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

Abstract

CD38 is expressed on malignant plasma cells. 4-1BB is a co-stimulatory molecule required for T cell activation and survival. Provided herein are novel multispecific antigen binding molecules (MABM) that bind to both CD38 and 4-1BB, for example, to provide a "signalling 2" in order to enhance T cell activation in the presence of a "signalling 1" provided by a tumor associated antigen (TAA) x CD3 bispecific antibody or an allogeneic reaction provided by an antigen presenting cell. In certain embodiments, the multispecific antigen binding molecules of the present invention are capable of inhibiting the growth of CD38 expressing tumors. The multispecific antigen-binding molecules of the present invention are useful in the treatment of diseases and conditions in which upregulation or induction of a CD38 targeted immune response is desirable and / or therapeutically beneficial. For example, the multispecific antigen binding molecules of the invention may be used to treat various cancers, including multiple myeloma, lymphoma, and leukemia.

Description

Technical Field

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

[0002] Sequence Listing

[0003] An official copy of the sequence listing is submitted electronically through the Patent Center at the same time as the specification. The contents of the electronic sequence listing (10927WO01_Sequence_Listing_ST26.xml; size: 151,552 bytes; and creation date: May 17, 2023) are incorporated herein by reference in their entirety. Background Art

[0004] Multiple myeloma (MM) is the second most common blood cancer after non-Hodgkin lymphoma, with a prevalence of about 120,000 in the United States, and about 30,000 new cases and 13,000 deaths each year. MM is characterized by the clonal expansion of malignant plasma cells that secrete cytokines in an unregulated manner. The production of cytokines, especially IL-6, causes local organ and tissue damage, resulting in many symptoms associated with myeloma. Subjects with MM suffer from bone pain and osteoporosis, anemia, impaired renal function and renal failure, bacterial infections and nerve damage. MM is rarely cured, with an average life expectancy of 4-5 years. Although progress has been made in the treatment of MM, new therapies have disproportionately benefited young patients. Relapsed MM patients have a poor prognosis and are in urgent need of new treatments.

[0005] CD38, also known as cyclic ADP ribose hydrolase, is a 45 kDa surface glycoprotein expressed on thymocytes, some activated peripheral blood T cells and B cells, plasma cells and dendritic cells. CD38 functions as an exoenzyme and is involved in the regulation of extracellular nicotinamide adenine dinucleotide (NAD + ) and the metabolism of cytoplasmic nicotinamide adenine dinucleotide phosphate (NADP) (Howard et al. Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyteantigen CD38. Science (1993) 262: 1056–9), leading to the production of Ca 2+Mobilizes compounds such as cyclic adenosine diphosphate (ADP) ribose, ADP ribose (ADPR), and nicotinic acid adenine dinucleotide phosphate. Calcium modulation leads to the activation of signaling pathways that control a wide range of physiological functions, including lymphocyte proliferation, insulin release from the pancreas, myocardial contraction, neutrophil chemotaxis, and T cell activation. CD38 enzyme activity regulates NAD + Levels and improve the function of proteasome inhibitors (Cagnetta et al. Intracellular NAD(+) depletion enhances bortezomib-induced anti-myeloma activity. Blood (2013) 122: 1243–55). In addition, ADPR can be metabolized by CD203a / PC-1 and CD73 to produce the immunosuppressive molecule adenosine (ADO), which promotes tumor cells to escape from the control of the immune system (Chillemi et al. Roles and modalities of ectonucleotidases in remodeling the multiple myeloma niche. Front Immunol. (2017) 8: 305). CD38 appears to contribute to the proliferative potential of B chronic leukemia / small lymphocytic lymphoma; malignant plasma cells in the bone marrow express high and uniform levels of CD38. Anti-CD38 monoclonal antibodies are believed to deplete CD38+ immunosuppressive cells, such as bone marrow-derived suppressor cells, regulatory T cells, and regulatory B cells, resulting in increased anti-tumor activity of immune effector cells. Daratumumab is an anti-CD38 antigen-binding molecule that has been approved for the treatment of patients with multiple myeloma that is resistant to conventional therapies.

[0006] T cell activation involves co-stimulation via the TNF receptor superfamily and is critical for survival, acquisition of effector function, and memory differentiation. 4-1BB (Tnfrsf9), also known as CD137, is a surface glycoprotein and member of the TNF receptor superfamily. Receptor expression is induced by TCR-mediated lymphocyte activation after priming, but its levels can be increased by CD28 co-stimulation. +Exposure of ligands or agonistic monoclonal antibodies on T cells produces co-stimulation of 4-1BB, which contributes to the clonal expansion, survival and development of T cells, the induced proliferation of peripheral monocytes, the activation of NF-κB, the activation-induced T cell apoptosis enhancement triggered by TCR / CD3, the generation of memory, and the regulation of CD28 co-stimulation (to promote Th1 cell response). Urerumab (BMS-663513) is a fully human IgG4 monoclonal antibody and the first anti-4-1BB therapeutic agent to enter clinical trials. When liver toxicity associated with the antibody was discovered, clinical development was stopped. Utorumab (PF-05082566) is a humanized IgG2 monoclonal antibody that activates 4-1BB while blocking binding to endogenous 4-1BBL. Therefore, there is a need in the art for alternative methods of treating cancer. Summary of the invention

[0007] The present invention relates in part to multispecific antigen binding molecules that bind CD38 and 4-1BB and their use in treating 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 comprise two antigen binding arms, A1 and A2. The A1 arm specifically binds to CD38. The A2 arm comprises a first antigen binding domain (R1) and a second antigen binding domain (R2), and A2 specifically binds to 4-1BB. R1 is connected to R2 via a linker to form a stacked antigen binding domain on the A2 arm. The combination of the A1 arm and the stacked A2 arm is referred to as a 1+2 format. The antigen binding domain of A2 may be contained in a Fab. See Figure 1 . In some aspects, R1 and R2 bind to different 4-1BB epitopes. In some aspects, R1 and R2 bind to the same 4-1BB epitope. In some aspects, the amino acid sequences of the R1 and R2 heavy chain variable regions are identical or substantially similar, that is, there are less than 5, less than 4, less than 3 amino acid differences in the heavy chain variable region or the heavy chain complementary determining region, or there are 2 or 1 amino acid differences in the heavy chain variable region or the heavy chain complementary determining region. In some aspects, the R1 and R2 heavy chain variable regions are different, that is, they have different antigen binding sequences. In some embodiments, R1 is included in the first Fab (Fab2), and R2 is included in the second Fab (Fab3). The Fab2 and Fab3 of the anti-CD38x anti-4-1BB1+2 construct are connected to the C-terminus of the CH1-3 "outer" Fab3 from the N-terminus of the VH-2 4-1BB "inner" Fab2 by a linker.

[0010] Multispecific antigen-binding molecules comprising anti-CD38 and anti-4-1BB antigen-binding domains

[0011] The present disclosure provides multispecific antigen binding molecules in combination with CD38 and 4-1BB. Such multispecific antigen binding molecules are also referred to herein as "anti-CD38 / anti-4-1BB multispecific antigen binding molecules". CD38 antigen binding arm A1 includes an antigen binding domain. 4-1BB antigen binding arm A2 includes two antigen binding domains, R1 and R2. R1 is referred to herein as 4-1BB "inner" binding domain, and R2 is referred to herein as 4-1BB "outer" domain. Multispecific antigen binding molecules with stacked antigen binding domains are referred to herein as anti-CD38 / anti-4-1BB 1+2 multispecific antigen binding molecules, or anti-CD38x anti-4-1BB 1+2 multispecific antigen binding molecules, etc.

[0012] The anti-CD38 part of anti-CD38 / anti-4-1BB multispecific molecule can be used for targeting tumor cells (such as plasma cells) expressing CD38, and the anti-4-1BB part of the multispecific molecule can be used for providing the co-stimulation of T cells activated by homologous MHC peptides or tumor-targeted CD3 multispecific antigen binding molecules.CD38 is combined with 4-1BB on tumor cells and promotes the directional killing (cell lysis) of activated T cells to targeted tumor cells by T cells.Therefore, the anti-CD38 / anti-4-1BB 1+2 multispecific molecules provided herein are particularly useful for treating diseases and disorders (for example, lymphoma, leukemia, multiple myeloma, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma or breast cancer) related to or caused by the tumor expressing CD38.

[0013] The multispecific antigen binding molecules provided herein include a first antigen binding arm A1 containing an antigen binding domain specifically binding to human CD38 and a second antigen binding arm A2 containing two antigen binding domains R1 and R2 specifically binding to 4-1BB. The present disclosure includes anti-CD38 / anti-4-1BB 1+2 multispecific molecules (e.g., multispecific antigen binding molecules), wherein each antigen binding domain includes a heavy chain variable region (HCVR) paired with a light chain variable region (LCVR). In certain exemplary embodiments of the present invention, the anti-CD38 antigen binding domain and the anti-4-1BB antigen binding domain each include a different, unique HCVR paired with a common LCVR or a universal LCVR. For example, as shown in Example 4 herein, a multispecific antigen binding molecule is constructed, which comprises 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; 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, wherein each HCVR is paired with a universal light chain LCVR. In such embodiments, the first, second, and third antigen binding domains comprise different anti-CD38 and anti-4-1BB HCVRs, respectively, but share a common light chain LCVR.

[0014] Provided herein are bispecific antigen binding molecules comprising:

[0015] (a) a first antigen-binding arm comprising three CDRs of a heavy chain variable region (HCVR) and three CDRs of a LCVR, wherein the first antigen-binding arm specifically binds to CD38; and

[0016] (b) a second antigen-binding arm comprising a first antigen-binding region (R1) comprising three CDRs of a HCVR (R1-HCVR) and three CDRs of a LCVR (R1-LCVR); and a second antigen-binding region (R2) comprising three CDRs of a HCVR (R2-HCVR) and three CDRs of a LCVR (R2-LCVR), wherein the second antigen-binding arm specifically binds to 4-1BB.

[0017] In some aspects, R1 and R2 bind to the same epitope on 4-1BB. In some aspects, R1 and R2 bind to different epitopes on 4-1BB.

[0018] In some aspects, R1 and R2 are connected by a peptide linker. An exemplary peptide linker comprises a peptide sequence of (GGGGS)n, wherein n is 1 to 6.

[0019] In some aspects, the bispecific antigen binding molecule comprises a first antigen binding arm, wherein the first antigen binding arm comprises three CDRs of a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40.

[0020] In some aspects, the bispecific antigen binding molecule comprises a first antigen binding arm, wherein the first antigen binding arm comprises three CDRs of a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0021] In some aspects, the bispecific antigen binding molecule comprises a first antigen binding arm, wherein the first antigen binding arm comprises three heavy chain complementarity determining regions (HCDR1-HCDR2-HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6-8 and 42-44-46, respectively.

[0022] In some aspects, 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) comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54, respectively.

[0023] In some aspects, 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.

[0024] In some aspects, the bispecific antigen binding molecule comprises a first antigen binding arm, wherein the first antigen binding arm comprises a LCVR, and the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0025] In some aspects, 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.

[0026] In some aspects, the bispecific antigen binding molecule comprises a first antigen binding arm, wherein the second antigen binding arm comprises a first antigen binding region (R1); and a second antigen binding region (R2).

[0027] In some aspects, R1 comprises three CDRs of a HCVR (R1-HCVR), wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94.

[0028] In some aspects, R1 comprises the three CDRs of a LCVR (R1-LCVR), wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0029] In some aspects, R1 comprises three heavy chain complementarity determining regions (R1-HCDR1-R1-HCDR2-R1-HCDR3), each of which comprises 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.

[0030] In some aspects, R1 comprises three light chain complementary determining regions (R1-LCDR1-R1-LCDR2-R1-LCDR3), each of which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54.

[0031] In some aspects, R1 comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94 (R1-HCVR).

[0032] In some aspects, R1 comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48 (R1-LCVR).

[0033] 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.

[0034] In some aspects, R2 comprises three CDRs of a HCVR (R2-HCVR), wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94.

[0035] In some aspects, R2 comprises the three CDRs of a LCVR (R2-LCVR), wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0036] In some aspects, R2 comprises three heavy chain complementarity determining regions (R2-HCDR1-R2-HCDR2-R2-HCDR3), each of which comprises 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.

[0037] In some aspects, R2 comprises three light chain complementary determining regions (R2-LCDR1-R2-LCDR2-R2-LCDR3), each of which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54.

[0038] In some aspects, R2 comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86, and 94 (R2-HCVR).

[0039] In some aspects, R2 comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48 (R2-LCVR).

[0040] 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.

[0041] In some aspects, the bispecific antigen binding molecule comprises:

[0042] (a) a first antigen-binding arm comprising three CDRs of a HCVR comprising the amino acid sequence of SEQ ID NO:40, and three CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO:48; and

[0043] (b) a second antigen-binding arm comprising:

[0044] (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 an amino acid sequence of SEQ ID NO: 48; and

[0045] (ii) a second antigen-binding region (R2) comprising three CDRs of an R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 84 and 94; and three CDRs of an R2-LCVR comprising an amino acid sequence of SEQ ID NO: 48.

[0046] In some aspects, the bispecific antigen binding molecule comprises:

[0047] (a) a first antigen-binding arm comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 respectively comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42-44-46-50-52-54; and

[0048] (b) a second antigen-binding arm comprising:

[0049] (i) a first antigen-binding region (R1) comprising R1-HCDR1-R1-HCDR2-R1-HCDR3-R1-LCDR1-R1-LCDR2-R1-LCDR3 comprising amino acid sequences 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, respectively; and

[0050] (ii) a second antigen-binding region (R2), which comprises R2-HCDR1-R2-HCDR2-R2-HCDR3-R2-LCDR1-R2-LCDR2-R2-LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 64-66-68-50-52-54, 74-76-78-50-52-54 and 88-90-92-50-52-54, respectively.

[0051] In some aspects, the bispecific antigen binding molecule comprises:

[0052] (a) a first antigen-binding arm comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 40, and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and

[0053] (b) a second antigen-binding arm comprising:

[0054] (i) a first antigen-binding region (R1) comprising an R1-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 62, and 72; and an R1-LCVR comprising an amino acid sequence of SEQ ID NO: 48; and

[0055] (ii) a second antigen-binding region (R2) comprising an R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:62, 86 and 94; and an R2-LCVR comprising an amino acid sequence of SEQ ID NO:48.

[0056] In some aspects, the bispecific antigen binding molecule comprises:

[0057] (a) a first antigen-binding arm comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 40, and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and

[0058] (b) a second antigen-binding arm comprising:

[0059] (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; and

[0060] (ii) a second antigen binding region (R2) comprising an R2-HCVR comprising SEQ ID NO:86; and an R2-LCVR comprising the amino acid sequence of SEQ ID NO:48.

[0061] In some aspects, the bispecific antigen binding molecule comprises:

[0062] (a) a first antigen-binding arm comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 40, and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and

[0063] (b) a second antigen-binding arm comprising:

[0064] (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; and

[0065] (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.

