral blood of a 52-year-old Japanese male with multiple myeloma in 2002. Obtained with DSMZ number: ACC569. Staining for confirmation of expression

[0226] OPM2 (ACL14164) Established from the peripheral blood of a 56-year-old female with multiple myeloma in the leukemic phase in 1982. Obtained with DSMZ number: ACC50. Staining for confirmation of expression

[0227] Example 1. Generation and screening of anti-CD38 antibody and anti-4-1BB antibody The anti-human CD38 antibody was obtained by immunizing a genetically engineered mouse containing DNA encoding the heavy and kappa light chain variable regions of human immunoglobulin with cells expressing CD38 or DNA encoding CD38. The immune response of the antibody was monitored by a CD38-specific immunoassay. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines producing CD38-specific antibodies. Using this technique, several anti-CD38 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained. Furthermore, as described in US2007 / 0280945A1, several fully human anti-CD38 antibodies were generated by directly isolating antigen-positive B cells without fusion with myeloma cells.

[0228] Similarly, the anti-4-1BB antibody was obtained by immunizing a genetically engineered mouse containing DNA encoding the human immunoglobulin heavy and kappa light chain variable regions with cells expressing 4-1BB or DNA encoding 4-1BB. The immune response of the antibody was monitored by a 4-1BB-specific immunoassay. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines producing 4-1BB-specific antibodies. Using this technique, several anti-4-1BB chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained. Furthermore, as described in US2007 / 0280945A1, several fully human anti-4-1BB antibodies were generated by directly isolating antigen-positive B cells without fusion with myeloma cells.

[0229] Antibodies were characterized and selected for desirable features including affinity, selectivity, etc. If necessary, the murine constant regions were replaced with the desired human constant regions, such as wild-type or modified IgG1 or IgG4 constant regions, to generate fully human anti-CD38 antigen-binding molecules or fully human anti-4-1BB antigen-binding molecules. The selected constant regions can vary according to the specific use, while the high-affinity antigen binding and target specificity characteristics reside in the variable regions.

[0230] Example 2. Amino Acid and Nucleic Acid Sequences of the Heavy and Light Chain Variable Regions of the Anti-CD38 Binding Arm Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-CD38 antigen-binding arms of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0231] (Table 1) Anti-CD38 Amino Acid Sequence Identifiers TIFF2025519057000004.tif47170

[0232] (Table 2) Anti-CD38 Nucleic Acid Sequence Identifiers TIFF2025519057000005.tif46170

[0233] The anti-CD38 binding arm can include the variable domains and CDR sequences described in Table 1, and a human Fc domain such as isotype IgG4, IgG1, etc. For a particular use or experiment, the Fc domain may be a murine Fc domain. As will be understood by those skilled in the art, an antigen-binding arm having a particular Fc isotype can be converted to an antigen-binding arm having a different Fc isotype (e.g., an antigen-binding molecule having a murine IgG4 Fc can be converted to an antigen-binding molecule having a human IgG1, etc.), but in any case, the variable domain (including the CDRs) - which is indicated by the numerical identifiers shown in Table 1 - remains the same and the binding characteristics are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0234] Example 3: Heavy and light chain variable region amino acids and nucleic acid sequences of the anti-4-1BB binding arm Table 3 describes the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-4-1BB binding arm of the bispecific antibody. The corresponding nucleic acid sequence identifiers are described in Table 4.

[0235] (Table 3) Anti-4-1bb amino acid sequence identifier TIFF2025519057000006.tif61170

[0236] (Table 4) Anti-4-1bb nucleic acid sequence identifier TIFF2025519057000007.tif61170

[0237] The anti-4-1BB antigen-binding arm may include the variable domains and CDR sequences described in Table 3, and a human Fc domain such as isotype IgG4, IgG1. For certain applications or experiments, the Fc domain may be a mouse Fc domain. As will be understood by those skilled in the art, an antigen-binding molecule having a particular Fc isotype can be converted to an antigen-binding molecule having a different Fc isotype (e.g., an antigen-binding molecule having a mouse IgG4 Fc can be converted to an antigen-binding molecule having a human IgG1, etc.), but in any case, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Table 3 - remain the same and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

[0238] Example 4: Generation of a multispecific antigen-binding molecule that binds to CD38 and 4-1BB Multispecific antigen-binding molecules that bind to CD38 and 4-1BB are also referred to herein as "anti-CD38×anti-4-1BB 1+2", or "anti-CD38×anti-4-1BB multispecific molecule", or "anti-CD38 / anti-4-1BB 1+2", or "CD38×4-1BB multispecific molecule". The anti-CD38 portion of the anti-CD38×anti-4-1BB multispecific molecule is useful for targeting tumor cells expressing CD38, and the anti-4-1BB portion of the multispecific molecule is useful for activating T cells.

[0239] Various 4-1BB Fabs (Fab3, heavy chain variable region (HCVR) having a heavy chain CH1 domain and a light chain) that bind to 4-1BB epitope 1 (ep1) or epitope 2 (ep2) were fused to the N-terminus of the 4-1BB VH domain from an existing IgG-like bispecific molecule that targets both 4-1BB and CD38.

[0240] (i) Various 4-1BB heavy chain variable regions (HCVRs), (ii) a heavy chain CH1 domain, followed by linkers of various lengths for connecting the heavy chain CH1 domain to a second 4-1BB heavy chain variable region (HCVR), (iii) a CD38 heavy chain variable region (HCVR), were synthesized by Integrated DNA Technologies, Inc. (San Diego, California).

[0241] Mammalian expression vectors for individual heavy chains were prepared by InFusion Cloning (Takara Bio USA Inc.) according to the protocol provided by Takara Bio USA Inc. The CD38 heavy chain variable region (HCVR) (VH-1) was cloned into a heavy chain expression plasmid (CH1-1_CH2_CH3). A 4-1BB heavy chain variable region (HCVR) (VH-3) fused to a CH1 domain (CH1-3) having linkers of various lengths (linker), followed by another 4-1BB heavy chain variable region (HCVR) (VH-2), was cloned into a heavy chain expression plasmid (CH1-2_CH2_CH3(*)) containing star mutations (H435R, Y436F, EU numbering).

[0242] The recombinant CD38×4-1BB×4-1BB 1+2 N-Fab MBM was produced in CHO cells after transfection with three expression plasmids: (i) the CD38 heavy chain plasmid, (ii) the 4-1BB+4-1BB heavy chain star plasmid, and (iii) the plasmid containing the universal light chain. The stably transfected CHO cell pool was isolated after selection with 400 μg / ml hygromycin for 12 days. The CHO cell pool was used to produce CD38×4-1BB×4-1BB 1+2 N-Fab MBM, which was then purified as described above (Sci Rep. 2015 Dec 11;5:17943).

[0243] An overview of the components of the selected multispecific antigen-binding molecules prepared according to this example is described in Table 5. The respective nucleic acid sequence identifiers of the component parts are provided in Table 6. Tables 7 and 8 provide the component parts, polypeptide sequences, and nucleic acid sequences of the control bispecific antigen-binding molecules, respectively.

[0244] (Table 5) TIFF2025519057000008.tif22699

[0245] (Table 6) TIFF2025519057000009.tif225111

[0246] (Table 7) TIFF2025519057000010.tif22752

[0247] (Table 8) TIFF2025519057000011.tif22952

[0248] Example 5: Screening of Multispecific Antigen-Binding Molecules Next, constructs that activate the 4-1BB signaling pathway were identified by screening.

[0249] Based on cell binding to human 4-1BB, utilization of the common light chain, and diversity of the CDR3 sequences, 34 4-1BB-binding antibodies were selected for inclusion in a 1+2 screening. For the screening, the 4-1BB variable domains were arrayed in two different 1+2 formats, split 4-1BB or tandem 4-1BB. See Figure 2B.

[0250] For the split format, the standard bivalent architecture of 4-1BB was maintained and tumor targeting was achieved by adding the variable domain and CH1 from the CD38 tumor targeting arm (26812) at the N-terminus on one side of the 4-1BB molecule separated by a G4S×3 spacer domain. These constructs were expressed as Knob in Hole (KiH) bispecifics, and thus only one arm of the expressed molecule contained the CD38 targeting motif.

[0251] For the tandem FAB format, the tandem 4-1BB sequences were joined in the same manner as the CD38×4-1BB described above, and tumor targeting was achieved by expression as a bispecific molecule with anti-CD38 present on the opposing arm. The 34 4-1BB variable domains were assembled in all possible combinations of tandem FABs that generated 1225 combinations together with one irrelevant control sequence (anti-BetV1). This included 34 molecules where the two tandem 4-1BB variable sequences were identical.