[0066] In some aspects, the bispecific antigen binding molecule comprises:

[0067] (a) the first antigen-binding arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 40 and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and

[0068] (b) the second antigen binding arm comprises:

[0069] (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

[0070] (ii) a second antigen binding region (R2) comprising an R2-HCVR comprising SEQ ID NO:62; and an R2-LCVR comprising the amino acid sequence of SEQ ID NO:48.

[0071] In some aspects, the bispecific antigen binding molecule is a bispecific antibody.

[0072] In some aspects, the bispecific antigen binding molecule is a bispecific antibody comprising a heavy chain constant region of the IgG1 or IgG4 isotype.

[0073] In some aspects, the bispecific antibody comprises a first heavy chain comprising a HCVR comprising a first antigen binding arm, and a second heavy chain comprising R1-HCVR and R2-HCVR comprising a second antigen binding arm, wherein the second heavy chain comprises mutations H435R and Y436F (EU numbering).

[0074] In some aspects, the bispecific antibody comprises a first heavy chain comprising a HCVR comprising a first antigen binding arm, paired with a light chain comprising a LCVR comprising a 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.

[0075] In some aspects, the bispecific antibody comprises a second heavy chain comprising R1-HCVR and R2-HCVR comprising a 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, wherein the second heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 70, 80, 82, and 84; and the 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.

[0076] In some aspects, the bispecific antibody comprises:

[0077] (a) a first antigen-binding arm that specifically binds to human CD38, the first antigen-binding arm comprising a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 60; and

[0078] (b) a second antigen-binding arm that specifically binds to human 4-1BB, the second antigen-binding arm 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.

[0079] In some aspects, the bispecific antibody comprises:

[0080] (a) a first antigen-binding arm that specifically binds to human CD38, the first antigen-binding arm comprising a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 60; and

[0081] (b) a second antigen-binding arm that specifically binds to human 4-1BB, the second antigen-binding arm 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.

[0082] In some aspects, the bispecific antibody comprises:

[0083] (a) a first antigen-binding arm that specifically binds to human CD38, the first antigen-binding arm comprising a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 60; and

[0084] (b) a second antigen-binding arm that specifically binds to human 4-1BB, the second antigen-binding arm 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.

[0085] In some aspects, the bispecific antibody comprises:

[0086] (a) a first antigen-binding arm that specifically binds to human CD38, the first antigen-binding arm comprising a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 60; and

[0087] (b) a second antigen-binding arm that specifically binds to human 4-1BB, the second antigen-binding arm 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.

[0088] In some aspects, the bispecific antibody comprises:

[0089] (a) a first antigen-binding arm that specifically binds to human CD38, the first antigen-binding arm comprising a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 60; and

[0090] (b) a second antigen-binding arm that specifically binds to human 4-1BB, the second antigen-binding arm 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.

[0091] Provided herein is 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:

[0092] (a) the first antigen-binding arm comprises three CDRs of a HCVR comprising the amino acid sequence of SEQ ID NO: 40, and three CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and

[0093] (b) the second antigen binding arm comprises:

[0094] (i) a first antigen-binding region (R1) comprising three CDRs of a HCVR (R1-HCVR) comprising the amino acid sequence of SEQ ID NO:62, and three CDRs of a LCVR (R1-LCVR) comprising the amino acid sequence of SEQ ID NO:48; and

[0095] (ii) a second antigen-binding region (R2) comprising three CDRs of a HCVR (R2-HCVR) comprising the amino acid sequence of SEQ ID NO:62, and three CDRs of a LCVR (R2-LCVR) comprising the amino acid sequence of SEQ ID NO:48.

[0096] In some aspects, the bispecific antigen binding molecule is a bispecific antibody.

[0097] In some aspects, the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen binding arm, wherein the first heavy chain is 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. In some aspects, the bispecific antibody comprises a second heavy chain comprising R1-HCVR and R2-HCVR of the second antigen binding arm, wherein the second heavy chain is paired with a first light chain comprising R1-LCVR and a second light chain comprising R2-LCVR, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NO: 70, 82, or 84, the 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.

[0098] In some aspects, the bispecific antigen binding molecule comprises:

[0099] (a) a first antigen-binding arm that specifically binds to human CD38, the first antigen-binding arm comprising a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 60; and

[0100] (b) a second antigen-binding arm that specifically binds to human 4-1BB, the second antigen-binding arm comprising:

[0101] (i) 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;

[0102] (ii) 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

[0103] (iii) 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.

[0104] In some embodiments, the multispecific antigen-binding molecule inhibits proliferation of a CD38+ tumor cell selected from the group consisting of a myeloma cell, a leukemia cell, a lymphoma cell, a hepatocellular carcinoma cell, a non-small cell lung cancer cell, a melanoma cell, a pancreatic ductal adenocarcinoma cell, a glioma cell, or a breast cancer cell.

[0105] In another aspect, a pharmaceutical composition is provided, the pharmaceutical composition comprising a multispecific antigen binding molecule and a pharmaceutically acceptable carrier or diluent. In related aspects, the present invention is characterized in that the composition 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 advantageously combined with an anti-CD38 / anti-4-1BB 1+2 multispecific antigen binding molecule.

[0106] In another aspect, a nucleic acid molecule comprising a nucleotide sequence encoding either A1 or A2 is provided. In some aspects, a nucleic acid molecule comprising a nucleotide sequence encoding any of HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, heavy chain and / or light chain is provided. In some embodiments, a nucleic acid molecule comprising one or more nucleotide sequences listed in Tables 2, 4, 6, 8 or 10 is provided. The nucleic acid molecules comprising the nucleic acid sequences may be in any functional combination or arrangement thereof.

[0107] In some aspects, provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region (HCVR) of the antigen-binding arm A1 of the multispecific antigen-binding molecule, wherein A1 binds to CD38, and wherein (a) the HCVR comprises three heavy chain complementarity determining regions (HCDR1-HCDR2-HCDR3) comprising the amino acid sequences of SEQ ID NO:42, 44 and 46, respectively, or (b) the HCVR comprises the amino acid sequence of SEQ ID NO:40.

[0108] In some aspects, provided herein are isolated nucleic acid molecules 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 wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:58.

[0109] In some aspects, provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region (HCVR) of an 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, and wherein (a) R1-HCVR comprises three heavy chain complementary 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 complementary 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.

[0110] 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 wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:56.

[0111] Also provided herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a first heavy chain variable region (HCVR) and a second HCVR of an antigen-binding arm A2 of a multispecific antigen-binding molecule, wherein A2 binds to 4-1BB, and wherein (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.

[0112] 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 the multispecific antigen binding molecule, wherein A2 binds to 4-1BB, and wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 82, and 84.

[0113] Provided herein are isolated nucleic acid molecules comprising nucleic acid sequences of a first heavy chain variable region (HCVR) and a second HCVR encoding an antigen binding arm A2 of a multispecific antigen binding molecule, wherein A2 binds to 4-1BB, and wherein (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.

[0114] 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 wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 80.

[0115] Also provided herein are isolated nucleic acid molecules comprising a nucleic acid sequence encoding a light chain variable region (LCVR) of a multispecific antigen-binding molecule, wherein (a) the LCVR comprises three heavy 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.

[0116] 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.

[0117] Provided herein is an expression vector or a group of expression vectors comprising one or more nucleic acid molecules of any of the nucleic acids described above. Also provided is a host cell, which comprises one or more expression vectors provided herein. In some aspects, the host cell is a mammalian cell or a prokaryotic cell. In some aspects, the host cell is a Chinese hamster ovary (CHO) cell or an Escherichia coli (E.coli) cell. Also provided is a composition comprising one or more nucleic acid molecules as described herein.

[0118] In some embodiments, a method for producing a multispecific antigen binding molecule is provided. As provided herein, the method includes growing a host cell under conditions allowing the production of a multispecific antigen binding molecule, wherein the host cell comprises a nucleic acid molecule containing a nucleic acid sequence encoding a heavy chain variable region (HCVR) of a multispecific antigen binding molecule antigen binding arm A1, a nucleic acid molecule encoding a nucleic acid sequence of a heavy chain variable region (HCVR) of a multispecific antigen binding molecule antigen binding arm A2, and / or a nucleic acid molecule containing 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 nucleic acid molecules are in different expression vectors. In some aspects, the host cell comprises a nucleic acid molecule containing a nucleic acid sequence encoding a heavy chain of a multispecific antigen binding molecule antigen binding arm A1, a nucleic acid molecule containing a nucleic acid molecule encoding a heavy chain of a multispecific antigen binding molecule antigen binding arm A2, and / or a nucleic acid molecule containing 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 nucleic acid molecules are in different expression vectors.

[0119] Provided herein are methods of inhibiting the growth of a plasma cell neoplasm 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 plasma cell neoplasm is multiple myeloma.

[0120] Also provided herein is the use of a multispecific antigen-binding molecule as described herein or a pharmaceutical composition as provided herein in the manufacture of a medicament for inhibiting the growth of a plasma cell tumor in a subject. The multispecific antigen-binding molecule or pharmaceutical composition can be administered to the subject. In some aspects, the plasma cell tumor is multiple myeloma.

[0121] Provided herein are methods of inhibiting the growth of a 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 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.

[0122] Also provided herein is the use of a multispecific antigen binding molecule as described herein or a pharmaceutical composition as provided herein in the manufacture of a medicament for inhibiting the growth of a tumor in a subject. The multispecific antigen binding molecule or pharmaceutical composition can be administered to the subject. In some aspects, 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.

[0123] Provided herein are methods for treating a patient with multiple myeloma or with another B-cell malignancy expressing BCMA, the method comprising administering to the subject a multispecific antigen-binding molecule provided herein or a pharmaceutical composition provided herein. In some aspects, the B-cell malignancy expressing BCMA is selected from the group consisting of: Waldenstrom's macroglobulinemia ( smacroglobulinemia), Burkitt's lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin's lymphoma.

[0124] 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 a patient with multiple myeloma or with another B-cell malignancy expressing BCMA. The multispecific antigen-binding molecule or pharmaceutical composition can be administered to the subject.

[0125] Provided herein are methods for treating patients with CD38+ tumors and / or tumors expressing BCMA. The method includes administering to the subject a multispecific antigen binding molecule described herein in combination with an anti-PD-1 antibody or its antigen-binding fragment or a pharmaceutical composition described herein. In some aspects, the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody, such as cemiplimab (bsAb2810).

[0126] 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 a patient having a CD38+ tumor and / or a tumor expressing BCMA.

[0127] In some embodiments, the method or use provided herein also includes administering a second therapeutic agent or treatment regimen. In some aspects, the second therapeutic agent is an antibody in conjunction with plasma cell tumors. In some aspects, the second therapeutic agent is an anti-BCMA / anti-CD3 bispecific antigen binding molecule. In some aspects, the second therapeutic agent is an anti-CD20 / anti-CD3 bispecific antigen binding molecule. In some aspects, the second therapeutic agent is an anti-CD28 / anti-4-1BB bispecific antigen binding molecule. In some aspects, the second therapeutic agent or treatment regimen includes chemotherapeutic drugs, DNA alkylating agents, immunomodulators, proteasome inhibitors, histone deacetylase inhibitors, radiotherapy, stem cell transplantation, oncolytic viruses, cancer vaccines, immunocytokines, CAR-T cells, different bispecific antibodies interacting with different tumor cell surface antigens and T cells or immune cell antigens, antibody drug conjugates, bispecific antibodies conjugated to anti-tumor agents, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 checkpoint inhibitors, CD22 inhibitors, BCMA inhibitors, CD28 agonists, CD20 inhibitors, or combinations thereof.

[0128] Also provided is the use of the multispecific antigen binding molecules provided herein or the pharmaceutical compositions provided herein in the treatment of diseases or conditions associated with CD38, CD20 and / or BCMA expression. In some aspects, the disease or condition is cancer. In some aspects, the cancer is multiple myeloma. In some aspects, the multispecific antigen binding molecules or pharmaceutical compositions are used in combination with anti-PD-1 antibodies or antigen-binding fragments thereof. The multispecific antigen binding molecules or pharmaceutical compositions comprising the multispecific antigen binding molecules are injected intravenously, intramuscularly or subcutaneously.

[0129] Anti-CD38 / anti-4-1BB 1+2 multispecific antigen binding molecules with modified glycosylation patterns are provided herein. In some applications, it may be useful to remove the modification of undesirable glycosylation sites, or the antigen binding molecules lacking fucose moieties are present on oligosaccharide chains, for example, to increase antigen binding molecule dependent cellular toxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylated modification can be performed to change complement dependent cytotoxicity (CDC).

[0130] In yet another aspect, provided herein is a therapeutic method for targeting / killing tumor cells expressing CD38 using the anti-CD38 / anti-4-1BB multispecific antigen-binding molecules of the present invention, wherein the therapeutic method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the anti-CD38 / anti-4-1BB multispecific antigen-binding molecules provided herein.

[0131] The present disclosure also includes the use of the anti-CD38 / anti-4-1BB multispecific antigen-binding molecules provided herein in the preparation of a medicament for treating a disease or condition associated with or caused by CD38 expression.

[0132] Other embodiments will become apparent by reference to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 It is a schematic diagram depicting the structure of an exemplary anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molecule. The A1 antigen binding arm comprises Fab1, which is specific for CD38, and the A2 antigen binding arm comprises two Fabs (Fab2 and Fab3), each of which is specific for 4-1BB. The Fab2 and Fab3 of the anti-CD38x anti-4-1BB 1+2 construct are connected to the C-terminus of CH1-3 "outer" Fab3 from the N-terminus of VH-2 4-1BB "inner" Fab2 via a linker. Fab1, Fab2 and Fab3 light chains comprise VL-1, VL-2 and VL-3 universal light chains, respectively.

[0134] Figure 2A The activation percentages of various formats of multispecific antigen-binding molecules against 4-1BB in a Jurkat reporter gene assay were compared, including identically split 4-1BB Fabs, identically stacked 4-1BB Fabs, and mixed-stacked 4-1BB Fabs. Figure 2B Various constructs tested in the screen are shown, and the target-dependent bioassays are depicted.

[0135] Figure 3 4x10 6 Immunodeficient NOD.Cg-Prkdc from peripheral blood mononuclear cells (PBMCs) of individuals scid Iq tm1Wjl In vivo tumor burden over time following administration of human multiple myeloma tumor cells in WT / SzJ(NSG) mice.

[0136] Figure 4A Shown is the tumor burden over time in mice treated with PBS relative to mice that did not receive tumor cells; Figure 4B Shown is the tumor burden over time in mice treated with a control bsAb binding CD3 (0.4 mg / kg) + a control bsAb binding 4-1BB (4 mg / kg) relative to mice that did not receive tumor cells; Figure 4C Shown is the tumor burden over time in mice treated with a control CD3-binding bsAb (0.4 mg / kg) + CD38x4-1BB (4 mg / kg) relative to mice that did not receive tumor cells; Figure 4D Shown is the tumor burden over time in mice treated with BCMAxCD3bsAb (0.4 mg / kg) + a control bsAb that binds 4-1BB (4 mg / kg) relative to mice that did not receive tumor cells; Figure 4E Shown is the tumor burden over time in mice treated with BCMAxCD3bsAb (0.4 mg / kg) + CD38x4-1BB (4 mg / kg) relative to mice that did not receive tumor cells. DETAILED DESCRIPTION

[0137] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims.

[0138] Unless defined otherwise, 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.

[0139] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0140] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0141] definition

[0142] As used herein, the expression "4-1BB" refers to the receptor of 4-1BBL. 4-1BB is cross-linked by 4-1BBL to enhance T cell activation. Human 4-1BB (or CD137) with UniProt accession number Q07011 comprises an amino acid sequence as shown in SEQ ID NO: 101; amino acid residues 1-23 are signal peptides. Residues 24-186 constitute the extracellular domain of the receptor.

[0143] Human 4-1BB (immunogen) amino acid sequence

[0144] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQK RGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:101)

[0145] All references to proteins, polypeptides and protein fragments herein are intended to represent the human forms of various proteins, polypeptides or protein fragments, unless explicitly stated to be from non-human species. Therefore, the expression "4-1BB" means human 4-1BB, unless specified as being from non-human species, for example, "mouse 4-1BB", "monkey 4-1BB" etc.

[0146] As used herein, "antigen binding molecules that bind 4-1BB" or "anti-4-1BB antigen binding molecules" include antigen binding molecules that specifically recognize 4-1BB expressed on the cell surface. The anti-4-1BB antigen binding molecules provided herein include VR and CDR as disclosed herein. In certain embodiments, the antigen binding molecule is an antibody. In certain embodiments, the antigen binding molecule is a bispecific antibody.

[0147] As used herein, the expression "CD38", also known as cyclic ADP ribose hydrolase, refers to a glycoprotein expressed on malignant plasma cells. CD38 plays an important role in regulating 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. The term "CD38" as used herein refers to human CD38 protein, unless specified as being from a non-human species (e.g., "mouse CD38", "monkey CD38", etc.). Human CD38 protein has an amino acid sequence as shown in SEQ ID NO: 102 (human CD38 extracellular domain (V43-I300). mFc), and / or has an amino acid sequence as shown in NCBI accession number NP_001766.2 or NM_001775.3.

[0148] Human CD38 extracellular domain (V43-I300).mFc (immunogen) amino acids

[0149] VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQ SCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLM ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDL PAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK *(SEQ ID NO: 102) mFc sequence is underlined

[0150] As used herein, “antigen binding molecules that bind to CD38” or “anti-CD38 antigen binding molecules” include antigen binding molecules that specifically recognize CD38.

[0151] The term "antigen binding molecule" includes multispecific antigen binding molecules, such as anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molecules. The anti-CD38 antigen binding molecules provided herein include VR and CDR, as disclosed herein. In certain embodiments, the antigen binding molecule is an antibody. In certain embodiments, the antigen binding molecule is a bispecific antibody.

[0152] As used herein, the term "antigen binding molecules" means any antigen binding molecules or molecular complexes comprising at least one complementary determining region (CDR) that specifically binds or interacts with a specific antigen (e.g., CD38 or 4-1BB). The term "antigen binding molecules" includes immunoglobulin molecules and multimers thereof (e.g., IgM) comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds. The term "antigen binding molecules" includes immunoglobulin molecules comprising two antigen binding arms A1 and A2. The term "antigen binding molecules" also includes immunoglobulin molecules consisting of four polypeptide chains (two heavy chains (H) and two light chains (L)) interconnected by disulfide bonds. Each antigen binding arm includes a heavy chain, which in turn includes 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 comprises a light chain variable region (abbreviated herein as LCVR; on the A1 arm, VL-1; on the A2 arm, VL-2 and VL-3) and a light chain constant region (on the A1 arm, CL-1, and on the A2 arm, CL-2 and CL-3). The VH and VL regions can also be subdivided into hypervariable regions (called complementary determining regions (CDRs)), which are interspersed with more conservative regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FR of the anti-CD38 antigen binding arm or the anti-4-1BB antigen binding arm (or its antigen binding portion) may be identical to the human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0153] As used herein, the term "antigen binding portion thereof", "antigen binding fragment" of antigen binding molecules, etc. include any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to antigen to form a complex. For example, any suitable standard technique (such as proteolytic digestion or the operation and expression of DNA encoding antigen binding molecule variable domains and optionally antigen binding molecule constant domains) can be used, for example, to derive the antigen binding fragment of antigen binding molecules from full antigen binding molecule molecules. Such DNA is known, and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antigen binding molecule libraries), or can be synthesized. DNA sequencing can be performed, and chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into suitable configurations, or to import codons, to produce cysteine ​​residues, to modify, add or delete amino acids, etc.

[0154] Anti-CD38 x anti-4-1BB 1+2 antigen binding molecules will generally include at least three variable domains. The variable domains can have any size or amino acid composition and will generally include at least one CDR adjacent to or in line with the frame of one or more framework sequences. When there is a VH domain associated with a VL domain, 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 contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding molecule can contain a monomer VH or VL domain.

[0155] In certain embodiments, an antigen binding molecule may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in multispecific antigen binding molecules of the invention include: (i) 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-CH 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 (xvi)V L -3-C L 3-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 H1-2-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 In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected via a full-length or partial hinge or linker region. The hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule.

[0156] For example, Figure 1 Shown is an A1 antigen binding arm comprising Fab1 specific for CD38 and an A2 antigen binding arm comprising two Fabs (Fab2 and Fab3) each specific for 4-1BB. The Fab1, Fab2, and Fab3 light chains can be universal light chains. In this example, V L -1 is connected to C L -1, which is connected to C on the A1 arm H 1-1. On the A2 arm, V L -3 connects to C L -3, which is connected to C H 1-3. Similarly, V L -2 connects to C L -2, which is connected to C H 1-2. Fab2 and Fab3 of anti-CD38xanti-4-1BB 1+2 constructs are linked from V H -2 4-1BB "inner" Fab2 N-terminus connected to C H 1-3 "outside" Fab3.

[0157] The antigen binding molecules of the present invention can act by complement dependent cytotoxicity (CDC) or antigen binding molecule dependent cell-mediated cytotoxicity (ADCC). "Complement dependent cytotoxicity" (CDC) refers to the cells expressing antigens by the antigen binding molecules of the present invention in the presence of complement. "Antigen binding molecule dependent cell-mediated cytotoxicity" (ADCC) refers to the cell-mediated reaction of the antigen binding molecules combined on the target cell and thus causing the cracking of the target cell in which the non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) expressing Fc receptors (FcR) recognize the antigen binding molecules combined on the target cell and thus cause the cracking of the target cell. CDC and ADCC can be measured using assays well known in the art and available. (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 for the ability of the antigen binding molecule to fix complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of an antigen-binding molecule can be selected based on whether the antigen-binding molecule is required for mediating cytotoxicity.

[0158] In certain embodiments of the present invention, the anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molecules provided herein are human antigen binding molecules. As used herein, the term "human antigen binding molecules" is intended to include antigen binding molecules with variable regions and constant regions derived from human germline immunoglobulin sequences. The human antigen binding molecules of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo), for example, in CDR, particularly CDR3. However, as used herein, the term "human antigen binding molecules" is not intended to include antigen binding molecules that have been implanted into human framework sequences from CDR sequences derived from germline of another mammalian species (such as mice).

[0159] In some embodiments, the multispecific antigen binding molecules provided herein can be recombinant human antigen binding molecules. As used herein, the term "recombinant human antigen binding molecules" is intended to include all human antigen binding molecules prepared, expressed, produced or separated by recombinant means, such as antigen binding molecules expressed by recombinant expression vectors transfected into host cells (described further below), antigen binding molecules separated from recombinant human antigen binding molecule combinatorial libraries (described further below), antigen binding molecules separated from transgenic animals (e.g., mice) (see, e.g., Taylor et al., (1992) Nucl. Acids Res. 20: 6287-6295) for human immunoglobulin genes or antigen binding molecules prepared, expressed, produced or separated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antigen binding molecules have variable regions and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antigen binding molecules are subjected to in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, in vivo somatic cell mutagenesis) and thus the V H and V L Although the amino acid sequence of the region is derived from human germline V H and V L Sequences that are related to but that may not naturally occur within the human antigen-binding molecule germline repertoire in vivo.

[0160] 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 about 150-160 kDa, in which the dimer is held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not connected via an interchain disulfide bond, and the molecule of about 75-80 kDa is composed of a covalently coupled light chain and a heavy chain (hapten binding molecule). Even after affinity purification, these forms are also extremely difficult to separate.

[0161] The frequency of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with 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 (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed with the human IgG1 hinge. The present invention encompasses substitutions in the hinge, C H 2 or C H Antigen-binding molecules having one or more mutations in region 3 may be desirable, for example, in production to increase the yield of the desired form of the antigen-binding molecule.

[0162] The multispecific antigen-binding molecules of the present invention may be isolated antigen-binding molecules. As used herein, "isolated multispecific antigen-binding molecules" means antigen-binding molecules that have been identified and separated and / or recovered from at least one component of the natural environment. For example, for the purposes of the present invention, antigen-binding molecules that have been separated or removed from at least one component of an organism or separated or removed from tissues or cells in which the antigen-binding molecules naturally exist or are naturally produced are "isolated antigen-binding molecules". Isolated antigen-binding molecules also include in situ antigen-binding molecules in recombinant cells. Isolated antigen-binding molecules are antigen-binding molecules that have been subjected to at least one purification or separation step. According to certain embodiments, the isolated antigen-binding molecules may be substantially free of other cellular substances and / or chemicals.

[0163] As compared to the corresponding germline sequence derived from the antigen binding molecule, the anti-CD38 x anti-4-1BB 1+2 antigen binding molecules disclosed herein may include one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR of the heavy chain and light chain variable domains. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, a public antigen binding molecule sequence database. The present disclosure includes multispecific antigen-binding molecules or antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more frameworks and / or CDRs are mutated relative to the sequence provided herein. In some aspects, mutations are made to reflect the corresponding residues of the germline sequence derived from the antigen binding molecule, or to reflect the corresponding residues of another human germline sequence, or to reflect the conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). A person of ordinary skill in the art can easily produce a variety of antigen binding molecules starting from the heavy chain and light chain variable region sequences disclosed herein, and the antigen binding molecules include one or more mutations, such as separate germline mutations or combinations thereof. In certain embodiments, V H and / or V L All frameworks and / or CDR residues in the domain are mutated back to the residue present in the original germline sequence derived from antigen binding molecules. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the mutated residues present in the first 8 amino acids of FR1 or the rear 8 amino acids of FR4, or only the mutated residues present in CDR1, CDR2 or CDR3. In other embodiments, one or more mutations in framework and / or CDR residues become one or more corresponding residues of different germline sequences (that is, different from the germline sequence of the germline sequence that initially derived antigen binding molecules).

[0164] In addition, the multispecific antigen-binding molecules of the present invention may contain any combination of two or more mutations within the framework and / or CDR regions, for example, where certain individual residues are mutated or mutated to the corresponding residues of a particular germline sequence relative to the sequences provided herein, while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. After obtaining an antigen-binding molecule containing one or more mutations, one or more desired properties of the antigen-binding domain can be easily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antigen-binding molecules obtained in this general manner are encompassed within the present invention.

[0165] Provided herein are anti-CD38 x anti-4-1BB antigen binding molecules, including variants with one or more substitutions of any HCVR, LCVR and / or CDR amino acid sequences disclosed herein. In some aspects, the substitutions are conservative amino acid substitutions. For example, the disclosure includes anti-CD38 x anti-4-1BB antigen binding molecules with HCVR, LCVR and / or CDR amino acid sequences, wherein the HCVR, LCVR and / or CDR amino acid sequences have, for example, 10 or less, 8 or less, 6 or less, 4 or less, 3 or less, 2 or 1 amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences shown in Tables 1, 3, 5 or 7 herein.

[0166] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (called a paratope) in the variable region of an antigen binding molecule. A single antigen may have more than one epitope. Therefore, different antigen binding molecules may bind to different regions on an antigen and may have different biological effects. An epitope may be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, a phosphoryl group, or a sulfonyl group moiety on an antigen.

[0167] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases are identical in nucleotide sequence as measured by any of the well-known sequence identity algorithms discussed below, such as FASTA, BLAST or Gap. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an identical or substantially similar amino acid sequence to a polypeptide encoded by the reference nucleic acid molecule.

[0168] When applied to polypeptides, the term "substantially similar" or "substantially similar" means that when two peptide sequences are optimally aligned, such as by the program GAP or BESTFIT using default gap weights, there is at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Amino acid substitutions that do not substantially change the functional properties of multispecific antigen-binding molecules are contemplated herein. In some aspects, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of proteins. In the case where the conservative substitutions of two or more amino acid sequences are different from each other, the sequence identity percentage or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups containing side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy 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, i.e., 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 that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445 (incorporated herein by reference). A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0169] Sequence analysis software is usually used to measure the sequence similarity of polypeptides, and the sequence similarity is also referred to as sequence identity. Protein analysis software uses similarity metrics about being assigned to various displacements, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, and these programs can be used with default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from organisms of different species), or between wild-type proteins and mutant proteins thereof. See, for example, GCG version 6.1. FASTA (the program in GCG version 6.1) can also be used to compare polypeptide sequences using default or recommended parameters. FASTA (for example, FASTA2 and FASTA3) provides comparison and sequence identity percentage (Pearson (2000) the same) of the best overlapping region between query and search sequence. When the sequence of the present invention is compared with a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., 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.

[0170] Binding properties of antigen-binding molecules

[0171] As used herein, the term "binding" in the context of the binding of an antigen binding molecule, an immunoglobulin, an antigen binding molecule binding fragment or an Fc-containing protein to, for example, a predetermined antigen (such as a cell surface protein) or a fragment thereof, generally refers to an interaction or association between two entities or molecular structures, such as an antigen binding molecule-antigen interaction.

[0172] For example, when measured by, for example, surface plasmon resonance (SPR) technology in a BIAcore instrument using an antigen as a ligand and an antigen-binding molecule, Ig, antigen-binding molecule binding fragment, or Fc-containing protein as an analyte (or anti-ligand), the binding affinity generally corresponds to a K D The value is about 10 -7 M or less, such as about 10 -8 M or less, such as about 10 -9M or less. Cell-based binding strategies, such as fluorescence activated cell sorting (FACS) binding assays are also frequently used, and FACS data correlate well with other methods such as radioligand competition 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).

[0173] Thus, the multispecific antigen-binding molecules or antigen-binding proteins of the invention bind to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to K D The affinity of the binding site is at least ten times lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). D An antigen binding molecule with an affinity value equal to or less than ten times that of a nonspecific antigen may be considered to have undetectable binding, however such an antigen binding molecule may be paired with a second antigen binding arm for generating a multispecific antigen binding molecule of the invention.