[0252] For screening, the 1+2 construct was transiently expressed in CHO cells and the supernatant containing bispecificity was harvested 4 days later. The supernatant was added to a co-culture of cells containing HEK cells overexpressing hCD38 and Jurkat cells carrying an NFkB luciferase reporter and overexpressing human 4-1BB. Binding and activation of 4-1BB by the test sample was compared to co-culture of Jurkat reporter cells with HEK cells (100%) expressing the 4-1BB ligand. Screening results from split FAB showed little or no activity in the Jurkat reporter assay, with a maximum activity of 3.7% observed. In contrast, robust activation was observed in the stacked FAB format, with over 25% of the samples tested resulting in over 30% activity. Activity was observed when the 4-1BB variable domains within the stacked FAB were of the same or unique sequence, with activity as high as approximately 66% observed. Introduction of an irrelevant control FAB anywhere within the stacked FAB resulted in loss of activity. See Figure 2A.

[0253] Example 6: Characterization of CD38×4-1BB Bispecific Antigen Binding Molecule Binding Using Flow Cytometry Binding of the CD38 arm was tested using MOLP8 cells that endogenously express CD38. Binding of the 4-1BB arm was evaluated using HEK293 cells engineered to express h4-1BB (HEK293 / h4-1BB). HEK293 cells were used to determine non-targeted cell binding as neither CD38 nor 4-1BB is expressed.

[0254] The ability of the multispecific antigen-binding molecule to bind to cells was evaluated using flow cytometry. Cell lines were selected to determine the ability of both the anti-CD38 and anti-4-1BB arms to bind to their targets. In the first experiment, test antibodies were incubated with MOLP8 (which endogenously expresses hCD38) and HEK293 (which does not express hCD38) cells. In the second experiment, test antibodies were incubated with HEK293 / h4-1BB (engineered to express h4-1BB) and HEK293 (which does not express h4-1BB) cells. Binding was detected by measuring fluorescence on a flow cytometer using a labeled secondary antibody.

[0255] Multispecific antigen-binding molecule: The multispecific antigen-binding molecules and controls tested in these two experiments are as shown in Table 9.

[0256] (Table 9) CD38×4-1BB binding molecules and controls TIFF2025519057000012.tif61128

[0257] Experiment 1 - CD38 binding: HEK293 cells were detached with trypsin, washed, and resuspended in staining buffer (2% FBS in PBS). MOLP8 cells were washed and resuspended in staining buffer. The cells were added to the wells of a 96-well V-bottom plate (3×10 5(Cells / Well). In staining buffer, the test antibody was diluted for dose titration at 1:4, 9 points to a final concentration in the range of 610 fM to 10 nM and added to the cells (including the "no antibody" control as the 9th point (153 fM) and labeled as "secondary only"). The cells and antibody were incubated on ice for 30 minutes and then washed with staining buffer. The cells were resuspended in 2 μg / ml allophycocyanin (APC)-conjugated goat anti-human secondary antibody and incubated on ice for 30 minutes. Then the cells were washed and resuspended in viability dye (according to the manufacturer's protocol) and incubated on ice for 30 minutes. Then the cells were washed with staining buffer, resuspended in 2% PFA, and left on ice for 30 minutes. After washing, the cells were filtered and analyzed by flow cytometry to determine the geometric mean fluorescence intensity (gMFI), which was then plotted using GraphPad Prism software. The EC 50 values of the antibody were determined from a four-parameter logistic equation over a 9-point dose-response curve (including the secondary-only control representing the 9th point). The results are shown in Table 10.

[0258] Dose-dependent binding of CD38×4-1BB 1+2 (REGN7633, REGN7647, and REGN7650) and 1+1 (REGN7150) multispecific antigen-binding molecules was observed in the presence of MOLP8 cells that endogenously express CD38. However, for the bivalent anti-4-1BB antibody REGN4249, no binding was observed. No binding was also observed for the isotype control antibodies (REGN7540 and REGN1945).

[0259] Binding of the CD38×4-1BB 1+2 antibody REGN7633 or the isotype control antibodies (REGN7540 and REGN1945) was not observed in the presence of HEK293 cells that do not express CD38. Weak binding (>100-fold lower gMFI than on MOLP8 cells) was observed with CD38×4-1BB 1+2 (REGN7647 and REGN7650) and 1+1 bispecific (REGN7150) antibodies, as well as the bivalent anti-4-1BB antibody REGN4249.

[0260] (Table 10) Maximum binding and EC50 values for binding to antibodies TIFF2025519057000013.tif76128 Abbreviations: ND: Not determined, NC: Not calculated because the data did not fit the four-parameter logistic equation * Most likely due to non-specific binding

[0261] Experiment 2 - 4-1BB binding: HEK293 and HEK293 / 4-1BB cells were detached with trypsin, washed, resuspended in staining buffer (2% FBS in PBS), and added to the wells of a 96-well V-bottom plate (3×10 5 cells / well). In staining buffer, the test antibodies were diluted 1:5 at 9 points for dose titration and added to the cells to a final concentration in the range of 1.3 pM to 100 nM (including the "no antibody" control as the 9th point (0.26 nM) and labeled as "secondary only"). The cells and antibodies were incubated on ice for 30 minutes and then washed with staining buffer. The cells were resuspended in 2 μg / ml allophycocyanin (APC)-conjugated goat anti-human secondary antibody and incubated on ice for 30 minutes. Then the cells were washed and resuspended in viability dye (according to the manufacturer's protocol) and incubated on ice for 30 minutes. Then the cells were washed with staining buffer, resuspended in 2% PFA, and left on ice for 30 minutes. After washing, the cells were filtered and analyzed by flow cytometry to determine the geometric mean fluorescence intensity (gMFI), which was then plotted using GraphPad Prism software. The EC 50 value of the antibody was determined from a four-parameter logistic equation over a 9-point dose-response curve (including the secondary-only control). The results are shown in Table 11.

[0262] The dose-dependent binding of CD38×4-1BB 1+2 (REGN7633, REGN7647, and REGN7650) and 1+1 (REGN7150) multispecific antigen-binding molecules was observed in the presence of HEK293 / h4-1BB cells engineered to express h4-1BB. Binding of the bivalent anti-4-1BB antibody REGN4249 was also observed. No binding was observed for isotype control antibodies (REGN7540 and REGN1945).

[0263] Binding of the CD38×4-1BB 1+2 antibody REGN7633 or isotype control antibodies (REGN7540 and REGN1945) was not observed in the presence of HEK293 cells that do not express h4-1BB. Slight binding (1,000-fold lower gMFI than on HEK293 / h4-1BB cells) was observed with the CD38×4-1BB 1+2 multispecific antigen-binding molecules (REGN7647 and REGN7650), CD38×4-1BB bispecific (REGN7150), and the bivalent anti-4-1BB antibody REGN4249.

[0264] (Table 11) Maximum binding and EC50 values for binding to antibodies TIFF2025519057000014.tif76128 Abbreviations: ND: not determined, NC: not calculated because the data did not fit the four-parameter logistic equation. *Most likely due to non-specific binding

[0265] Example 7: Characterization of CD38×4-1BB bispecific antibodies in a T cell activation assay using HEK293 / hCD20 / hCD38, HEK293 / hCD20, MOLP8, and human primary T cells Proper T cell activation requires two signals, "Signal 1" and "Signal 2". "Signal 1" is induced by binding of the T cell receptor (TCR) on the T cell to the peptide-bound major histocompatibility complex (MHC) molecule on the antigen-presenting cell (APC). On the other hand, "Signal 2" is provided by binding a costimulatory receptor to the T cell. One such costimulatory receptor is the 4-1BB receptor, which is an inducible type I membrane protein and a member of the tumor necrosis factor receptor (TNFR) superfamily. Expression of the 4-1BB receptor is induced on the surface of T cells after antigen or mitogen-induced activation. Activation of 4-1BB occurs through binding to 4-1BB ligand (4-1BBL) present on the APC. Thus, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0266] The CD38×4-1BB (1+1 and 1+2) bispecific antibody targets + cells to crosslink 4-1BB receptor-positive T cells, designed to mimic the natural ligand of 4-1BB, to provide "Signal 2" in the presence of "Signal 1" provided by a tumor-associated antigen (TAA)×CD3 bispecific antibody or an allogeneic response provided by an APC to enhance T cell activation.

[0267] Multispecific antigen-binding molecules: The multispecific antigen-binding molecules and controls tested in this experiment are as shown in Table 12.

[0268] (Table 12) CD38×4-1BB binding molecules and controls TIFF2025519057000015.tif75128

[0269] The ability of a CD38×4-1BB multispecific antigen-binding molecule to activate human primary T cells by binding to CD38 and 4-1BB receptors and delivering "Signal 2", as determined by IL-2 release, was evaluated in the presence of a human embryonic kidney cancer cell line engineered to express hCD20 and hCD38 (HEK293 / hCD20 / hCD38) using REGN1979 (CD20×CD3), which functions as "Signal 1". HEK293 cells expressing only hCD20 were included as a control to measure the activity that can occur in the absence of CD38 on APCs. Additionally, the multiple myeloma cell line, MOLP8, which endogenously expresses hCD38, was included in the testing of the CD38×4-1BB bispecific antibody. Since MOLP8 cells endogenously express BCMA, REGN5458 (BCMA×CD3) was used to function as "Signal 1". Notably, unlike HEK293 cells, MOLP8 cells can provide a detectable allogeneic stimulation of T cells that function as "Signal 1" in the absence of CD3 stimulation provided by REGN5458.

[0270] Human primary CD3 + Isolation of T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor leukocyte pack (Donor 555105) from Precision for Medicine using density gradient centrifugation. Briefly, 15 ml of Ficoll-Paque PLUS was added to a 50 ml conical tube, and then 30 ml of blood diluted 1:1 with PBS containing 2% FBS was layered on top. After centrifugation at 400×g for 30 minutes without braking, the buffy coat (containing mononuclear cells) was transferred to a fresh tube, diluted 5-fold with PBS containing 2% FBS, and centrifuged at 300×g for 8 minutes. Subsequently, CD3 + T cells were isolated from PBMCs using StemCell Technologies' EasySep™ Human CD3 + T Cell Isolation Kit according to the manufacturer's recommended instructions.

[0271] IL-2 Release Assay: Concentrated CD3 resuspended in stimulation medium + T cells were added to 96-well round-bottom plates at a concentration of 1 × 10 5 cells / well. Growth-arrested HEK293 / hCD20 / hCD38 or HEK293 / hCD20 were added to CD3 4 T cells at a final concentration of 1 × 10 + cells / well. Growth-arrested MOLP8 cells were added to CD3 4 T cells at a final concentration of 5 × 10 + cells / well. After addition of the cells, a constant 0.1 nM REGN1979 or its corresponding isotype control (REGN7540) was added to the wells containing HEK293 / hCD20 / hCD38 or HEK293 / hCD20. A constant 0.5 nM REGN5458 or isotype control was added to the wells containing MOLP8 cells. Subsequently, CD38×4-1BB (1+1 or 1+2), bivalent 4-1BB (REGN4249), or isotype control (REGN7540 or REGN1945) was diluted 1:4 for titration from 3 pM to 200 nM and added to the wells. The final point of the 10-point dilution did not contain the antibody for titration. The plates were incubated at 37 °C, 5% CO2 for 72 hours, and 5 μL of the total supernatant was used to measure IL-2. The amount of cytokine in the assay supernatant was determined using the PerkinElmer AlphaLisa kit according to the manufacturer's protocol. The cytokine measurements were obtained using the Perkin Elmer multi-label plate reader Envision, and the values were reported as pg / mL. All serial dilutions were tested in duplicate.

[0272] The EC 50 values of the antibodies were determined from the four-parameter logistic equation for the 10-point dose-response curve using GraphPad Prism™ software. The maximum IL-2 is given as the mean maximum response detected within the tested dose range. The results are provided in Table 13.

[0273] Results with HEK293 / hCD20 and HEK293 / hCD20 / hCD38 cell lines In the presence of target and "Signal 1" provided by REGN1979, 4-1BB antibody treatment resulted in a higher IL-2 response compared to the corresponding isotype controls (REGN7540 and REGN1945). Notably, the 1+2 format of CD38×4-1BB resulted in higher maximum IL-2 and higher potency compared to the 1+1 CD38×4-1BB (REGN7150) and bivalent 4-1BB (REGN4249) antibodies. In the absence of the CD38 target, only the bivalent 4-1BB (REGN4249) exhibited a dose-dependent increase in IL-2 release. In the absence of "Signal 1", none of the antibodies led to a dose-dependent enhancement of IL-2 release.

[0274] Results with MOLP8 cell line In the presence of allogeneic MOLP8 cells and in the absence of REGN5458, a dose-dependent increase in IL-2 was observed for the 1+2 CD38×4-1BB antibodies REGN7647 and REGN7650, and to a lesser extent for the 1+2 CD38×4-1BB antibody REGN7633 and the bivalent 4-1BB antibody REGN4249. The 1+1 CD38×4-1BB and isotype control antibodies did not exhibit a dose-dependent enhancement of IL-2 release. "Signal 1" can be provided by allogeneic MOLP8 cells, while the addition of REGN5458 was also evaluated. Under these conditions, all 4-1BB antibodies resulted in a dose-dependent increase in IL-2 release compared to the corresponding isotype controls, and the 1+2 CD38×4-1BB antibodies exhibited the maximum potency and maximum increase in IL-2.

[0275] (Table 13) Maximum IL-2 release and potency values of antibodies TIFF2025519057000016.tif129165 Abbreviations: ND: Not determined, NC: Not calculated because the data did not fit the four-parameter logistic equation.

[0276] Example 8: Characterization of the CD38×4-1BB(1+2) multispecific antigen-binding molecule in a T cell activation assay using HEK293 / hCD20 / hCD38, HEK293 / hCD20, MOLP8, NALM6, and human primary T cells Proper T cell activation requires two signals, "signal 1" and "signal 2". "Signal 1" is induced by binding of the T cell receptor (TCR) on T cells to peptide-bound major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). "Signal 2" is provided by binding a costimulatory receptor to the T cell. One such costimulatory receptor is the 4-1BB receptor, an inducible type I membrane protein and a member of the tumor necrosis factor receptor (TNFR) superfamily. Expression of the 4-1BB receptor is induced on the surface of T cells after antigen or mitogen-induced activation. Activation of 4-1BB occurs via binding to 4-1BBL present on APCs. Thus, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0277] The CD38×4-1BB(1+1 and 1+2) bispecific antibodies crosslink target cells to 4-1BB receptor-positive T cells and are designed to mimic the natural ligand of 4-1BB to provide "signal 2" to enhance T cell activation in the presence of "signal 1" provided by a tumor-associated antigen (TAA)×CD3 bispecific antibody or an allogeneic response provided by APCs. + by crosslinking target cells to 4-1BB receptor-positive T cells, designed to mimic the natural ligand of 4-1BB, to enhance T cell activation in the presence of "signal 1" provided by a tumor-associated antigen (TAA)×CD3 bispecific antibody or an allogeneic response provided by APCs.

[0278] Multispecific antigen-binding molecules: The multispecific antigen-binding molecules and controls tested in this experiment are as shown in Table 14.

[0279] (Table 14) CD38×4-1BB binding molecules and controls TIFF2025519057000017.tif83128

[0280] The ability of a CD38×4-1BB 1+2 multispecific antigen-binding molecule carrying different linkages between two tandem 4-1BB-binding Fab domains to activate human primary T cells by binding to CD38 and 4-1BB receptors and delivering "Signal 2" as determined by IL-2 or IFNγ release was evaluated in the presence of a human embryonic kidney cancer cell line engineered to express hCD20 and hCD38 (HEK293 / hCD20 / hCD38) using REGN2281 (CD20×CD3) which functions as "Signal 1". HEK293 cells expressing only hCD20 were included as a control to measure the activity that can occur in the absence of CD38 on APCs. Further, the testing of the CD38×4-1BB(1+2) bispecific antibody included the multiple myeloma cell line, MOLP8, which endogenously expresses hCD38. Since MOLP8 cells endogenously express BCMA, REGN5458 (BCMA×CD3) was used to function as "Signal 1". Finally, the state of including either MOLP8 or NALM-6 (an acute lymphoblastic leukemia cell line that endogenously expresses CD38) as target cells in the absence of CD3 bispecificity was tested. Notably, unlike HEK293 cells, MOLP8 and NALM-6 cells can provide a detectable allogeneic stimulation of T cells that function as "Signal 1" in the absence of CD3 antibody stimulation.

[0281] Human primary CD3 + Isolation of T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor leukocyte pack (Donor 555114) from Precision for Medicine using the EasySep Human T-Cell Isolation kit from StemCell Technologies according to the manufacturer's recommendations. Subsequently, CD3 + T cells were isolated from PBMCs using the EasySep™ Human CD3 + T Cell Isolation Kit from StemCell Technologies according to the instructions recommended by the manufacturer.

[0282] Primary T cell activation assay: Concentrated CD3 resuspended in stimulation medium + T cells were added to a 96-well round-bottom plate at a concentration of 1×10 5 cells / well. Target cells were added at 1×10 4 cells / well for HEK293 / hCD20 / hCD38 or HEK293 / hCD20 cells, or 5×10 4 cells / well at the final concentration for MOLP8 and NALM-6 cells, and added to the CD3 + T cells. After cell addition, a constant 0.25 nM of REGN2281 was added to wells containing HEK293 / hCD20 / hCD38 or HEK293 / hCD20 cells. A constant 0.5 nM REGN5458 or isotype control was added to wells containing MOLP8 cells. CD3 bispecificity was not added to wells containing NALM-6 target cells. Subsequently, CD38×4-1BB (1+1 or 1+2), bivalent 4-1BB (REGN4249), or isotype control (REGN7540 or REGN1945) was diluted for titration at 1:5 from 128 fM to 50 nM and added to the wells. The final point of the 10-point dilution did not contain the antibody for titration. The plate was incubated at 37 °C, 5% CO2 for 72 hours, and 5 μL of the total supernatant was used to measure IL-2 or IFNγ. The amount of cytokine in the assay supernatant was determined using the PerkinElmer AlphaLisa kit according to the manufacturer's protocol. Cytokine measurements were obtained using the Perkin Elmer multi-label plate reader Envision, and the values were reported as pg / mL. All serial dilutions were tested in duplicate.