[0174] The term "K D "(M)" refers to the dissociation equilibrium constant of a specific antigen-binding molecule-antigen interaction, or the dissociation equilibrium constant of the binding of an antigen-binding molecule or antigen-binding molecule binding fragment to an antigen. K D There is an inverse relationship between K and binding affinity, so K D The smaller the value, the higher the affinity, i.e. the stronger it is. Therefore, the term "higher affinity" or "stronger affinity" relates to a higher ability to form an interaction, and thus K D In contrast, the term "lower affinity" or "weaker affinity" relates to a lower ability to form an interaction, so K D In some cases, the same as the numerator ( 例如 A higher binding affinity (or KD) of a particular molecule (e.g., an antigen binding molecule) to its interacting partner molecule (e.g., antigen X) compared to the binding affinity of an antigen binding molecule (e.g., antigen Y) to another interacting partner molecule (e.g., antigen Y) can be expressed by substituting a larger KD for D value (lower or weaker affinity) divided by the smaller K D The binding ratio is determined by comparing the binding affinity of the target protein to that of the target protein (higher or stronger affinity), for example, expressed as a 5-fold or 10-fold greater binding affinity, as the case may be.

[0175] The term “k d "(seconds-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 koff value.

[0176] The term “k a "(M-1 x second-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.

[0177] The term "K A "(M-1 or 1 / M) refers to the binding equilibrium constant of a specific antigen-binding molecule-antigen interaction, or the binding equilibrium constant of an antigen-binding molecule or antigen-binding molecule binding fragment. The binding equilibrium constant is calculated by dividing k a Divide by k d To obtain.

[0178] The term "EC50" or "EC 50 ” refers to the half-maximal effective concentration, which includes the concentration of the antigen-binding molecule that induces a response halfway between baseline and maximum after a specified exposure time. EC 50 Essentially represents the concentration of antigen binding molecule at which 50% of the maximal effect is observed. 50 The value is equal to the concentration of the antigen binding molecule of the present invention that produces half-maximal binding to cells expressing 4-1BB or tumor-associated antigens (e.g., CD38), as determined by, for example, a FACS binding assay. 50 or an increase in the half-maximal effect concentration value, a decrease or weakening of binding was observed.

[0179] In one embodiment, reduced binding can be defined as the EC being able to bind to half the maximal number of target cells. 50 The concentration of antigen binding molecules increases.

[0180] In another embodiment, EC 50 The value represents the concentration of the antigen-binding molecule of the invention that causes half-maximal depletion of target cells through T cell cytotoxic activity. 50 or a decrease in the half-maximal effect concentration value, an increase in cytotoxic activity (e.g., T cell-mediated tumor cell killing) was observed.

[0181] Multispecific antigen binding molecules

[0182] The antigen binding molecules of the present invention bind both CD38 and 4-1BB. Multispecific antigen binding molecules can be specific for different epitopes of a target polypeptide, or can contain antigen binding domains that are specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244.

[0183] According to certain exemplary embodiments, the present invention includes multispecific antigen binding molecules that specifically bind to 4-1BB and CD38. Such molecules may be referred to herein as, for example, "anti-CD38 x anti-4-1BB 1+2" or "anti-CD38 / anti-4-1BB1+2" or "anti-CD38x4-1BB 1+2" or "CD38x4-1BB 1+2" multispecific molecules or other similar terms.

[0184] The present disclosure includes multispecific antigen binding molecules, wherein one arm A2 of the immunoglobulin has two antigen binding domains that bind to human 4-1BB, 4-1BB "inner" domain, and 4-1BB "outer" domain, and the other arm A1 of the immunoglobulin has specificity for binding to human CD38. The 4-1BB binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Table 3 (anti-4-1BB "inner") or Table 5 (anti-4-1BB "outer"), in a stacked form.

[0185] In certain embodiments, the 4-1BB binding arm is combined with human 4-1BB and promotes human T cell activation. In certain embodiments, the 4-1BB binding arm is combined with human 4-1BB and induces human T cell activation. In other embodiments, the 4-1BB binding arm is combined with human 4-1BB, and induces tumor-related antigen-expressing cell killing in the context of multispecific or multispecific antigen binding molecules. The CD38 binding arm may include any of the HCVR / LCVR or CDR amino acid sequences shown in Table 1 herein.

[0186] As used herein, the expression "antigen binding molecule" means a protein, polypeptide or molecular complex comprising or consisting of at least one complementary determining region (CDR) that specifically binds to a specific antigen, either alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, an antigen binding molecule is an antigen binding molecule or a fragment of an antigen binding molecule, such as those terms defined elsewhere herein.

[0187] As used herein, the expression "multi-specific antigen binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain within a multi-specific antigen binding molecule comprises at least one CDR that specifically binds to a specific antigen 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 (eg, CD38), and the second antigen binding arm A2 specifically binds to a second and third antigen (eg, 4-1BB) different from the first antigen.

[0188] The multispecific antigen-binding molecules discussed herein can comprise a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is of isotype IgG1. In some cases, the human IgG heavy chain constant region is of isotype IgG4. In various embodiments, the multispecific antigen-binding molecules comprise a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0189] The first antigen binding arm A1 and the second antigen binding arm A2 can be directly or indirectly connected to each other to form a multi-specific 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 multi-domain. The combination of a multi-domain with another multi-domain promotes the combination between the two antigen binding domains, thereby forming a multi-specific antigen binding molecule. As used herein, a "multi-domain" is any macromolecule, protein, polypeptide, peptide or amino acid that has the ability to bind to a second multi-domain of the same or similar structure or structure. For example, a multi-domain can be a domain comprising immunoglobulin C. H A non-limiting example of a multimeric component is the Fc portion of an immunoglobulin (comprising C H 2-C H 3 domains), such as an Fc domain of IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype of IgG within each isotype group.

[0190] The multispecific antigen-binding molecules of the present invention will generally comprise two multimerization domains, such as two Fc domains, each of which is individually part of a separate antigen-binding molecule heavy chain. The first multimerization domain and the second multimerization domain may have the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimerization domain and the second multimerization domain may have different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0191] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length, which comprises at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short peptide containing cysteine. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helical loop motif, or a coiled coil motif.

[0192] Any multispecific antigen binding molecule format or technology can be used to prepare the multispecific antigen binding molecules of the present invention. For example, an antigen binding molecule or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities, such as another antigen binding molecule or antigen binding molecule fragment having a second antigen binding specificity, to produce a multispecific antigen binding molecule. Specific exemplary multispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or dual-body multispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs into holes, common light chains (e.g., common light chains with knobs into holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab 2 Multispecific formats (for review of the aforementioned formats, see, eg, Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein).

[0193] In the context of the multispecific antigen-binding molecules of the invention, a multimerization domain (e.g., an Fc domain) can comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of an Fc domain. For example, the invention includes multispecific antigen-binding molecules comprising one or more modifications in an Fc domain that result in a modified binding interaction (e.g., enhanced or weakened) between the Fc and FcRn of the modified Fc domain. In one embodiment, the multispecific antigen-binding molecule comprises one or more modifications in an Fc domain. H 2 or C HIn the 3rd region, the modification is included, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome with a pH range of 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); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., or T) and 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 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 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0194] The present disclosure also includes a first C H 3 domain and the second Ig C H A multispecific antigen-binding molecule with 3 domains, wherein the first and second Ig C H At least one amino acid in the first Ig C domains differs from the first Ig C domains, and wherein at least one amino acid difference reduces binding of the multispecific antigen-binding molecule to protein A compared to a bispecific antigen-binding molecule lacking the amino acid difference. H 3 domains bind protein A and the second Ig C H The 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may also contain a Y96F modification (according to IMGT; Y436F according to EU). H 3 may also contain the L105P modification (according to IMGT; L455P according to EU). See, e.g., U.S. Pat. No. 8,586,713. HOther modifications within 3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antigen-binding molecules; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) in the case of IgG2 antigen-binding molecules; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antigen-binding molecules.

[0195] In certain embodiments, the Fc domain can be chimeric, combining Fc sequences from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise Fc sequences from human IgG1, human IgG2, or human IgG4. H C in Zone 2 H 2 sequence, and C derived from human IgG1, human IgG2 or human IgG4 H 3 sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, combined with a "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule described herein include, from N-terminus to C-terminus: [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any antigen-binding molecule described herein comprises, from N-terminus to 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 can be included in any antigen binding molecule of the invention are described in U.S. Patent No. 9,359,437, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector function.

[0196] Sequence variants

[0197] Compared to the corresponding germline sequence from which a separate antigen binding domain is derived, 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 region and / or CDR region of the heavy chain variable domain and the light chain variable domain. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, a public antigen binding molecule sequence database. The antigen binding molecules of the present invention may include antigen binding domains derived from exemplary amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions are mutated into corresponding residues of the germline sequence from which the antigen binding molecule is derived, or corresponding residues of another human germline sequence, or conservative amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). A person of ordinary skill in the art can easily produce a variety of antigen binding molecules starting from the heavy chain and light chain variable region sequences disclosed herein, the antigen binding molecules comprising one or more separate germline mutations or combinations thereof. In certain embodiments, V H and / or V L All framework and / or CDR residues in the domain are mutated back to the residues present in the original germline sequence from which the antigen-binding domain was initially derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues present in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues present in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated into different germline sequences (i.e., different from the germline sequence from which the antigen-binding domain was initially derived) corresponding to one or more residues.

[0198] In addition, the antigen binding domain may contain any combination of two or more germline mutations within the framework region and / or CDR region, for example, where certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues different from the original germline sequence are retained or mutated to the corresponding residues of a different germline sequence. After obtaining an antigen binding domain containing one or more germline mutations, one or more desired properties of the antigen binding domain can be easily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Multispecific antigen binding molecules comprising one or more antigen binding domains obtained in this general manner are encompassed in the present invention.

[0199] pH-dependent binding

[0200] The present invention includes anti-CD38 x anti-4-1BB multispecific antigen binding molecules with pH-dependent binding properties. For example, compared with at neutral pH, the anti-CD38 antigen binding arm of the present invention may show reduced binding to CD38 at acidic pH. Alternatively, compared with at neutral pH, the anti-CD38 antigen binding arm of the present invention enhances binding to CD38 at acidic pH. The expression "acidic pH" includes 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 pH value. As used herein, the expression "neutral pH" refers to a pH of 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.

[0201] In certain instances, "reduced binding at acidic pH compared to at neutral pH" refers to the K of an antigen-binding molecule binding to its antigen at acidic pH. D The K value of the antigen-binding molecule binding to its antigen at neutral pH D For example, for the purposes of the present invention, if an antigen-binding molecule exhibits an acidic / neutral K D 0 or greater, then the antigen-binding molecule can be considered to exhibit "reduced binding to CD38 at acidic pH compared to at neutral pH". In certain exemplary embodiments, the acidic / neutral K of the antigen-binding molecule of the invention is D The ratio may 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.

[0202] Can for example by with respect to compared with neutral pH at acidic pH and specific antigen binding reduction (or enhancement) and screening antigen binding molecule colony to obtain the antigen binding molecule with pH dependency binding characteristics.In addition, the antigen binding molecule with pH dependency characteristics can be produced at amino acid level modification antigen binding domain.For example, by one or more amino acids (for example, inside CDR) of antigen binding domain are replaced with histidine residues, can obtain relative to neutral pH at acidic pH antigen binding action reduction antigen binding molecule.

[0203] Antigen binding molecules comprising Fc variants

[0204] According to certain embodiments of the present invention, there is provided an anti-CD38 x anti-4-1BB multispecific antigen-binding molecule comprising an Fc domain having one or more mutations, wherein the mutations enhance or weaken the binding of the antigen-binding molecule to the FcRn receptor at an acidic pH compared to at a neutral pH. For example, the present invention includes an antigen-binding molecule wherein the antigen-binding molecule has a C H 2 or C H In the 3rd region, mutations are included, wherein one or more mutations increase the affinity of the Fc domain to FcRn under an acidic environment (e.g., in an endosome with a pH range of from about 5.5 to about 6.0). When applied to an animal, such mutations can result in an increase in the serum half-life of the antigen binding molecule. Non-limiting examples of such Fc modifications include, for example, position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., or T) and 256 (e.g., S / R / Q / E / D or T); or position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y) modifications; or position 250 and / or 428 modifications; or position 307 or 308 (e.g., 308F, V308F) and 434 modifications. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0205] For example, the present disclosure includes anti-CD38 x anti-4-1BB 1+2 multispecific antigen-binding molecules comprising an Fc domain, the Fc domain comprising one or more pairs or one or more 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 aforementioned Fc domain mutations and other mutations within the variable domains of the antigen-binding molecules disclosed herein are contemplated within the scope of the present invention.

[0206] Biological properties of antigen-binding molecules and multispecific antigen-binding molecules

[0207] Provided herein are anti-CD38 x anti-4-1BB multispecific antigen binding molecules that bind to CD38 expressed on MOLP8 cells. As shown in Example 6, dose-dependent binding of the CD38 x 4-1BB 1+2 (REGN7633, REGN7647, and REGN7650) and 1+1 (REGN7150) bispecific antibodies was observed in the presence of MOLP8 cells, with a maximum gMFI range of 8.8 x 10 4 Up to 1.4x10 5 , E.C. 50 The range is 4.23x10 -9 M to 9.27x10 -9 M.

[0208] Provided herein are anti-CD38xanti-4-1BB multispecific antigen binding molecules that bind to 4-1BB expressed on HEK293 cells engineered to express 4-1BB. Dose-dependent binding of the CD38x4-1BB 1+2 (REGN7633, REGN7647, and REGN7650) and 1+1 (REGN7150) bispecific antibodies was observed in the presence of HEK293 / h4-1BB cells, with a maximum gMFI in the range of 7.4x10 5 Up to 2.4x10 6 , E.C. 50 The range is 1.47x10 -8 M to 9.97x10 -10 M.

[0209] CD38x4-1BB (1+1 and 1+2) multispecific antigen binding molecules simulate the natural ligand of 4-1BB by connecting CD38+ target cells to 4-1BB receptor-positive T cells.When doing so, construct provides "signal 2" and enhances the activation of T cells in the presence of "signal 1" provided by tumor-associated antigen (TAA) x CD3 bispecific antibody or allogeneic reactions provided by APC.As shown in Example 7, the multispecific antigen binding molecules provided herein cause a maximum IL-2 reaction and greater efficacy higher than the matched isotype control in the presence of target and signal 1 (provided by REGN1979) in T cell activation assays.As shown in Example 8, even if the length of the linker between Fab2 and Fab3 changes, the multispecific antigen binding molecules also have a dose-dependent increase of IL-2 and IFN and greater efficacy.

[0210] 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 shown in an engineered reporter gene assay. As shown in Example 9, 4-1BB activation was achieved using constructs with different linker lengths.

[0211] In certain embodiments, the multispecific antigen binding molecules provided herein result in a dose-dependent increase in IL-2 and IFNγ release. As shown in Example 10, in the presence of allogeneic NALM-6 cells or NALM-6 cells engineered to express PD-L1, CD38x4-1BB 1+2 antibody treatment (REGN7633, REGN7647, and REGN7650) results in a dose-dependent increase in IL-2 and IFNγ release and higher efficacy.