[0283] The EC 50 value of the antibody was determined from a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism™ software. The maximum cytokine is given as the mean maximum response detected within the tested dose range. The results are provided in Tables 15 and 16.

[0284] HEK293 / hCD20 and HEK293 / hCD20 / hCD38 In the presence of the target and "Signal 1" provided by REGN2281, 4-1BB antibody therapy resulted in higher IL-2 and IFNγ responses compared to the corresponding isotype IgG4 P-PVA , IgG4 P , or bispecific antibody controls (REGN7540, REGN1945, and REGN13168, respectively). Notably, the 1+2 format of CD38×4-1BB resulted in higher maximum cytokine release and higher potency compared to the 1+1 CD38×4-1BB (REGN7150) and bivalent 4-1BB (REGN4249) antibodies, and similar levels of cytokine release and potency were observed regardless of the binding between the two tandem 4-1BB-binding Fab domains. In the absence of the CD38 target, only bivalent 4-1BB (REGN4249) exhibited a dose-dependent increase in cytokine release.

[0285] MOLP8 In the presence of syngeneic MOLP8 cells and in the absence of CD3 bispecific antibody stimulation, the 1+2 CD38×4-1BB antibodies REGN7647, REGN9682, and REGN9686 mediated a dose-dependent increase in IL-2 and IFNγ. In comparison, the 1+1 CD38×4-1BB antibody REGN7150, and the bivalent 4-1BB antibody REGN4249, resulted in a slight dose-dependent increase in IL-2 but not IFNγ. For isotype and non-targeted 1+2 4-1BB control antibodies, no dose-dependent increase in either IL-2 or IFNγ was observed. The addition of a fixed amount of REGN5458 (BCMA×CD3) under conditions using MOLP8 cells and primary human T cells resulted in a dose-dependent increase in IL-2 and IFNγ for all CD38-targeted 4-1BB antibodies and REGN4249. As described above, the 1+2 CD38×4-1BB antibodies exhibited greater potency and maximal cytokine increases compared to REGN7150 and REGN4249, and the binding format between 4-1BB-binding Fabs had little effect on potency or maximal cytokine release. Isotype control and non-TAA×4-1BB 1+2 control antibodies did not result in dose-dependent cytokine release.

[0286] NALM-6 In the presence of syngeneic NALM-6 cells and in the absence of CD3 bispecific antibody stimulation, all CD38-targeted 4-1BB antibodies (REGN7647, REGN9682, REGN9686, and REGN7150), as well as the bivalent 4-1BB antibody (REGN4249), resulted in a dose-dependent increase in IFNγ and IL-2. The 1+2 CD38×4-1BB antibodies REGN7647, REGN9682, and REGN9686 exhibited greater potency and maximal cytokine release compared to the 1+1 CD38×4-1BB antibody REGN7150, and the bivalent 4-1BB antibody REGN4249. As described above, all 1+2 CD38×4-1BB multispecific antigen-binding molecules were similarly performed regardless of the linker between 4-1BB-binding Fabs. Isotype control and non-TAA×4-1BB 1+2 control antibodies did not result in dose-dependent cytokine release.

[0287] (Table 15) TIFF2025519057000018.tif22894

[0288] (Table 16) TIFF2025519057000019.tif22996

[0289] Example 9: Characterization of a CD38×4-1BB(1+2) multispecific antigen-binding molecule in an engineered reporter assay using HEK293 / hCD20 / hCD38, HEK293 / hCD20, MOLP8, OPM2, and HEK293 / NFkB-Luc / h4-1BB cells The ability of a CD38×4-1BB multispecific antigen-binding molecule to specifically activate the 4-1BB receptor in the presence of target cells expressing CD38 was measured in an engineered reporter assay. In this assay, engineered HEK293 cells express the reporter gene luciferase together with the costimulatory receptor 4-1BB (HEK293 / NFkB-Luc / h4-1BB) under the control of the transcription factor NF-KB (NFKB-Luc). The target cells used in this assay were HEK293 cells engineered to express CD20 alone or in combination with CD38, or cell lines that endogenously express CD38, namely, OPM2 and MOLP8. The ability of the 4-1BB arm to stimulate 4-1BB activity was evaluated by combining reporter cells with target cells and titrating the CD38×4-1BB 1+2 antibody. Activation of 4-1BB results in NFKB-driven luciferase production, which is then measured via a luminescence readout. In these assays, the effect of different types of linkages between two tandem 4-1BB targeting domains was also evaluated.

[0290] Multispecific antigen-binding molecule: The multispecific antigen-binding molecules and controls tested in this experiment are as shown in Table 17.

[0291] (Table 17) CD38×4-1BB binding molecules and controls TIFF2025519057000020.tif61156

[0292] One day before the experiment, HEK293 reporter cells were split into 5×10 5 cells / ml in DMEM + 10% FBS + P / S / G + 500 μg / ml G418 growth medium.

[0293] On the day of the experiment, adherent HEK293 reporter and target cells were trypsinized, washed, and resuspended in assay medium (DMEM + 10% FBS + P / S / G). Reporter HEK293 / NFKB-Luc / h4-1BB cells were added to wells of a 96-well white microtiter plate at a final concentration of 5×10 3 cells / well, followed by the addition of target cells, either HEK293 / hCD20 or HEK293 / hCD20 / hCD38, at a final concentration of 1×10 4 cells / well, or MOLP8 and OPM2 target cells were added at a final concentration of 2.5×10 4 cells / well.

[0294] CD38×4-1BB 1+2 and control antibody were titrated in a serial dilution in the range of final concentrations from 1:3, 10 points, 3.0 pM to 20 nM, and the last point was included as a control without antibody. After addition of the antibody, the 96-well white microtiter plate was incubated at 37°C / 5% CO2 for 5 hours, followed by addition of an equal volume of ONE-Glo™ (Promega) reagent to lyse the cells and detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer). The EC 50 value of the antibody was determined from a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism software (the 10th point was without antibody). The results are provided in Table 18.

[0295] HEK293 / hCD20 and HEK293 / hCD20 / hCD38 In the presence of HEK293 / hCD20 / hCD38 target cells, the CD38×4-1BB 1+2 bispecific antibodies (REGN7647, REGN9682, REGN9686, and REGN7650) resulted in a similar increase in luciferase activity regardless of the binding between the two anti-4-1BB binding Fab domains. The anti-4-1BB bivalent antibody (REGN4249) also resulted in a dose-dependent increase in luciferase activity, while the isotype control antibody did not.

[0296] In the presence of HEK293 / hCD20 target cells lacking CD38, no response was seen with either the CD38×4-1BB bispecific antibody or the isotype control. Only the anti-4-1BB bivalent antibody (REGN4249) resulted in a dose-dependent increase in luciferase activity.

[0297] MOLP8 and OPM2 cells In the presence of either MOLP8 or OPM2 target cells, the CD38×4-1BB 1+2 (REGN7647, REGN7650, REGN9682, and REGN9686) antibodies, as well as the bivalent anti-4-1BB antibody (REGN4249), resulted in a dose-dependent increase in luciferase activity, and 1+2 CD38×4-1BB variants with different linker lengths resulted in similar activity. The isotype control antibody did not result in any signal.

[0298] (Table 18) Maximum luciferase activity and potency values of the antibodies TIFF2025519057000021.tif75170 Abbreviations: ND: Not determined because no concentration-dependent response was observed, NC: Not calculated because the data did not fit the four-parameter logistic equation.