[0212] According to certain embodiments, treatment with the multispecific antigen binding molecule when administered in combination with the BCMAxCD3 bispecific antibody in vivo produces more effective anti-tumor efficacy than either treatment alone. 6 Immunodeficient NOD.Cg-Prkdc from peripheral blood mononuclear cells (PBMCs) of individuals scid Iq tm1Wjl / SzJ (NSG) mice were administered human multiple myeloma cells. After receiving tumor cells, mice were treated with 0.4 mg / kg of a control bispecific Ab or BCMAxCD3 (REGN5458) bsAb binding to CD3 and 4 mg / kg of a control bispecific Ab (1+2 form) or CD38x4-1BB (1+2 form; REGN9686) binding to 4-1BB. The treatment combination was applied twice more on the 7th and 14th days, for a total of three doses. Combination therapy with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb showed a more effective combined anti-tumor efficacy than either therapy alone.

[0213] Epitope mapping and related technologies

[0214] The epitope on CD38 and / or 4-1BB that the antigen binding molecules of the present invention are combined can be composed of 3 or more (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) single continuous sequence of amino acids of corresponding CD38 or 4-1BB protein.Alternately, the epitope can be composed of multiple non-adjacent amino acids (or amino acid sequences) of CD38 or 4-1BB.

[0215] As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (pm is called a paratope) in the variable region of an antigen binding molecule. A single antigen may have more than one epitope. Therefore, different antigen binding molecules may bind to different regions on an antigen and may have different biological effects. An epitope may be conformational or linear. Conformational epitopes are produced by spatially juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, a phosphoryl group, or a sulfonyl group moiety on an antigen.

[0216] Various techniques known to those of ordinary skill in the art can be used to determine whether an 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 Antigen binding molecules , Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248: 443-463) and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9: 487-496). Another method that can be used to identify amino acids in polypeptides that interact with the antigen binding domains of antigen binding molecules is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange methods involve deuterium labeling of the protein of interest, and then antigen binding molecules are combined with deuterium-labeled proteins. Then, the protein / antigen binding molecule complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except the residues protected by the antigen binding molecules (still deuterium-labeled). After the antigen binding molecule dissociates, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to specific amino acids that interact with the antigen binding molecule. See, for example, Ehring (1999) Analytical Biochemistry 267 (2): 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A. X-ray crystallography of antigen / antigen binding molecule complexes can also be used for epitope mapping purposes.

[0217] Provided herein 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 shown in Table 1 herein). Likewise, 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 shown in Table 1 herein).

[0218] Provided herein is an anti-4-1BB antigen binding arm A2 comprising a first antigen binding domain (R1) and a second antigen binding domain (R2), wherein R1 or R2 binds to the same epitope as any specific exemplary antigen binding domain described herein (e.g., an antigen binding arm comprising any amino acid sequence as shown 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 specific exemplary antigen binding domain described herein (e.g., an antigen binding arm comprising any amino acid sequence as shown in Table 3 or Table 5 herein).

[0219] Likewise, provided herein are multispecific antigen-binding molecules comprising a first antigen-binding arm (Fab1) that specifically binds to human CD38 and a second antigen-binding arm (Fab2 and Fab3) that specifically binds to human 4-1BB, wherein the first antigen-binding domain competes with any specific exemplary CD38-specific antigen-binding arm described herein for binding to CD38, and / or wherein the second antigen-binding arm competes with any specific exemplary 4-1BB-specific antigen-binding Fab described herein for binding to 4-1BB.

[0220] By using conventional methods known in the art, it is easy to determine whether a specific antigen binding molecule (e.g., multispecific 1+2 antigen binding molecules) or its antigen binding fragment is bound to the same epitope as the reference antigen binding molecules of the present invention or competes with the reference antigen binding molecules disclosed herein. For example, in order to determine whether the test antigen binding molecule is bound to the same epitope on CD38 (or 4-1BB) with the reference multispecific antigen binding molecules of the present invention, first allow the reference multispecific molecule to bind to CD38 protein (or 4-1BB protein). Next, the ability of the test antigen binding molecule to bind to CD38 (or 4-1BB) molecules is assessed. If the test antigen binding molecule can be bound 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 is bound to different epitopes of CD38 (or 4-1BB) with the reference multispecific antigen binding molecule. In another aspect, if the test antigen binding molecule cannot bind to CD38 (or 4-1BB) molecules after saturation binding with a reference multispecific antigen binding molecule, the test antigen binding molecule can be bound to the same CD38 (or 4-1BB) epitope as the epitope bound by the reference multispecific antigen binding molecule of the present invention.Other conventional experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antigen binding molecule is in fact due to binding to the same epitope as the reference multispecific antigen binding molecule, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding.This 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 certain embodiments of the invention, if, for example, a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antigen-binding protein inhibits the binding of another antigen-binding protein by at least 50%, but preferably 75%, 90% or even 99%, as measured in a competitive binding assay, then 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 the antigen that reduce or eliminate the binding of one antigen-binding protein reduce or eliminate the binding of another antigen-binding protein, then the two antigen-binding proteins are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antigen-binding protein reduces or eliminates the binding of another antigen-binding protein, then the two antigen-binding proteins are considered to have "overlapping epitopes".

[0221] In order to determine whether antigen binding molecules or their antigen binding domains compete with reference antigen binding molecules for binding, the binding method described above is carried out in two directions: in the first direction, reference antigen binding molecules are allowed to bind to CD38 protein (or 4-1BB protein) under saturation conditions, and then the combination of test antigen binding molecules and CD38 (or 4-1BB) molecules is evaluated. In the second direction, test antigen binding molecules are allowed to bind to CD38 (or 4-1BB) under saturation conditions, and then the combination of reference antigen binding molecules and CD38 (or 4-1BB) molecules is evaluated. If only the first (saturated) antigen binding molecules can be bound to CD38 (or 4-1BB) molecules in two directions, the following conclusions are drawn: test antigen binding molecules and reference antigen binding molecules compete with CD38 (or 4-1BB) for binding. As understood by those of ordinary skill in the art, the antigen binding molecules that compete with the combination of reference antigen binding molecules may not necessarily be bound to the same epitope as the reference antigen binding molecules, but may block the combination of reference antigen binding molecules spatially by overlapping or adjacent epitopes.

[0222] Preparation of antigen binding domains and construction of multispecific molecules

[0223] Antigen binding domains with specificity for a particular antigen can be prepared by any antigen binding molecule production technology known in the art.Once obtained, different antigen binding domains provided herein with specificity for different antigens (e.g., CD38 and 4-1BB) can be appropriately arranged relative to each other to produce the multispecific antigen binding molecules of the present invention using conventional methods.(Discussion of exemplary multispecific antigen binding molecule forms that can be used to construct the multispecific antigen binding molecules of the present invention is provided elsewhere herein). In certain embodiments, one or more individual components (e.g., heavy chain and light chain) of the multispecific antigen binding molecules of the present invention are derived from chimeric, humanized or fully human antigen binding molecules.Methods for preparing such antigen binding molecules are well known in the art.For example, VELOCIMMUNE can be used TM One or more heavy chains and / or light chains of the multispecific antigen-binding molecules of the invention are prepared using VELOCIMMUNE TM Technology (or any other human antigen binding molecule production technology), first separate the high-affinity chimeric antigen binding molecules for specific antigens (e.g., CD38 or 4-1BB) with human variable regions and mouse constant regions. For the desired characteristics, including affinity, selectivity, epitope, etc. characterize and select antigen binding molecules. The mouse constant region is replaced by the desired human constant region to produce fully human heavy chains and / or light chains that can be incorporated into the multispecific antigen binding molecules of the present invention.

[0224] Genetically engineered animals can be used to prepare human multispecific antigen-binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein the mice only express one or two human light chain variable domains encoded by human immunoglobulin sequences, and the human immunoglobulin sequences are operably connected to the mouse κ constant gene at the endogenous mouse κ locus. Such genetically modified mice can be used to produce fully human antigen-binding molecules comprising two different heavy chains, and the heavy chains are associated with the same light chains comprising the variable domains derived from one of two different human light chain variable region gene segments. (See, for example, US 2011 / 0195454). Fully human refers to an antigen-binding molecule or its antigen-binding fragment or its immunoglobulin domains, and the amino acid sequence encoded by the DNA derived from a human sequence is included in the total length of each polypeptide of the antigen-binding molecule or its antigen-binding fragment or its immunoglobulin domains. In some cases, fully human sequences are derived from endogenous proteins of people. In other cases, fully human proteins or protein sequences comprise chimeric sequences, and each component sequence is from a human sequence. While not 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, compared to any wild-type human immunoglobulin region or domain.

[0225] Provided herein are antigen binding molecules having amino acid sequences different from those of exemplary molecules disclosed herein but retaining the ability to bind CD38 and / or 4-1BB. When compared to the parent sequence, such variant molecules may include one or more amino acid additions, deletions or substitutions, but exhibit biological activity substantially equivalent to the multispecific antigen binding molecules described.

[0226] Antigen binding molecules that are bioequivalent to any exemplary antigen binding molecules shown herein are drug equivalents or drug substitutes, and their absorption rate and degree do not show significant differences when applied with the same molar dose (single dose or multiple doses) under similar experimental conditions. If some antigen binding proteins are equivalent in absorption degree, but are not equivalent in absorption rate, then they will be considered as equivalents or drug substitutes, but because this difference in absorption rate is intentional and reflected in the labeling, so it can be considered as bioequivalent, these antibodies are not necessary for, for example, reaching effective in vivo drug concentrations in long-term use, and are considered to have no clinical significance for the specific medicine studied.

[0227] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in the safety, purity, and potency of the two antigen binding proteins.

[0228] In one embodiment, two antigen binding proteins are bioequivalent if patients can make one or more switches between the reference product and the biological product without an expected increase in risk of adverse reactions, including clinically significant changes in immunogenicity, or decreased effectiveness, compared to continuous therapy without such switching.

[0229] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action for one or more conditions of use, to the extent such mechanisms are known.

[0230] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence metrics include, for example, (a) in vivo tests in humans or other mammals, in which the concentration of the antigen binding molecule or its metabolites in blood, plasma, serum or other biological fluids over time is measured; (b) in vitro tests that are associated with and can reasonably predict the bioavailability data in humans; (c) in vivo tests in humans or other mammals, in which the appropriate acute pharmacological effects of the antigen binding molecule (or its target) over time are measured; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0231] Bioequivalent variants of the exemplary multispecific antigen-binding molecules of the antigen-binding molecules described herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not required for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen-binding proteins can include variants of the exemplary multispecific antigen-binding molecules described herein that include amino acid changes that modify the glycosylation characteristics of the molecule (e.g., mutations that eliminate or remove glycosylation).

[0232] Species selectivity and cross-species reactivity

[0233] According to certain embodiments of the present invention, there is provided an antigen binding molecule that is combined with human 4-1BB but not combined with 4-1BB from other species.Antigen binding molecules that are combined with human CD38 but not combined with CD38 from other species are also provided.The present invention also includes antigen binding molecules combined with human 4-1BB and CD38 from one or more non-human species; and / or antigen binding molecules combined with human 4-1BB and 4-1BB from one or more non-human species.

[0234] According to certain exemplary embodiments of the present invention, there is provided an antigen binding molecule, which binds to human CD38 and / or human 4-1BB and can bind or not bind (as the case may be) to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus monkey or chimpanzee CD38 and / or 4-1BB. For example, in a specific exemplary embodiment disclosed herein, there is provided a multispecific antigen binding molecule, the multispecific antigen binding molecule comprising a first antigen binding arm binding to human CD38 or cynomolgus CD38 and a second antigen binding arm containing 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, the multispecific antigen binding molecule comprising a second antigen binding arm and a first antigen binding arm specifically binding to human CD38, the second antigen binding arm comprising a first antigen binding domain and a second antigen binding domain binding to human 4-1BB and / or cynomolgus 4-1BB.

[0235] Therapeutic formulation and administration

[0236] The present invention provides pharmaceutical compositions comprising the multispecific antigen-binding molecules disclosed herein. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, and the like. Numerous suitable formulations can be found in the formulary known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0237] The dosage of the multispecific antigen-binding molecule administered to a patient may vary according to the patient's age and size, target disease, condition, route of administration, and the like. Preferred dosages are generally calculated based on body weight or body surface area. When the multispecific antigen-binding molecules of the present invention are used for therapeutic purposes in adult patients, it may be advantageous to administer the multispecific antigen-binding molecules of the present invention intravenously in a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the disease, the frequency and duration of treatment may be adjusted. The effective dosage and schedule of administration of the multispecific antigen-binding molecule may be determined empirically; for example, patient progress may be monitored by regular assessment, and the dosage may be adjusted accordingly. In addition, interspecies scaling of dosages may be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[0238] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endosomes (see, e.g., Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration can be systemic or local.

[0239] The pharmaceutical composition of the present invention can be delivered subcutaneously, intramuscularly or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to deliver the pharmaceutical composition of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge becomes empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. In fact, a disposable pen delivery device is pre-filled with the pharmaceutical composition in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0240] A variety of reusable pen delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN TM(Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany) etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM pen (Abbott Labs, Abbott Park IL), etc.

[0241] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, a polymer material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, so only a small portion of the systemic dose is required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review of Langer, 1990, Science 249: 1527-1533.

[0242] The injectable preparation can include the dosage form for intravenous, subcutaneous, intradermal and intramuscular injection, drip infusion, etc. These injectable preparations can be prepared by well-known methods. For example, the injectable preparation can be prepared by, for example, dissolving, suspending or emulsifying the antigen binding molecules described above or their salts in the sterile aqueous medium or oily medium routinely used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizing agents (such as alcohol (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)], etc.). As an oily medium, using, for example, sesame oil, soybean oil, etc., it can be used in combination with solubilizing agents, such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in suitable ampoule.

[0243] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared into a dosage form suitable for a unit dose that meets the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antigen binding molecules contained is generally about 1 to about 1000mg per dosage form in a unit dose; especially in injection form, for other dosage forms, the amount of the aforementioned antigen binding molecules contained is about 1 to about 100mg and about 10 to about 250mg is preferred. In some embodiments, the unit dose can be up to about 750mg, 800mg, 900mg or 1000mg.

[0244] Therapeutic uses of antigen binding molecules

[0245] The present invention includes methods comprising administering a therapeutic composition to a subject in need, the therapeutic composition comprising a multispecific antigen binding molecule that specifically binds CD38 and 4-1BB. The therapeutic composition may include any multispecific antigen binding molecule as disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "subject in need" refers to a human or non-human animal (e.g., a subject expressing a tumor or suffering from any cancer mentioned herein below) that exhibits one or more symptoms or signs of cancer, or otherwise will benefit from the inhibition or reduction of CD38 activity or the consumption of CD38+ cells (e.g., multiple myeloma cells).

[0246] The multispecific antigen binding molecules of the present invention (and therapeutic compositions comprising the multispecific antigen binding molecules) are particularly useful for treating any disease or condition in which stimulation, activation and / or targeted immune responses would be beneficial. Specifically, the anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molecules of the present invention can be used to treat, prevent and / or improve any disease or condition associated with CD38 and / or BCMA expression or activity or CD38+ and / or BCMA+ cell proliferation or mediated by it. The mechanism of action of the treatment method of the present invention includes killing cells expressing CD38 in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing CD38 that can be inhibited or killed using the multispecific antigen binding molecules of the present invention include, for example, multiple myeloma cells.

[0247] The multispecific antigen-binding molecules of the present disclosure can be used to treat a disease or disorder 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.

[0248] According to certain embodiments, the anti-CD38 x anti-4-1BB 1+2 antigen binding molecule can be used to inhibit the growth of a plasma cell tumor in a subject. In some aspects, the plasma cell tumor is multiple myeloma.

[0249] The multispecific antigen-binding molecules of the present disclosure can be used to inhibit tumor growth 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.

[0250] According to certain embodiments, anti-CD38 x anti-4-1BB 1+2 antigen binding molecules can be used to treat tumor cells expressing, for example, BCMA or CD20. Antigen binding molecules provided herein can also be used to treat diseases or conditions associated with BCMA expression, including, for example, cancer, including multiple myeloma or other B cell or plasma cell cancers, such as Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and diffuse large B cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma and Hodgkin's lymphoma. According to certain embodiments of the present invention, anti-CD38 x anti-4-1BB antigen binding molecules can be used to treat patients with multiple myeloma. According to other related embodiments of the present invention, a method is provided, comprising administering to a patient with cancer cells expressing BCMA or CD20 a combination of an anti-CD38 x anti-4-1BB multispecific antigen-binding molecule provided herein and an anti-BCMA antigen-binding molecule, or an anti-BCMA x anti-CD3 multispecific antigen-binding molecule, or an anti-CD20 x anti-CD3 multispecific antigen-binding molecule, or an anti-CD28 x anti-4-1BB multispecific antigen-binding molecule as disclosed herein. Analysis / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has multiple myeloma or another B cell lineage cancer.

[0251] In some embodiments, the anti-CD38 x 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 drug, a DNA alkylating agent, an immunomodulator, 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, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a PD-1 inhibitor (such as an anti-PD-1 antibody, e.g., cemiprilimab), a PD-L1 inhibitor, a CTLA-4 checkpoint inhibitor, or a combination thereof.

[0252] The present invention also includes methods for treating residual cancer in a subject.As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0253] According to certain aspects, the present invention provides a method for treating a disease or condition (e.g., multiple myeloma) associated with CD38 expression, the method comprising administering to the subject one or more anti-CD38 x anti-4-1BB 1+2 antigen binding molecules described herein after it has been determined that the subject suffers from multiple myeloma. For example, the present invention includes a method for treating multiple myeloma, the method comprising administering to the patient anti-CD38 x anti-4-1BB multispecific antigen binding molecules 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the subject has received other immunotherapy or chemotherapy.

[0254] Combination therapies and formulations

[0255] The present invention provides methods, the methods 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 combined or administered in combination with the antigen binding molecules of the present invention include, for example, anti-tumor agents (e.g., chemotherapeutic agents, including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (adriamycin), liposomal doxorubicin (Doxil), obendamustine (obendamustine) (Treanda) or any other drugs known to be effective in treating plasma cell tumors in subjects). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes targeted therapy, including thalidomide (thalidomide), lenalidomide (lenalidomide) and bortezomib (bortezomib), which are approved for the treatment of newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents that are effective in treating relapsed myeloma.

[0256] In some embodiments, the second therapeutic agent is an anti-BCMAxCD3 bispecific antigen binding molecule. Illustrative anti-BCMAxCD3 bispecific antigen binding molecules are disclosed in US2020 / 0024356 (incorporated herein by reference). As disclosed in US2020 / 0024356, exemplary anti-BCMAxCD3 bispecific antigen binding molecules are REGN5458. In some embodiments, the second therapeutic agent is an anti-CD20xCD3 bispecific antigen binding molecule. Illustrative anti-CD20xCD3 bispecific antigen binding molecules are disclosed in U.S. Patent No. 9,657,102 (incorporated herein by reference). Exemplary anti-CD20xCD3 bispecific antigen binding molecules are REGN1979 (U.S. Patent No. 9,657,102).

[0257] In certain embodiments, the second therapeutic agent is a scheme including radiotherapy or stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulator. In certain embodiments, the second therapeutic agent can be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor, such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody drug conjugate, a multispecific / bispecific / monospecific antigen binding molecule conjugated to an anti-tumor agent, a checkpoint inhibitor, an oncolytic virus, a cancer vaccine, a CAR-T cell, or a combination thereof. Other agents that can be advantageously administered in combination with the antigen binding molecules of the present invention include cytokine inhibitors, including small molecule cytokine inhibitors and 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 their respective receptors. The pharmaceutical composition of the present invention (e.g., a pharmaceutical composition comprising anti-CD38 x anti-4-1BB multispecific antigen binding molecules as disclosed herein) can also be administered as part of a treatment regimen comprising one or more treatment combinations, wherein the one or more treatment combinations are selected from monoclonal antigen binding molecules other than those described herein that may interact with different antigens on the surface of plasma cells; multispecific antigen binding molecules having one arm that binds to an antigen on the surface of a tumor cell and another arm that binds to an antigen on a T cell; antibody drug conjugates; bispecific antibodies conjugated to anti-tumor agents, checkpoint inhibitors (e.g., checkpoint inhibitors targeting PD-1 or CTLA-4) or a combination thereof. 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 may be used in combination with the antigen binding molecules of the present invention are described above.

[0258] The present invention also includes a therapeutic combination comprising any antigen binding molecule mentioned herein and an inhibitor of one or more of the following: VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, urine soluble protein or any of the above cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, siRNA, a peptibody, a nanobody or an antigen binding molecule fragment (e.g., a Fab fragment; a F(ab')2 fragment; a Fd fragment; a Fv fragment; a scFv; a dAb fragment; or other engineered molecules, such as double antibodies, three antibodies, four antibodies, mini antibodies and minimum recognition units). The antigen binding molecules of the present invention can also be administered and / or co-formulated in combination with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs. The antigen binding molecules of the invention may also be administered as part of a treatment regimen that additionally comprises radiation therapy and / or conventional chemotherapy.

[0259] The additional therapeutically active component may be administered prior to, concurrently with, or immediately after administration of the antigen binding molecules of the invention; (for purposes of this disclosure, such administration regimens are considered administration of the antigen binding molecules "in combination" with the additional therapeutically active component).

[0260] The invention includes pharmaceutical compositions in which the antigen binding molecules of the invention are co-formulated together with additional therapeutically active ingredients as described elsewhere herein.

[0261] Administration regimen

[0262] According to certain embodiments of the present invention, multiple doses of antigen binding molecules (e.g., multispecific antigen binding molecules that specifically bind CD38 and 4-1BB) can be administered to a subject within a defined time course. The method according to this aspect of the present invention includes applying multiple doses of the multispecific antigen binding molecules of the present invention to a subject in sequence. As used herein, "sequential administration" means that the antigen binding molecules of each dose are applied to a subject at different time points, for example, at different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes such a method: the method includes sequentially administering to a patient a single initial dose of antigen binding molecules, followed by one or more second doses of antigen binding molecules, and optionally followed by one or more third doses of antigen binding molecules.

[0263] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the temporal order of the administration of the antigen binding molecules of the present invention. Therefore, "initial dose" is the dosage (also referred to as "baseline amount") administered at the beginning of the treatment regimen; "secondary dose" is the dosage administered after the initial dose; and "tertiary dose" is the dosage administered after the second dose. Initial dose, second dose and third dose can all contain the same amount of antigen binding molecules, but can generally be different from each other in terms of frequency of administration. However, in certain embodiments, the amount of antigen binding molecules contained in initial dose, second dose and / or third dose is different from each other during the treatment process (for example, appropriately raised or lowered). In certain embodiments, two or more (for example, 2, 3, 4 or 5) dosages are administered as "loading doses" at the beginning of the treatment regimen, followed by subsequent doses (for example, "maintenance doses") administered at a lower frequency.

[0264] In an exemplary embodiment of the invention, each second and / or third dose is 1 to 26 (e.g., 1, 1 1 / 2,2,2 1 / 2,3,3 1 / 2,4,4 1 / 2,5,5 1 / 2,6,6 1 / 2,7,7 1 / 2,8,8 1 / 2,9,9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 As used herein, the phrase "immediately preceding dose" refers to a sequence of multiple administrations in which the dose of the antigen binding molecule is administered to the patient before the next dose immediately following in the sequence, without an intermediate dose.

[0265] The method according to this aspect of the invention may include administering any number of second and / or third doses of antigen binding molecules (e.g., multi-specific 1+2 antigen binding molecules that specifically bind CD38 and 4-1BB) to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, the second dose is administered twice or more (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or more). Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, the third dose is administered twice or more (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or more).

[0266] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, during the course of the treatment regimen, the frequency of the second dose and / or third dose administered to the patient can vary. The physician can also adjust the frequency of administration during treatment, depending on the needs of each patient after clinical examination.

[0267] Diagnostic Uses of Antigen Binding Molecules

[0268] The anti-CD38 antigen binding molecules disclosed herein can be used to detect and / or measure CD38 or cells expressing CD38 in a sample, for example, for diagnostic purposes. For example, anti-CD38 antigen binding molecules or fragments thereof can be used to diagnose a disorder 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, wherein the anti-CD38 antigen binding molecule is labeled with a detectable marker or reporter molecule. Alternatively, unlabeled anti-CD38 antigen binding molecules can be combined with a second antigen binding molecule that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S or 125 I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-CD38 antigen binding molecules of the invention includes 89Zr-labeled (such as 89 Zr-deferoxamine-labeled) antigen binding molecules for the purpose of non-invasively identifying and tracking tumor cells in a subject (e.g., positron emission tomography (PET) imaging). (See, e.g., Tavare, R. et al. Cancer Res. 2016 Jan 1; 76(1):73-82; and Azad, BB. 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).

[0269] Samples that can be used for CD38 diagnostic assays according to the present invention include any tissue or fluid sample that can be obtained from a patient under normal or pathological conditions, and the sample contains a detectable amount of CD38 protein or fragments thereof. Typically, the CD38 level in a specific sample obtained from a healthy patient (e.g., a patient who does not suffer from a disease or condition associated with abnormal CD38 levels or activity) is measured to preliminarily establish a baseline or standard CD38 level. This baseline level of CD38 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 a condition.

[0270] Device

[0271] The present invention also provides a vessel (e.g., a vial or chromatographic column) or an injection device (e.g., a syringe, a prefilled syringe, or an automatic injector) comprising the multispecific antigen-binding molecules described herein (e.g., their pharmaceutical preparations). The vessel or injection device can be packaged into a kit.

[0272] An injection device is a device for introducing a substance into a subject (e.g., a human) via a parenteral route (e.g., intraocular, intravitreal, intramuscular, subcutaneous, or intravenous route). For example, an injection device can be a syringe (e.g., a syringe prefilled with a pharmaceutical preparation, such as an automatic syringe), the syringe, for example, comprising a barrel or barrel for accommodating a fluid to be injected (e.g., containing an antigen-binding molecule or fragment thereof or a pharmaceutical preparation), a needle for puncturing the skin, blood vessels, or other tissues to inject the liquid; a plunger for pushing the fluid out of the barrel and into the subject through the pinhole.

[0273] The pharmaceutical compositions provided herein can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are easily applicable to the delivery of the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable barrel containing the pharmaceutical composition. Once all of the pharmaceutical composition in the barrel has been administered and the barrel becomes empty, the empty barrel can be easily discarded and replaced with a new barrel containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable barrel. In fact, the disposable pen delivery device is pre-filled with the pharmaceutical composition in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0274] A variety of reusable pen delivery devices and auto-injector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk) and KWIKPENTM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM Pen (Abbott Labs, Abbott Park IL), etc.

[0275] Provided herein are methods of administering the multispecific antigen-binding molecules of the disclosure comprising introducing (eg, injecting) the molecules into the body of a subject, eg, using an injection device.

[0276] Expression

[0277] Provided herein are recombinant methods for preparing multispecific antigen-binding molecules of the invention or immunoglobulin chains thereof, comprising (i) introducing one or more polynucleotides encoding immunoglobulin light chains and / or heavy chains of such multispecific antigen-binding molecules into a host cell, e.g., wherein the one or more polynucleotides are contained in one or more vectors; and / or integrated into a host cell chromosome and / or operably linked to a promoter; (ii) culturing the host cell (e.g., mammal, fungus, Chinese hamster ovary (CHO), Pichia or Pichia pastoris) under conditions favorable for expression of the polynucleotides, and (iii) optionally, isolating the multispecific antigen-binding molecule or immunoglobulin chain from the host cell and / or the culture medium in which the host cell is grown. The products of such methods and pharmaceutical compositions thereof also form part of the present disclosure.

[0278] In some aspects, step (i) includes cloning a single A1 heavy chain, A2 heavy chain and universal light chain into a single expression vector. For example, the heavy chain variable region (HCVR) (VH-1) with CD38 can be cloned into a heavy chain expression plasmid (CH1-1_CH2_CH3). The heavy chain variable region (HCVR) (VH-3) with 4-1BB is fused to the CH1 domain (CH1-3) with various lengths of joints (joints), followed by another heavy chain variable region (HCVR) (VH-2) with 4-1BB, which can be cloned into a heavy chain expression plasmid (CH1-2_CH2_CH3 (*)) containing mutations H435R and Y436F (EU numbering) (US 8,586,713). Together with a plasmid containing a universal light chain, expression plasmids can be transfected into a host cell (such as a CHO cell). Then, host cells can produce multispecific antigen binding molecules as described herein.

[0279] In one embodiment, the method of preparing the multispecific antigen-binding molecule includes a method of purifying the molecule, for example, by column chromatography, precipitation and / or filtration. The products of such methods and pharmaceutical compositions thereof also form part of the present disclosure.

[0280] Host cells comprising the multispecific antigen-binding molecules of the disclosure and / or polynucleotides encoding the immunoglobulin chains of such molecules (e.g., in vectors) are also part of the invention. Host cells include, for example, mammalian cells (such as Chinese hamster ovary (CHO) cells) and fungal cells (such as Pichia cells (e.g., Pichia pastoris)).

[0281] Example

[0282] The following examples are provided to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but certain experimental errors and deviations should be considered. 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.

[0283] The antigen-binding molecules used as controls in Examples 6 to 10 include:

[0284] • CD20xCD3 bispecific antibody (REGN1979) (US 10,550,193) was used as signal 1 along with a matched isotype control (REGN7540). Both REGN1979 and REGN7540 contain hIgG4 P-PVA Isoform (US 9,359,437).

[0285] A second CD20xCD3 bispecific antibody (REGN2281) was used as signal 1. REGN2281 contained the same variable regions as REGN1979, with the hIgG4 isotype having an S108P substitution (hIgG4 P )

[0286] BCMAxCD3 (REGN5458) is also used as signal 1 (US 11,384,153).

[0287] Comparator 1: A 4-1BB bivalent agonist (REGN4249) comprising the variable regions of antibody "10C7" (US 7,288,638).

[0288] The anti-PD-1 antibody cemiplimab (REGN2810; )(US 9,987,500).

[0289] • Isotype control for 4-1BB bivalent agonist (hIgG4P) (REGN1945); REGN1945 was also used as an isotype control for cemiplimab.