[0299] Example 10: Characterization of CD38×4-1BB 1+2 bispecific antibodies in a T cell allogeneic semi-primed combination assay using NALM-6, NALM-6 / PDL1, and human primary T cells Proper T cell activation requires two signals, "signal 1" and "signal 2". "Signal 1" is induced by binding of the T cell receptor (TCR) on the T cell to a peptide-bound major histocompatibility complex (MHC) molecule on the antigen-presenting cell (APC). "Signal 2" is provided by binding a costimulatory receptor to the T cell. One such costimulatory receptor is the 4-1BB receptor, an inducible type I membrane protein and a member of the tumor necrosis factor receptor (TNFR) superfamily. Expression of the 4-1BB receptor is induced on the surface of T cells after antigen- or mitogen-induced activation. Activation of 4-1BB occurs through binding to 4-1BB ligand (4-1BBL) present on the APC. Thus, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0300] The CD38×4-1BB(1+2) bispecific antibody binds to CD38 +By crosslinking the target cells with 4-1BB receptor-positive T cells, a "signal 2" is provided to enhance T cell activation in the presence of "signal 1" provided by a tumor-associated antigen (TAA)×CD3 bispecific antibody or an allogeneic response provided by an APC, which is designed to mimic the natural ligand of 4-1BB. However, T cell activation can be inhibited by ligation of the programmed cell death protein 1 receptor (PD-1) on T cells to the ligand PD-L1 on APCs. Ligation of PD-1 leads to the recruitment of phosphatases to CD28 and the TCR complex (Zou et al. Inhibitory B7-family molecules in the tumor microenvironment. Nature Reviews Immunology 2008, 8:467-477, Francisco et al. 2010, Hui et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017, 355(6332):1428-33), resulting in the suppression of TCR signaling and 4-1BB stimulation. Therefore, blockade of the PD-1 / PD-L1 interaction with semiprimab, a PD-1 antagonist in combination with a CD38×4-1BB bispecific antibody, can promote T cell function.

[0301] Multispecific antigen-binding molecule: The multispecific antigen-binding molecules and controls tested in this experiment are as shown in Table 19.

[0302] (Table 19) CD38×4-1BB binding molecules and controls TIFF2025519057000022.tif61130

[0303] The ability of a CD38×4-1BB 1+2 multispecific antigen-binding molecule to activate human primary T cells by delivering "Signal 2" by binding CD38 and 4-1BB, as determined by IL2 and IFNγ release, was evaluated in the presence of CD38 engineered to express PD-L1 + It was evaluated in the presence of the human acute lymphoblastic leukemia cancer cell line (NALM6 / hPD-L1). NALM6 cells provide an allogeneic TCR response sufficient to function as "Signal 1". The addition of the PD-1 antagonist antibody semiprimab, at a fixed concentration, was evaluated in the presence of titrations of CD38×4-1BB 1+2 or control antibody

[0304] Human primary CD3 + Isolation of T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocyte packs from Precision for Medicine (donor 555192) using the EasySep™ Direct Human PBMC Isolation Kit and frozen, according to the protocol recommended by the manufacturer. CD3 + T cells were isolated by thawing vials of frozen PBMCs. Donor PBMCs were depleted of CD3 + T cells using the StemCell Technologies EasySep™ Human CD3 + T Cell Isolation Kit, according to the manufacturer's recommended instructions

[0305] IL-2 and IFNγ release assay: Concentrated CD3 + T cells resuspended in stimulation medium were added to 96-well round-bottom plates at a concentration of 1×10 5 cells / well. NALM6 cells or NALM-6 cells engineered to express hPD-L1 were added to CD3 4 at a final concentration of 5×10 +It was added to T cells. Then, REGN7633, REGN7647, REGN7650, REGN4249, and REGN7540 were diluted for titration to 0.76 pM to 50 nM at a ratio of 1:4 and added to the wells. The final point of the 10-point dilution did not contain the antibody for titration. After the addition of the titrated antibody, either a constant 20 nM of semi-primab or its corresponding isotype control (REGN1945) was added to the wells. The plate was incubated at 37 °C and 5% CO2 for 72 hours, and 5 μL from the supernatant was used to measure IL-2 and IFNγ. The amount of cytokine in the assay supernatant was determined using the PerkinElmer AlphaLisa kit according to the manufacturer's protocol. The cytokine measurements were obtained with the Perkin Elmer multi-label plate reader Envision, and the values were reported as pg / mL. All serial dilutions were tested in triplicate.

[0306] Results The EC 50 values were determined from a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism™ software. The maximum cytokine is given as the mean maximum response detected within the tested dose range. The results are provided in Tables 20 and 21.

[0307] In the presence of syngeneic NALM6 cells or NALM6 cells engineered to express PD-L1, CD38×4-1BB 1+2 antibody therapy (REGN7633, REGN7647, and REGN7650) resulted in a dose-dependent increase in IL-2 and IFNγ release compared to the corresponding isotype control (REGN7540). Maximum IL-2 and IFNγ release was lower in conditions using NALM6 / PD-L1 cells compared to NALM-6 (which does not express PD-L1). In the presence of NALM6 cells expressing PD-L1, semiprimab increased maximum cytokine release compared to the corresponding isotype control of semiprimab, REGN1945. Notably, the 1+2 format of the CD38×4-1BB antibody resulted in higher maximum cytokine release and higher potency compared to the bivalent 4-1BB (REGN4249) antibody.

[0308] (Table 20) Maximum IL-2 release and potency values TIFF2025519057000023.tif68170 Abbreviations: ND: Not determined, NC: Not calculated because the data did not fit the four-parameter logistic equation.

[0309] (Table 21) Maximum IFNγ release and potency values TIFF2025519057000024.tif68170 Abbreviations: ND: Not determined, NC: Not calculated because the data did not fit the four-parameter logistic equation.

[0310] Example 11: In Vivo Antitumor Efficacy of CD38×4-1BB Bispecific Antibody in Combination with BCMA×CD3 Bispecific Antibody The multispecific antigen-binding molecules and controls tested in this experiment are as shown in Table 22.

[0311] (Table 22) CD38×4-1BB binding molecules and controls TIFF2025519057000025.tif31129

[0312] To determine the in vivo antitumor efficacy of a CD38×4-1BB 1+2 format bispecific antibody (bsAb) in combination with a BCMA×CD3 bsAb, xenograft studies were performed. On day -11, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were intraperitoneally injected with 4×10 6 human peripheral blood mononuclear cells (PBMCs) from normal healthy donors. 2×10 6 BCMA + CD38 + MOLP-8 human multiple myeloma tumor cells that had also been engineered to express firefly luciferase (MOLP-8-luciferase cells) were intravenously administered to the mice on day 0. Then, mice (n = 4 - 5 per group) were immediately administered either a CD3-binding control bispecific Ab or a BCMA×CD3 bispecific antibody (REGN5458, U.S. Patent Application Publication No. 2020 / 0024356) at 0.4 mg / kg in combination with a 4-1BB-binding control bispecific Ab (1+2 format) or a CD38×4-1BB bispecific antibody (1+2 format, REGN9686) at 4 mg / kg. The mice were administered these antibodies two more times on days 7 and 14 for a total of three administrations. Tumor growth was evaluated over 53 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n = 5) was given only MOLP-8 luciferase cells and PBMCs, but no antibody (PBS-treated group). To measure background BLI levels, one group of mice (n = 5) was left untreated and received no tumor, PBMCs, or antibody (tumor-free group).

[0313] These studies demonstrated that BCMA×CD3 bispecific antibody monotherapy demonstrated only modest antitumor efficacy, CD38×4-1BB 1+2 bispecific antibody monotherapy demonstrated little or no antitumor activity, and combination therapy with a BCMA×CD3 bispecific antibody and a CD38×4-1BB 1+2 bispecific antibody resulted in a more potent combination antitumor efficacy that was superior to either therapy alone.

[0314] Heterologous tumor transplantation and measurement - On day - 11, 4×10 scid Il2rg tm1Wjl human peripheral blood mononuclear cells (PBMCs) from normal healthy donors were intraperitoneally injected into NOD.Cg - Prkdc 6 / SzJ (NSG) mice. 2×10 6 BCMA + CD38 + MOLP - 8 human multiple myeloma tumor cells were intravenously administered to the mice on day 0. Then, the mice (n = 4 - 5 per group) were immediately administered either a 0.4 mg / kg CD3 - binding control bispecific antibody or a BCMA×CD3 bispecific antibody (REGN5458), in combination with a 4 mg / kg 4 - 1BB - binding control bispecific antibody (1 + 2 format) or a CD38×4 - 1BB bispecific antibody (1 + 2 format, REGN9686). The mice were administered these Abs two more times on days 7 and 14, for a total of three administrations. Tumor growth was evaluated over 53 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n = 5) was given only MOLP - 8 luciferase cells and PBMCs, but no antibody (PBS - treated group). To measure the background BLI level, a group of mice (n = 5) was left untreated and received no tumor, PBMCs, or antibody (tumor - free group).

[0315] Measurement of heterologous tumor growth Tumor burden was measured using BLI imaging. Mice were intraperitoneally injected with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of the mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image acquisition was performed at a field of view in D, a subject height of 1.5 cm, and a medium binning level, with an autoexposure time determined by Living Image Software. The BLI signal was extracted using Living Image software. Regions of interest were drawn around each tumor mass, and the photon intensity was recorded as the total flux (photons / second - p / s).