[0290] • Bispecific 4-1BB x non-TAA isotype control (REGN13168).

[0291] Correct T cell activation requires two signals, i.e., "signal 1" and "signal 2". "Signal 1" is induced by the binding of the major histocompatibility complex (MHC) molecules of the T cell receptor (TCR) on the T cell to the peptide on the antigen presenting cell (APC). "Signal 2" is provided by engaging the co-stimulatory receptors on the T cell. One such co-stimulatory 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. After antigen or mitogen-induced activation, the expression of 4-1BB receptors on the T cell surface is induced. The activation of 4-1BB occurs by engaging with the 4-1BBL presented on the APC. Therefore, the activation of 4-1BB signaling provides a targeted method for enhancing existing TCR signaling.

[0292] Cell line description

[0293] The characteristics of the cell lines used in the following examples are provided below:

[0294] NALM6 (ACL14036)

[0295] NALM6 clone is an acute lymphoblastic leukemia (ALL) cell line isolated from a 19-year-old male [NALM6 clone G5 (ATCC, #CRL-3273)]. NALM6 cells were maintained in RPMI 1640 + 10% FBS + P / S / G; 37°C 5% CO2.

[0296] Staining to confirm expression

[0297] NALM6 / hPD-L1(ACL16389)

[0298] NALM6 cells genetically engineered to stably express human PD-L1 (amino acids M1-T290 of accession number NP_054862.1). Cells were maintained in RPMI 1640 + 10% FBS + P / S / G + 1 ug / ml Puro; 37°C 5% CO2.

[0299] Viral infection and transduction

[0300] Staining to confirm expression

[0301] HEK293 parent (ACL2397)

[0302] The human embryonic kidney cell line [HEK-293 (ATCC, #CRL-1573)] isolated from a fetus is referred to as the HEK293 parental cell line. HEK293 cells were maintained in DMEM + 10% FBS + P / S / G.

[0303] HEK293 / hCD20 (ACL14268)

[0304] Cell line prepared by stably transducing HEK293 (HZ) cells with human CD20 (Uniprot accession number: P11836, amino acids M1 to P297). The engineered cell line was maintained in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 100 μg / mL hygromycin; 5% CO2.

[0305] Virus transfection / production

[0306] Cell line transduction

[0307] Staining to confirm expression

[0308] HEK293 / hCD20 / hCD38 (ACL14270)

[0309] Cell line prepared by stably transducing HEK293 (HZ) cells with human CD20 (Uniprot accession number: P11836, amino acids M1 to P297) and human CD38 (Uniprot accession number: P28907, amino acids M1 to I300). The engineered cell line was maintained in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 500 μg / mL G418 + 100 μg / mL hygromycin; 5% CO2.

[0310] Virus transfection / production

[0311] Cell line transduction

[0312] Staining to confirm expression

[0313] HEK293 / h4-1BB(ACL7888)

[0314] Cell line prepared by stably transducing HEK293 (HZ) cells with human TNFRSF9 (4-1BB) (Accession No.: NM_001561, amino acids M1 to L255). The engineered cell line was maintained in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 500μg / mL G418; 5% CO2.

[0315] Virus transfection / production

[0316] Cell line transduction

[0317] Staining to confirm expression

[0318] HEK293 / NFkB-Luc(ACL5021)

[0319] The cell line was generated by transfection with the firefly luciferase-IRES-GFP gene driven by five copies of the NF-κB response element located upstream of a minimal TATA promoter. Positive cell lines were selected by flow cytometry and a single clone, D9, was isolated.

[0320] Plasmid production.

[0321] Stain to confirm expression.

[0322] HEK293 / NFkB-Luc / h4-1BB(ACL11090)

[0323] Cell lines prepared by stably transducing HEK293 / NFkB-Luc cells with human TNFRSF9 (4-1BB) (Accession No.: NM_001561, amino acids M1 to L255). The engineered cell lines were maintained in DME + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) + 500 μg / mL G418; 5% CO2.

[0324] Virus transfection / production

[0325] Cell line transduction

[0326] Staining to confirm expression

[0327] MOLP8(ACL14198)

[0328] Human multiple myeloma line established in 2002 from the peripheral blood of a 52-year-old Japanese male with multiple myeloma. Obtained through DSMZ number: ACC 569.

[0329] Staining to confirm expression

[0330] OPM2(ACL14164)

[0331] Established in 1982 from the peripheral blood of a 56-year-old woman with multiple myeloma in the leukemic stage. Obtained through DSMZ number: ACC50.

[0332] Staining to confirm expression

[0333] Example 1. Production and screening of anti-CD38 antibodies and anti-4-1BB antibodies

[0334] Anti-CD38 antibodies are obtained by immunizing genetically engineered mice containing DNA encoding human immunoglobulin heavy chains and kappa light chain variable regions with cells expressing CD38 or with DNA encoding CD38. The antibody immune response is monitored by CD38-specific immunoassay. When the desired immune response is reached, splenocytes are harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines are screened and selected to identify cell lines that produce CD38-specific antibodies. Using this technology, several anti-CD38 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) have been obtained. In addition, as described in US2007 / 0280945A1, several fully human anti-CD38 antibodies are produced by separation directly from antigen-positive B cells that are not fused with myeloma cells.

[0335] Similarly, anti-4-1BB antibodies are obtained by conferring genetically engineered mouse immunity comprising DNA encoding human immunoglobulin heavy chain and kappa light chain variable region with cells expressing 4-1BB or with DNA encoding 4-1BB. Antibody immune response is monitored by 4-1BB specific immunoassay. When the desired immune response is reached, splenocytes are harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines are screened and selected to identify cell lines producing 4-1BB specific antibodies. Using this technology, several anti-4-1BB chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) are obtained. In addition, as described in US2007 / 0280945A1, several fully human anti-4-1BB antibodies are produced by separation directly from antigen-positive B cells that are not fused with myeloma cells.

[0336] For desired features, including affinity, selectivity, etc. characterization and selection of antibodies. If necessary, replace the mouse constant region with the desired human constant region (such as wild-type or modified IgG1 or IgG4 constant region), to produce fully human anti-CD38 antigen binding molecules or fully human anti-4-1BB antigen binding molecules. Although the selected constant region can vary according to specific use, high-affinity antigen binding and target-specific characteristics are present in the variable region.

[0337] Example 2. Heavy and light chain variable region amino acid and nucleic acid sequences of the anti-CD38 binding arm.

[0338] Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-CD38 antigen binding arms of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0339]

[0340]

[0341] The anti-CD38 binding arm may comprise the variable domain and CDR sequences as shown in Table 1 and a human Fc domain of isotype IgG4, IgG1, etc. For certain applications or experiments, the Fc domain may be a mouse Fc domain. As will be appreciated by one of ordinary skill in the art, an antigen binding arm having a particular Fc isotype may be converted into an antigen binding arm having a different Fc isotype (e.g., an antigen binding molecule having a mouse IgG4 Fc may be converted into an antigen binding molecule having a human IgG1, etc.), but in any case, the variable domains (including CDRs) (which are represented by the numerical identifiers shown in Table 1) will remain the same, and the binding properties are expected to be the same or substantially similar, regardless of the nature of the Fc domain.

[0342] Example 3: Heavy and light chain variable region amino acid and nucleic acid sequences of the anti-4-1BB binding arm.

[0343] Table 3 shows the amino acid sequence identifiers of the heavy chain and light chain variable regions and CDRs of the selected anti-4-1BB binding arms of the bispecific antibodies. The corresponding nucleic acid sequence identifiers are shown in Table 4.

[0344]

[0345]

[0346] Anti-4-1BB antigen binding arms can include variable domains and CDR sequences as shown in Table 3 and human Fc domains of isotypes IgG4, IgG1, etc. For some applications or experiments, the Fc domain may be a mouse Fc domain. As will be appreciated by those of ordinary skill in the art, antigen binding molecules with specific Fc isotypes can be converted into antigen binding molecules with different Fc isotypes (for example, antigen binding molecules with mouse IgG4 Fc can be converted into antigen binding molecules with human IgG1, etc.), but in any case, variable domains (including CDR) (which are represented by the digital identifiers shown in Table 3) will remain the same, and it is expected that the binding properties are the same or substantially similar, regardless of the nature of the Fc domain.

[0347] Example 4: Generation of multispecific antigen-binding molecules that bind CD38 and 4-1BB

[0348] Multispecific antigen-binding molecules that bind CD38 and 4-1BB are also referred to herein as "anti-CD38 x anti-4-1BB 1+2" or "anti-CD38 x anti-4-1BB multispecific molecules" or "anti-CD38 / anti-4-1BB 1+2" or "CD38 x 4-1BB multispecific molecules". The anti-CD38 portion of the anti-CD38 x anti-4-1BB multispecific molecule can be used to target tumor cells expressing CD38, and the anti-4-1BB portion of the multispecific molecule can be used to activate T cells.

[0349] Various 4-1BB Fabs (Fab3; heavy chain variable region (HCVR) with heavy chain CH1 domain and 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 of existing IgG-like bispecific molecules targeting both 4-1BB and CD38.

[0350] DNA fragments encoding (i) various 4-1BB heavy chain variable regions (HCVRs); (ii) a heavy chain CH1 domain followed by linkers of varying lengths for connecting the heavy chain CH1 domain to a second 4-1BB heavy chain variable region (HCVR); and (iii) a CD38 heavy chain variable region (HCVR) were synthesized by Integrated DNA Technologies, Inc. (San Diego, California).

[0351] InFusion Cloning (Takara Bio USA Inc.) creates a mammalian expression vector for a single heavy chain according to a protocol provided by Takara Bio USA Inc. The CD38 heavy chain variable region (HCVR) (VH-1) is cloned into a heavy chain expression plasmid (CH1-1_CH2_CH3). The 4-1BB heavy chain variable region (HCVR) (VH-3) is fused to the CH1 domain (CH1-3) with various lengths of linkers (linkers), followed by another 4-1BB heavy chain variable region (HCVR) (VH-2), which is cloned into a heavy chain expression plasmid (CH1-2_CH2_CH3 (*)) containing a star mutation (H435R, Y436F, EU numbering).

[0352] After transfection with 3 expression plasmids (i) CD38 heavy chain plasmid, (ii) 4-1BB + 4-1BB heavy chain star plasmid, (iii) plasmid containing universal light chain, recombinant CD38 x4-1BB x 4-1BB 1 + 2N-Fab MBM was produced in CHO cells. After selection for 12 days with 400 μg / ml hygromycin, a stable transfected CHO cell pool was isolated. The CHO cell pool was used to produce CD38 x 4-1BB x 4-1BB 1 + 2N-Fab MBM, which was then purified as previously described (Sci Rep. 2015 December 11; 5: 17943).

[0353] A summary of the component parts of the antigen binding domains of selected multispecific antigen binding molecules prepared according to this example is shown in Table 5. The corresponding 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 control bispecific antigen binding molecules, respectively.

[0354]

[0355] Example 5: Screening of multispecific antigen-binding molecules

[0356] Next, screening was performed to identify constructs that activated the 4-1BB signaling pathway.

[0357] 34 4-1BB binding antibodies were selected for inclusion in a 1+2 screen based on cell-mediated binding to human 4-1BB, utilization of common light chains, and diversity of CDR3 sequences. For the screen, the 4-1BB variable domains were arranged in two different 1+2 formats (split 4-1BB or stacked 4-1BB). Figure 2B .

[0358] For the split format, the standard bivalent structure of 4-1BB was maintained, and tumor targeting was achieved by adding the variable domains from the CD38 tumor targeting arm (26812) and CH1 (separated by the G4Sx3 spacer domain) to the N-terminus of one side of the 4-1BB molecule. These constructs are expressed as knob-into-hole (KiH) bispecifics, so only one arm of the expressed molecule contains the CD38 targeting motif.

[0359] For the stacked FAB format, the tandem 4-1BB sequence is connected in the same manner as CD38 x 4-1BB described above, and tumor targeting is achieved by expressing it as a bispecific molecule, where anti-CD38 is present on the opposite arm. 34 4-1BB variable domains and an unrelated control sequence (anti-BetV1) are combined in all possible tandem FAB combinations that produce 1225 combinations. This includes 34 molecules in which two stacked 4-1BB variable sequences are identical.

[0360] For screening, the 1+2 construct was transiently expressed in CHO cells and the bispecific-containing supernatant was harvested 4 days later. The supernatant was added to a co-culture containing HEK cells overexpressing hCD38 and Jurkat cells carrying an NFkB luciferase reporter and overexpressing human 4-1BB. 4-1BB engagement and activation of the test samples were compared to a co-culture of Jurkat reporter cells and HEK cells expressing the 4-1BB ligand (100%). Screening results from the split FABs showed little to no activity in the Jurkat reporter assay, with the highest activity observed being 3.7%. In contrast, robust activation was observed with the stacked FAB format, with more than 25% of the tested samples producing greater than 30% activity. Activity was observed when the 4-1BB variable domains within the stacked FABs were the same or unique sequence, and up to 66% activity was observed. Introduction of an unrelated control FAB at any position within the stacked FAB resulted in a loss of activity. See Figure 2A .

[0361] Example 6: Characterization of CD38x4-1BB multispecific antigen binding molecule binding using flow cytometry

[0362] The combination of CD38 arms is tested using MOLP8 cells endogenously expressing CD38. The combination of 4-1BB arms is assessed using HEK293 cells (HEK293 / h4-1BB) engineered to express h4-1BB. HEK293 cells are used to determine non-targeted cell binding because they do not express CD38 or 4-1BB.

[0363] The ability of multispecific antigen binding molecules to bind cells is assessed using flow cytometry.Cell lines are selected to determine the ability of anti-CD38 arms and anti-4-1BB arms to bind to their targets.In a first experiment, test antibodies are incubated with MOLP8 (endogenously expressed hCD38) and HEK293 (no hCD38) cells.In a second experiment, test antibodies are incubated with HEK293 / h4-1BB (engineered to express h4-1BB) and HEK293 (no h4-1BB) cells.Binding is detected by using labeled secondary antibodies and measuring fluorescence on a flow cytometer.

[0364] Multispecific antigen-binding molecules:

[0365] The multispecific antigen-binding molecules and controls tested in these two experiments are shown in Table 9.