[0316] Results: Tables 23 - 32 provide the results of treatment combinations on tumor burden and subject survival at days 6, 10, 13, 17, 20, 24, 27, 31, 34, and 38 after administration of human multiple myeloma tumor cells. Figure 3 is a graphical representation of the data shown in the table over 38 days. Figure 4A illustrates the tumor burden over time in mice treated with PBS compared to mice not receiving tumor cells. Figure 4B illustrates the tumor burden over time in mice treated with CD3-binding control bsAb (0.4 mg / kg) + 4-1BB-binding control bsAb (4 mg / kg) compared to mice not receiving tumor cells. Figure 4C illustrates the tumor burden over time in mice treated with CD3-binding control bsAb (0.4 mg / kg) + CD38×4-1BB (4 mg / kg) compared to mice not receiving tumor cells. Figure 4D illustrates the tumor burden over time in mice treated with BCMA×CD3 bsAb (0.4 mg / kg) + 4-1BB-binding control bsAb (4 mg / kg) compared to mice not receiving tumor cells. Figure 4E illustrates the tumor burden over time in mice treated with BCMA×CD3 bsAb (0.4 mg / kg) + CD38×4-1BB (4 mg / kg) compared to mice not receiving tumor cells.

[0317] BCMA×CD3 monotherapy: BCMA×CD3 bsAb (REGN5458) + 4-1BB binding control bsAb provided some anti-tumor efficacy, with a reduction in mean BLI measurements at day 24 (p < 0.0001) and day 26 (p = 0.0015) by two-way ANOVA compared to mice receiving CD3 binding control bsAb + 4-1BB binding negative control bsAb.

[0318] CD38×4-1BB monotherapy: Treatment with CD3 binding control bsAb + CD38×4-1BB 1+2 bsAb (REGN9686) did not significantly reduce the mean BLI readings compared to mice receiving CD3 binding control bsAb + 4-1BB binding control bsAb.

[0319] BCMA×CD3 + CD38×4-1BB 1+2: The combination of BCMA×CD3 bsAb (REGN5458) and CD38×4-1BB 1+2 bsAb (REGN9686) resulted in lower mean BLI measurements than mice receiving BCMA×CD3 bsAb and 4-1BB binding control bsAb (p < 0.0001 at day 38 by two-way ANOVA).

[0320] Therefore, these studies demonstrate that BCMA×CD3 bsAb monotherapy demonstrated only modest anti-tumor efficacy, CD38×4-1BB 1+2 bsAb monotherapy demonstrated little or no anti-tumor activity, and combination therapy with BCMA×CD3 bsAb and CD38×4-1BB 1+2 bsAb resulted in superior and more potent combination anti-tumor efficacy compared to either therapy alone.

[0321] (Table 23) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 6 TIFF2025519057000026.tif97163

[0322] (Table 24) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 10 TIFF2025519057000027.tif98163

[0323] (Table 25) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 13 TIFF2025519057000028.tif97163

[0324] (Table 26) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 17 TIFF2025519057000029.tif97162

[0325] (Table 27) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 20 TIFF2025519057000030.tif97162

[0326] (Table 28) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 24 TIFF2025519057000031.tif99165

[0327] (Table 29) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 27 TIFF2025519057000032.tif98165

[0328] (Table 30) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 31 TIFF2025519057000033.tif99167

[0329] (Table 31) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 34 TIFF2025519057000034.tif99165

[0330] (Table 32) Anti-tumor efficacy through combination therapy with BCMA×CD3 bsAb + CD38×4-1BB 1+2 bsAb - Day 38 TIFF2025519057000035.tif97162

[0331] Example 12: Biacore binding kinetics of anti-CD38×4-1BB antigen-binding molecules The binding kinetics of the anti-CD38×4-1BB antibody were determined by the antibody capture format Biacore binding kinetics of the anti-CD38×4-1BB 1+2 antibody that binds to monomeric and dimeric human 4-1BB reagents, and the antigen capture format Biacore binding kinetics of the anti-CD38×4-1BB 1+2 antibody that binds to the dimeric human 4-1BB reagent. Both experiments were performed at 25°C.

[0332] Antibody capture format method: The equilibrium dissociation constant (K of human 4-1BB expressed with a C-terminal myc-myc-hexahistidine tag (h4-1BB.mmH, REGN3584) or human 4-1BB expressed with a C-terminal mouse Fc tag (h4-1BB.mFc, REGN3585), which binds to the purified anti-CD38×4-1BB 1+2 antibody DThe value) was determined using a real-time surface plasmon resonance biosensor using a Biacore 8k instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567). All Biacore binding tests were performed in a buffer composed of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20 (HBS-EP running buffer). Different concentrations of h4-1BB.mmH (REGN3584) (range of 100 - 6.25 nM in 4-fold serial dilutions) prepared in HBS-EP running buffer, or h4-1BB.mFc (REGN3585) (range of 100 - 1.56 nM in 4-fold serial dilutions) prepared in HBS-EP running buffer were injected onto the captured anti-CD38×4-1BB 1+2 antibody at a flow rate of 30 μL / min. Antibody-reagent association was monitored for 5 minutes while dissociation in HBS-EP running buffer was monitored for 10 minutes. At the end of each cycle, the anti-CD38×4-1BB 1+2 antibody capture surface was regenerated using a 10-second injection of 20 mM phosphoric acid. All binding kinetics experiments were performed at 25°C. The results are presented in Tables 33 and 34.

[0333] Antigen capture format method: Equilibrium dissociation constant (K of human 4-1BB expressed with a C-terminal mouse Fc tag (h4-1BB.mFc, REGN3585) that binds to the purified anti-CD38×4-1BB 1+2 antibody DThe value) was determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a polyclonal rabbit anti-mouse Fc antibody (GE, #BR-1008-38). All Biacore binding tests were performed in a buffer composed of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20 (HBS-EP running buffer). Different concentrations of the CD38×4-1BB 1+2 construct (in the range of 100 - 6.25 nM in 4-fold serial dilutions) prepared in HBS-EP running buffer were injected onto the h4-1BB.mFc (REGN3585) capture surface at a flow rate of 30 μL / min. The association of the antibody-reagent was monitored for 5 minutes, while the dissociation in HBS-EP running buffer was monitored for 10 minutes. At the end of each cycle, the h4-1BB.mFc capture surface was regenerated using a 40-second injection of 10 mM glycine, pH 1.5. All binding kinetics experiments were performed at 25 °C. The results are presented in Table 35.

[0334] Data analysis for both formats: Using Cytiva Insight curve fitting software, the real-time sensorgrams were fitted to a 1:1 binding model to determine the kinetic association rate constant (k a ) and dissociation rate constant (k d ). The binding dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) were calculated from the reaction rate constants as follows: Calculated as in TIFF2025519057000036.tif9128.

[0335] (Table 33) Kinetic and equilibrium binding parameters of monomeric human 4-1BB to surface-captured anti-CD38×4-1BB 1+2 antibody at 25 °C TIFF2025519057000037.tif56160* Dose-dependent binding of h4-1bb.mmH was observed **IC: The observed binding did not conform to the binding simulation model, and the binding kinetic parameters were not determined under the current experimental conditions.

[0336] (Table 34) Kinetic and equilibrium binding parameters of dimeric human 4-1BB to surface-captured anti-CD38×4-1BB 1+2 antibody at 25 °C TIFF2025519057000038.tif55160

[0337] (Table 35) Kinetic and equilibrium binding parameters of anti-CD38×4-1BB 1+2 antibody to surface-captured dimeric human 4-1BB at 25 °C TIFF2025519057000039.tif55144

[0338] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

Claims

1. (a) A first antigen-binding arm that specifically binds to CD38, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) within the HCVR, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 40 and 42, and the LCVR comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3) within the LCVR, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 48 and 418, and (b) A second antigen-binding arm that specifically binds to 4-1BB, comprising a first antigen-binding region (R1) and a second antigen-binding region (R2), (i) R1 comprises a heavy chain variable region (R1-HCVR) and a light chain variable region (R1-LCVR), the R1-HCVR comprises three heavy chain CDRs (R1-HCDR1, R1-HCDR2, and R1-HCDR3), and the R1-LCVR comprises three light chain CDRs (R1-LCDR1, R1-LCDR2, and R1-LCDR3), (ii) R2 comprises a heavy chain variable region (R2-HCVR) and a light chain variable region (R2-LCVR), the R2-HCVR comprises three heavy chain CDRs (R2-HCDR1, R2-HCDR2, and R2-HCDR3), and the R2-LCVR comprises three light chain CDRs (R2-LCDR1, R2-LCDR2, and R2-LCDR3), Second antigen-binding arm A bispecific antigen-binding molecule containing this molecule.

2. The bispecific antigen-binding molecule according to claim 1, wherein R1 and R2 bind to the same epitope on 4-1BB.

3. The bispecific antigen-binding molecule according to claim 1, wherein R1 and R2 bind to different epitopes on 4-1BB.

4. The bispecific antigen-binding molecule according to claim 1, wherein R1 and R2 are connected via a peptide linker.

5. The bispecific antigen-binding molecule according to claim 4, wherein the peptide linker comprises the peptide sequence (GGGGS)n, and n is 1 to 6.

6. The bispecific antigen-binding molecule according to claim 1, wherein the first antigen-binding arm comprises a heavy chain and a light chain.

7. The bispecific antigen-binding molecule according to claim 1, wherein the second antigen-binding arm comprises a heavy chain and two light chains.

8. The first antigen-binding arm described above, Three heavy chain complementarity-determining regions (HCDR1-HCDR2-HCDR3), each containing a set of amino acid sequences selected from the groups consisting of SEQ ID NOs: 42-44-46 and 4-6-8. A bispecific antigen-binding molecule according to claim 1, comprising:

9. The first antigen-binding arm described above, Three light chain complementarity-determining regions (LCDR1-LCDR2-LCDR3), each containing a set of amino acid sequences selected from the groups consisting of SEQ ID NOs: 50-52-54 and 20-22-24. A bispecific antigen-binding molecule according to claim 1, comprising:

10. The bispecific antigen-binding molecule according to claim 1, wherein the first antigen-binding arm comprises an HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 40 and 42.

11. The bispecific antigen-binding molecule according to claim 1, wherein the first antigen-binding arm comprises an LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 18.

12. The bispecific antigen-binding molecule according to claim 1, wherein the first antigen-binding arm comprises an HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 and 42, and an LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 48.

13. The bispecific antigen-binding molecule according to claim 1, wherein R1 comprises an R1-HCVR containing three heavy chain CDRs within an R1-HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 10, 32, 72, 86, and 94.

14. The bispecific antigen-binding molecule according to claim 1, wherein R1 comprises an R1-LCVR containing three light chain CDRs within an R1-LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 48 and 18.

15. R1 is Each of the three heavy chain complementarity-determining regions (R1-HCDR1-R1-HCDR2-R1-HCDR3) contains a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 64-66-68, 12-14-16, 34-36-38, 74-76-78, 88-90-92, and 96-98-100. A bispecific antigen-binding molecule according to claim 1, comprising:

16. R1 is Each of the three light chain complementarity-determining regions (R1-LCDR1-R1-LCDR2-R1-LCDR3) contains a set of amino acid sequences selected from the groups consisting of SEQ ID NOs: 50-52-54 and 20-22-24. A bispecific antigen-binding molecule according to claim 1, comprising:

17. The bispecific antigen-binding molecule according to claim 1, wherein R1 comprises R1-HCVR, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 10, 32, 72, 86, and 94.

18. The bispecific antigen-binding molecule according to claim 1, wherein R1 comprises R1-LCVR, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 18.

19. The bispecific antigen-binding molecule according to claim 1, wherein R1 comprises R1-HCVR, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 10, 32, 72, 86, and 94, and R1-LCVR, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 18.

20. The bispecific antigen-binding molecule according to claim 1, wherein R2 comprises an R2-HCVR containing three heavy chain CDRs within an R2-HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 10, 32, 72, 86, and 94.

21. The bispecific antigen-binding molecule according to claim 1, wherein R2 comprises an R2-LCVR containing three light chain CDRs within an R2-LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 48 and 18.

22. R2, Each of the three heavy chain complementarity-determining regions (R2-HCDR1-R2-HCDR2-R2-HCDR3) contains a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 64-66-68, 12-14-16, 34-36-38, 74-76-78, 88-90-92, and 96-98-100. A bispecific antigen-binding molecule according to claim 1, comprising:

23. R2, Each of the three light chain complementarity-determining regions (R2-LCDR1-R2-LCDR2-R2-LCDR3) contains a set of amino acid sequences selected from the groups consisting of SEQ ID NOs: 50-52-54 and 20-22-24. A bispecific antigen-binding molecule according to claim 1, comprising:

24. The bispecific antigen-binding molecule according to claim 1, wherein R2 comprises R2-HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 10, 32, 72, 86, and 94.

25. The bispecific antigen-binding molecule according to claim 1, wherein R2 comprises R2-LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 18.

26. The bispecific antigen-binding molecule according to claim 1, wherein R2 comprises R2-HCVR, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 10, 32, 72, 86, and 94, and R2-LCVR, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and 18.

27. (a) The first antigen-binding arm comprises an HCVR containing three heavy chain CDRs contained within an HCVR containing the amino acid sequence of SEQ ID NO: 40, and an LCVR containing three light chain CDRs contained within an LCVR containing the amino acid sequence of SEQ ID NO: 48, (b) The second antigen-binding arm (i) R1-HCVR containing three heavy chain CDRs contained within R1-HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 62, 32, and 72, and R1-LCVR containing three light chain CDRs contained within R1-LCVR containing the amino acid sequence of SEQ ID NO. 48, (ii) R2-HCVR containing three heavy chain CDRs contained within R2-HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 62, 86, and 94, and R2-LCVR containing three light chain CDRs contained within R2-LCVR containing the amino acid sequence of SEQ ID NO.

48. including, The bispecific antigen-binding molecule according to claim 1.

28. (a) Each of the first antigen-binding arms comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, which each contains the amino acid sequence set of SEQ ID NOs: 42-44-46-50-52-54 (b) The second antigen-binding arm (i) A first antigen-binding region (R1) comprising R1-HCDR1-R1-HCDR2-R1-HCDR3-R1-LCDR1-R1-LCDR2-R1-LCDR3, each containing a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 64-66-68-50-52-54, 34-36-38-50-52-54, and 74-76-78-50-52-54, (ii) A second antigen-binding region (R2) comprising R2-HCDR1-R2-HCDR2-R2-HCDR3-R2-LCDR1-R2-LCDR2-R2-LCDR3, each containing a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 64-66-68-50-52-54, 74-76-78-50-52-54, and 88-90-92-50-52-54. including, The bispecific antigen-binding molecule according to claim 1.

29. (a) The first antigen-binding arm comprises an HCVR containing the amino acid sequence of SEQ ID NO: 40 and an LCVR containing the amino acid sequence of SEQ ID NO: 48, (b) The second antigen-binding arm (i) R1-HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 62, 32, and 72, and R1-LCVR containing the amino acid sequence of SEQ ID NO. 48, (ii) R2-HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 86, and 94, and R2-LCVR containing the amino acid sequence of SEQ ID NO: 48 including, The bispecific antigen-binding molecule according to claim 1.

30. (a) The first antigen-binding arm comprises an HCVR containing the amino acid sequence of SEQ ID NO: 40 and an LCVR containing the amino acid sequence of SEQ ID NO: 48, (b) The second antigen-binding arm (i) R1-HCVR containing the amino acid sequence of SEQ ID NO: 62, and R1-LCVR containing the amino acid sequence of SEQ ID NO: 48, (ii) R2-HCVR containing the amino acid sequence of SEQ ID NO: 62, and R2-LCVR containing the amino acid sequence of SEQ ID NO: 48 including, The bispecific antigen-binding molecule according to claim 1.

31. (a) The first antigen-binding arm comprises an HCVR containing the amino acid sequence of SEQ ID NO: 40 and an LCVR containing the amino acid sequence of SEQ ID NO: 48, (b) The second antigen-binding arm (i) R1-HCVR containing the amino acid sequence of SEQ ID NO: 32, and R1-LCVR containing the amino acid sequence of SEQ ID NO: 48, (ii) R2-HCVR containing the amino acid sequence of SEQ ID NO: 86, and R2-LCVR containing the amino acid sequence of SEQ ID NO: 48 including, The bispecific antigen-binding molecule according to claim 1.

32. (a) The first antigen-binding arm comprises an HCVR containing the amino acid sequence of SEQ ID NO: 40 and an LCVR containing the amino acid sequence of SEQ ID NO: 48, (b) The second antigen-binding arm (i) R1-HCVR containing the amino acid sequence of SEQ ID NO: 72, and R1-LCVR containing the amino acid sequence of SEQ ID NO: 48, (ii) R2-HCVR containing the amino acid sequence of SEQ ID NO: 94, and R2-LCVR containing the amino acid sequence of SEQ ID NO: 48 including, The bispecific antigen-binding molecule according to claim 1.

33. The bispecific antigen-binding molecule according to claim 1, wherein the molecule is a bispecific antibody.

34. The bispecific antibody according to claim 33, wherein the bispecific antibody comprises a heavy chain constant region of an IgG1 or IgG4 isotype.

35. The bispecific antibody according to claim 33, wherein the second heavy chain comprises mutants H435R and Y436F (EU numbering).

36. The bispecific antibody according to claim 33, wherein the first antigen-binding arm comprises a first heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO:

60.

37. The bispecific antibody according to claim 33, wherein the second antigen-binding arm comprises a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 56, 70, 80, and 82, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60.

38. (a) The first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 60, (b) The second antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 84, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60. The bispecific antibody according to claim 33.

39. (a) The first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 60, (b) The second antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 56, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60. The bispecific antibody according to claim 33.

40. (a) The first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 60, (b) The second antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 70, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60. The bispecific antibody according to claim 33.

41. (a) The first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 60, (b) The second antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 80, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60. The bispecific antibody according to claim 33.

42. (a) The first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 60, (b) The second antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 82, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60. The bispecific antibody according to claim 33.

43. It is a bispecific antigen-binding molecule, (a) A first antigen-binding arm that specifically binds to CD38, comprising three heavy chain CDRs contained within an HCVR containing the amino acid sequence of SEQ ID NO: 40, and three light chain CDRs contained within an LCVR containing the amino acid sequence of SEQ ID NO: 48, (b) A second antigen-binding arm that specifically binds to 4-1BB, wherein the second antigen-binding arm includes a first antigen-binding region (R1) and a second antigen-binding region (R2), (i) R1 comprises a heavy chain variable region (R1-HCVR) and a light chain variable region (R1-LCVR), and the R1-HCVR comprises three heavy chain CDRs (R1-HCDR1, R1-HCDR2, and R1-HCDR3) contained within the R1-HCVR which contains the amino acid sequence of SEQ ID NO: 62, and the R1-LCVR comprises three light chain CDRs (R1-LCDR1, R1-LCDR2, and R1-LCDR3) contained within the R1-LCVR which contains the amino acid sequence of SEQ ID NO: 48, (ii) R2 comprises a heavy chain variable region (R2-HCVR) and a light chain variable region (R2-LCVR), and the R2-HCVR comprises three heavy chain CDRs (R2-HCDR1, R2-HCDR2, and R2-HCDR3) contained within the R2-HCVR containing the amino acid sequence of SEQ ID NO: 62, and the R2-LCVR comprises three light chain CDRs (R2-LCDR1, R2-LCDR2, and R2-LCDR3) contained within the R2-LCVR containing the amino acid sequence of SEQ ID NO: 48, The second antigen-binding arm, A bispecific antigen-binding molecule containing this molecule.

44. The bispecific antigen-binding molecule according to claim 43, wherein the molecule is a bispecific antibody.

45. The bispecific antigen-binding molecule according to claim 44, wherein the bispecific antibody is a human antibody.

46. The bispecific antigen-binding molecule according to claim 44, wherein the bispecific antibody is an immunoglobulin molecule comprising five polypeptide chains, a first heavy chain paired with a light chain, and a second heavy chain paired with the first and second light chains, wherein the heavy chains and light chains are interconnected by disulfide bonds, each heavy chain comprises a heavy chain variable region and a heavy chain constant region, and each light chain comprises a light chain variable region and a light chain constant region.

47. The bispecific antigen-binding molecule according to claim 44, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, and the two heavy chains are of isotype IgG1 or IgG4.

48. The bispecific antigen-binding molecule according to claim 44, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, and the first heavy chain or the second heavy chain comprises a CH3 domain having H435R (EU numbering) modification and Y436F (EU numbering) modification, but not both.

49. The bispecific antigen-binding molecule according to claim 44, wherein the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

50. The bispecific antigen-binding molecule according to claim 43, wherein the first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 paired with a light chain containing the amino acid sequence of SEQ ID NO:

60.

51. The bispecific antigen-binding molecule according to claim 50, wherein the second antigen-binding arm comprises a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 70, or 82, paired with a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO:

60.

52. (a) The first antigen-binding arm comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 60, (b) The second antigen-binding arm (i) comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 84, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO: 60, (ii) comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 70, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO: 60, or (iii) A heavy chain containing the amino acid sequence of SEQ ID NO: 82, a first light chain containing the amino acid sequence of SEQ ID NO: 60, and a second light chain containing the amino acid sequence of SEQ ID NO: 60, The bispecific antigen-binding molecule according to claim 43.

53. A pharmaceutical composition comprising a bispecific antigen-binding molecule according to claim 1 and a pharmaceutically acceptable carrier.

54. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the HCVR of the first antigen-binding arm of the bispecific antigen-binding molecule according to claim 1.

55. An isolated nucleic acid molecule comprising nucleic acid sequences encoding R1-HCVR and R2-HCVR of the second antigen-binding arm of the bispecific antigen-binding molecule according to claim 1.

56. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the LCVR of the bispecific antigen-binding molecule described in claim 1.

57. An expression vector comprising an isolated nucleic acid molecule according to any one of claims 54 to 56.

58. A host cell comprising the expression vector described in claim 57.

59. The host cell according to claim 58, which is Escherichia coli (E. coli) or a CHO cell.

60. A method for producing a bispecific antigen-binding molecule, The method includes a step of growing the host cells described in claim 58 under conditions that allow the production of the bispecific antigen-binding molecule, The host cell comprises a first nucleic acid molecule containing a nucleic acid sequence encoding the heavy chain variable region (HCVR) of the bispecific antigen-binding molecule antigen-binding arm A1, a second nucleic acid molecule containing a nucleic acid sequence encoding the heavy chain variable region (HCVR) of the bispecific antigen-binding molecule antigen-binding arm A2, and a third nucleic acid molecule containing a nucleic acid sequence encoding the common light chain variable region (LCVR). method.

61. The method according to claim 60, wherein the host cell comprises a first nucleic acid molecule containing a nucleic acid sequence encoding the heavy chain of the bispecific antigen-binding molecule antigen-binding arm A1, a second nucleic acid molecule encoding the heavy chain of the bispecific antigen-binding molecule antigen-binding arm A2, and a third nucleic acid molecule containing a nucleic acid sequence encoding a common light chain.

62. A composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 52, or the pharmaceutical composition according to claim 53, for inhibiting the proliferation of plasma cell tumors in a target.

63. The composition according to claim 62, wherein the plasma cell tumor is multiple myeloma.

64. A composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 52, or a pharmaceutical composition according to claim 53, for inhibiting the proliferation of a tumor in a target, wherein the tumor is selected from the group consisting of multiple myeloma, lymphoma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer partially characterized by having CD38+ cells.

65. A composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 52, or the pharmaceutical composition according to claim 53, for treating a patient suffering from a BCMA-expressing B-cell malignancy.

66. The composition according to claim 65, wherein the BCMA-expressing B-cell malignancy is selected from the group consisting of Waldenström macroglobulinemia, Burkitt lymphoma, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, multiple myeloma, and Hodgkin lymphoma.

67. The composition according to claim 62, further comprising the step of providing a second therapeutic agent or therapeutic regimen.

68. The composition according to claim 67, wherein the second therapeutic agent or therapeutic regimen is an antibody that binds to plasma cell tumors.

69. The composition according to claim 67, wherein the second therapeutic agent is an anti-BCMA / anti-CD3 bispecific antigen-binding molecule.

70. The composition according to claim 67, wherein the second therapeutic agent is an anti-CD20 / anti-CD3 bispecific antigen-binding molecule.

71. The composition according to claim 67, wherein the second therapeutic agent is a CD28 agonist or a 4-1BB agonist.

72. The composition according to claim 67, wherein the second therapeutic agent or therapeutic regimen comprises a chemotherapeutic agent, a DNA alkylating agent, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, radiotherapy, stem cell transplantation, different bispecific antibodies that interact with different tumor cell surface antigens and T cells or immune cell antigens, an antibody-drug conjugate, a bispecific antibody conjugated to an antitumor agent, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 checkpoint inhibitor, a CD28 agonist, a 4-1BB agonist, an anti-BCMA / anti-CD3 bispecific antigen-binding molecule, an anti-CD20 / anti-CD3 bispecific antigen-binding molecule, a cancer vaccine, an oncolytic virus, an immunocytokine, a CD22 inhibitor, an IL4 inhibitor, an IL6 inhibitor, a T cell containing a chimeric antigen receptor (CAR-T cell), or a combination thereof.

73. A composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 52, or the pharmaceutical composition according to claim 53, for treating a patient suffering from a CD38+ tumor and / or a BCMA-expressing tumor, comprising the step of administering the composition to a target in combination with a PD-1 inhibitor.

74. The composition according to claim 73, wherein the PD-1 inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

75. The composition according to claim 74, wherein the anti-PD-1 inhibitor is selected from semiprimab, nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab.

76. The composition according to claim 74, wherein the anti-PD-1 inhibitor is semiprimab.

77. A composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 52, or the pharmaceutical composition according to claim 53, for the treatment of a disease or disorder related to the expression of CD38, CD20, and / or BCMA.

78. The composition according to claim 77, wherein the disease or disorder is cancer.

79. The composition according to claim 78, wherein the cancer is multiple myeloma, lymphoma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer partially characterized by having CD38+ cells.

80. The composition according to claim 77, in which a further combination is made with an anti-PD-1 antibody or an antigen-binding fragment thereof.

81. The composition according to claim 77, wherein the bispecific antigen-binding molecule or pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.

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