[0366] Table 9: CD38x4-1BB binding molecules and controls

[0367] MABM Identifier describe REGN7633 <![CDATA[CD38x4-1BB(1+2),(hIgG4 P-PVA )]]> REGN7647 <![CDATA[CD38x4-1BB(1+2),(hIgG4 P-PVA )]]> REGN7650 <![CDATA[CD38x4-1BB(1+2),(hIgG4 P-PVA )]]> REGN7150 <![CDATA[CD38x4-1BB(1+1),(hIgG4 P-PVA )]]> REGN4249 Comparison 1 REGN7540 <![CDATA[Isotype control (hIgG4 P-PVA )]]> REGN1945 <![CDATA[Isotype control (hIgG4 P )]]>

[0368] Experiment 1 – CD38 binding:

[0369] HEK293 cells were lifted with trypsin, washed and resuspended in staining buffer (2% FBS in PBS). MOLP8 cells were washed and resuspended in staining buffer. Cells were added to wells of a 96-well V-bottom plate (3x10 5Cells / well). 1:4, 9-point, dose-titrated test antibodies were diluted in staining buffer and added to cells at final concentrations ranging from 610fM to 10nM (a "no antibody" control was included as the ninth point (153fM), labeled "Secondary Only"). Cells and antibodies were incubated on ice for 30 minutes and then washed in staining buffer. Cells were resuspended in 2ug / ml allophycocyanin (APC)-conjugated goat anti-human secondary antibody and incubated on ice for 30 minutes. Cells were then washed and suspended in viability dye (according to the manufacturer's protocol) and incubated on ice for 30 minutes. Cells were then washed with staining buffer and resuspended in 2% PFA on ice for 30 minutes. After washing, cells were filtered and analyzed by flow cytometry to determine the geometric mean fluorescence intensity (gMFI), which was then plotted using GraphPad Prism software. EC values ​​for antibodies were determined according to a 4-parameter logistic equation on a 9-point dose-response curve (including a secondary only control, representing the 9th point) 50 The results are shown in Table 10.

[0370] Dose-dependent binding of CD38x4-1BB 1+2 (REGN7633, REGN7647 and REGN7650) and 1+1 (REGN7150) multispecific antigen binding molecules was observed in the presence of MOLP8 cells endogenously expressing CD38. However, no binding was observed for the bivalent anti-4-1BB antibody REGN4249. No binding was observed for isotype control antibodies (REGN7540 and REGN1945).

[0371] No binding was observed for CD38x4-1BB 1+2 antibody REGN7633 or isotype control antibodies (REGN7540 and REGN1945) in the presence of HEK293 cells that do not express CD38. Weak binding (>100-fold lower than gMFI on MOLP8 cells) was observed using CD38x4-1BB 1+2 (REGN7647 and REGN7650) and 1+1 bispecific (REGN7150) antibodies and bivalent anti-4-1BB antibody REGN4249.

[0372] Table 10: Maximum binding to antibodies and EC50 values ​​of binding

[0373]

[0374] Abbreviations: ND: not determined; NC: not calculated because data did not fit the 4-parameter logistic equation.

[0375] *Most likely due to nonspecific binding

[0376] Experiment 2 – 4-1BB binding:

[0377] HEK293 and HEK293 / 4-1BB cells were lifted with trypsin, washed and resuspended in staining buffer (2% FBS in PBS) and added to wells of a 96-well V-bottom plate (3x10 5 Cells / well). A 1:5, 9-point, dose-titrated test antibody was diluted in staining buffer and added to the cells at final concentrations ranging from 1.3 pM to 100 nM (a "no antibody" control was included as the ninth point (0.26 nM), labeled "Secondary Only"). The cells and antibodies were incubated on ice for 30 minutes and then washed in staining buffer. The cells were resuspended in 2 ug / ml allophycocyanin (APC)-conjugated goat anti-human secondary antibody and incubated on ice for 30 minutes. The cells were then washed and suspended in a viability dye (according to the manufacturer's protocol) and incubated on ice for 30 minutes. The cells were then washed with staining buffer and resuspended in 2% PFA 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 values ​​of the antibodies were determined according to a 4-parameter logistic equation on the 9-point dose-response curves (including the secondary only control). 50 The results are shown in Table 11.

[0378] Dose-dependent binding of CD38x4-1BB 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 divalent anti-4-1BB antibody REGN4249 was also observed. No binding was observed for isotype control antibodies (REGN7540 and REGN1945).

[0379] No binding was observed for CD38x4-1BB 1+2 antibody REGN7633 or isotype control antibodies (REGN7540 and REGN1945) in the presence of HEK293 cells that do not express h4-1BB. Slight binding (greater than 1,000-fold lower than gMFI on HEK293 / h4-1BB cells) was observed with CD38x4-1BB 1+2 multispecific antigen binding molecules (REGN7647 and REGN7650), CD38x4-1BB bispecific (REGN7150), and bivalent anti-4-1BB antibody REGN4249.

[0380] Table 11: Maximum binding to antibodies and EC50 values ​​for binding

[0381]

[0382] Abbreviations: ND: not determined; NC: not calculated because data did not fit the 4-parameter logistic equation.

[0383] *Most likely due to nonspecific binding

[0384] Example 7: Characterization of CD38x4-1BB bispecific antibodies in T cell activation assays using HEK293 / hCD20 / hCD38, HEK293 / hCD20, MOLP8 and human primary T cells.

[0385] Correct T cell activation requires two signals, i.e., "signal 1" and "signal 2". "Signal 1" is induced by the binding of the major histocompatibility complex (MHC) molecules of the T cell receptor (TCR) on the T cell to the peptide on the antigen presenting cell (APC). However, "signal 2" is provided by engaging the co-stimulatory receptors on the T cell. One such co-stimulatory 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. After antigen or mitogen-induced activation, the expression of 4-1BB receptors on the T cell surface is induced. The activation of 4-1BB occurs by engaging with the 4-1BBL presented on the APC. Therefore, the activation of 4-1BB signaling provides a targeted method for enhancing existing TCR signaling.

[0386] CD38x4-1BB (1+1 and 1+2) bispecific antibodies were designed by + Target cells are bridged with 4-1BB receptor-positive T cells to mimic the natural ligand of 4-1BB to provide "signal 2" in order to enhance T cell activation in the presence of "signal 1" provided by tumor-associated antigen (TAA) x CD3 bispecific antibody or allogeneic response provided by APC.

[0387] Multispecific antigen-binding molecules:

[0388] The multispecific antigen-binding molecules and controls tested in this experiment are shown in Table 12.

[0389] Table 12: CD38x4-1BB binding molecules and controls

[0390]

[0391]

[0392] Using REGN1979 (CD20xCD3) to act as "signal 1", in the presence of human embryonic renal cancer cells engineered to express hCD20 and hCD38 (HEK293 / hCD20 / hCD38), CD38x4-1BB multispecific antigen binding molecules are evaluated by engaging CD38 and 4-1BB receptors to deliver "signal 2" (as determined by IL-2 release) to activate human primary T cells. HEK293 cells expressing only hCD20 are included as controls to measure the activity that may occur when CD38 is lacking on APC. In addition, multiple myeloma cell line MOLP8 endogenously expressing hCD38 is included in the test CD38x4-1BB bispecific antibody. Because MOLP8 cells endogenously express BCMA, REGN5458 (BCMAxCD3) is included to act as "signal 1". Notably, unlike HEK293 cells, MOLP8 cells were able to provide detectable T cell allogeneic stimulation in the absence of CD3 stimulation provided by REGN5458, acting as a “signal 1”.

[0393] Human primary CD3 + Isolation of T cells:

[0394] Density gradient centrifugation was used to separate human peripheral blood mononuclear cells (PBMCs) from a healthy donor leukocyte bank (donor 555105) from Precision for Medicine. Briefly, 15 ml of Ficoll-Paque PLUS was added to a 50 ml conical tube, followed by 30 ml of blood diluted 1:1 with PBS containing 2% FBS layered on top. After centrifugation at 400 x g for 30 minutes under braking, the buffy coat (containing mononuclear cells) was transferred to a new tube, diluted 5 times with PBS containing 2% FBS and centrifuged at 300 x g for 8 minutes. Subsequently, EasySepTM human CD3 + T cell isolation kit and follow the manufacturer's instructions to isolate CD3 + T cells were isolated from PBMCs.

[0395] IL-2 release assay:

[0396] Resuspend the enriched CD3 + T cells were 1×10 5 Cells were added to a 96-well round-bottom plate at a concentration of 1x10 cells / well. Growth-arrested HEK293 / hCD20 / hCD38 or HEK293 / hCD20 4 The final concentration of cells / well was added to CD3 +T cells. Growth-arrested MOLP8 cells were cultured at 5x10 4 The final concentration of cells / well was added to CD3 + T cells. After adding cells, a constant 0.1nM REGN1979 or its matching isotype control (REGN7540) was added to the wells containing HEK293 / hCD20 / hCD38 or HEK293 / hCD20. A constant 0.5nM REGN5458 or isotype control was added to the wells containing MOLP8 cells. Subsequently, CD38x4-1BB (1+1 or 1+2), bivalent 4-1BB (REGN4249) or isotype control (REGN7540 or REGN1945) were titrated from 3pm to 200nm with a 1:4 dilution and added to the wells. The end point of the 10-point dilution did not contain titrated antibodies. The plate was incubated at 37°C, 5% CO2 for 72 hours, and 5 μL of total supernatant was used to measure IL-2. According to the manufacturer's protocol, the amount of cytokines in the assay supernatant was determined using the AlphaLisa kit from PerkinElmer. Cytokine measurements were obtained on the Perkin Elmer multilabel microplate reader Envision and reported in pg / mL. All serial dilutions were tested in duplicate.

[0397] Using GraphPad Prism TM The software determines the EC of the antibody based on a four-parameter logistic equation on a 10-point dose-response curve. 50 Values. Maximum IL-2 is given as the average maximum response detected in the dose range tested. Results are provided in Table 13.

[0398] Results using HEK293 / hCD20 and HEK293 / hCD20 / hCD38 cell lines

[0399] Compared to matched isotype controls (REGN7540 and REGN1945), 4-1BB antibody treatment resulted in a higher IL-2 response in the presence of the target and "signal 1" provided by REGN1979. Notably, the 1+2 form of CD38x4-1BB resulted in higher maximum IL-2 and greater efficacy compared to 1+1CD38x4-1BB (REGN7150) and bivalent 4-1BB (REGN4249) antibodies. In the absence of the CD38 target, only the bivalent 4-1BB (REGN4249) showed a dose-dependent increase in IL-2 release. In the absence of 'signal 1', no antibody resulted in a dose-dependent enhancement of IL-2 release.

[0400] Results using the MOLP8 cell line

[0401] In the presence of allogeneic MOLP8 cells and the absence of REGN5458, a dose-dependent increase in IL-2 was observed for 1+2CD38x4-1BB antibodies REGN7647 and REGN7650 and to a lesser extent for 1+2CD38x4-1BB antibodies REGN7633 and bivalent 4-1BB antibodies REGN4249. 1+1CD38x4-1BB and isotype control antibodies did not show a dose-dependent enhancement of IL-2 release. Although allogeneic MOLP8 cells can provide "signal 1", 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 matched isotype controls, with 1+2CD38x4-1BB antibodies showing the greatest potency and greatest increase in IL-2.

[0402] Table 13: Maximum IL-2 release and potency values ​​of antibodies

[0403]

[0404]

[0405] Abbreviations: ND: not determined; NC: not calculated because data did not fit the 4-parameter logistic equation.

[0406] Example 8: Characterization of CD38x4-1BB(1+2) multispecific antigen-binding molecules in T cell activation assays using HEK293 / hCD20 / hCD38, HEK293 / hCD20, MOLP8, NALM6 and human primary T cells.

[0407] Correct T cell activation requires two signals, i.e., "signal 1" and "signal 2". "Signal 1" is induced by the binding of the major histocompatibility complex (MHC) molecules of the T cell receptor (TCR) on the T cell to the peptide on the antigen presenting cell (APC). "Signal 2" is provided by engaging the co-stimulatory receptors on the T cell. One such co-stimulatory 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. After antigen or mitogen-induced activation, the expression of 4-1BB receptors on the T cell surface is induced. The activation of 4-1BB occurs by engaging with the 4-1BBL presented on the APC. Therefore, the activation of 4-1BB signaling provides a targeted method for enhancing existing TCR signaling.

[0408] CD38x4-1BB (1+1 and 1+2) bispecific antibodies were designed by +Target cells are bridged with 4-1BB receptor-positive T cells to mimic the natural ligand of 4-1BB to provide "signal 2" in order to enhance T cell activation in the presence of "signal 1" provided by tumor-associated antigen (TAA) x CD3 bispecific antibody or allogeneic response provided by APC.

[0409] Multispecific antigen-binding molecules:

[0410] The multispecific antigen-binding molecules and controls tested in this experiment are shown in Table 14.

[0411] Table 14: CD38x4-1BB binding molecules and controls

[0412]

[0413]

[0414] Using REGN2281 (CD20xCD3) to act as "signal 1", in the presence of human embryonic renal cancer cells engineered to express hCD20 and hCD38 (HEK293 / hCD20 / hCD38), the CD38x4-1BB 1+2 multispecific antigen binding molecules with different connections between 2 series 4-1BB binding Fab domains are evaluated by engaging CD38 and 4-1BB receptors to deliver "signal 2" (as determined by IL-2 or IFNγ release) to activate human primary T cells. HEK293 cells expressing only hCD20 are included as controls to measure the activity that may occur when CD38 is lacking on APC. In addition, multiple myeloma cell lines MOLP8 endogenously expressing hCD38 are included in the test CD38x4-1BB (1+2) bispecific antibodies. Since MOLP8 cells endogenously express BCMA, REGN5458 (BCMAxCD3) is included to act as "signal 1". Finally, the inclusion of MOLP8 or NALM-6 (an acute lymphoblastic leukemia cell line that endogenously expresses CD38) as target cells was tested in the absence of CD3 bispecific antibodies. Notably, unlike HEK293 cells, MOLP8 and NALM-6 cells were able to provide detectable T cell allogeneic stimulation in the absence of CD3 antibody stimulation, acting as “signal 1”.

[0415] Human primary CD3 + Isolation of T cells:

[0416] 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 and frozen. + T cell isolation kit and follow the manufacturer's instructions to isolate CD3 + T cells were isolated from PBMCs.

[0417] Primary T cell activation assay:

[0418] Resuspend the enriched CD3 + T cells were 1×10 5 For HEK293 / hCD20 / hCD38 or HEK293 / hCD20 cells, add 1x 10 cells / well to a 96-well round-bottom plate. 4 cells / well and for MOLP8 and NALM-6 cells, 5 x 10 4 Target cells were added to CD3 + T cells. After adding cells, a constant 0.25nM REGN2281 was added to wells containing HEK293 / hCD20 / hCD38 or HEK293 / hCD20 cells. A constant 0.5nM REGN5458 or isotype control was added to wells containing MOLP8 cells. CD3 bispecific antibodies were not added to wells containing NALM-6 target cells. Subsequently, CD38x4-1BB (1+1 or 1+2), bivalent 4-1BB (REGN4249) or isotype control (REGN7540 or REGN1945) were titrated from 128fM to 50nM at a 1:5 dilution and added to the wells. The endpoint of the 10-point dilution did not contain titrated antibodies. The plate was incubated at 37°C, 5% CO2 for 72 hours, and 5 μL of total supernatant was used to measure IL-2 or IFNγ. According to the manufacturer's protocol, the amount of cytokines in the assay supernatant was determined using the AlphaLisa kit from PerkinElmer. Cytokine measurements were obtained on the Perkin Elmer multilabel microplate reader Envision and reported in pg / mL. All serial dilutions were tested in duplicate.

[0419] Using GraphPad Prism TM The software determines the EC of the antibody based on a four-parameter logistic equation on a 10-point dose-response curve.50 Values. Maximum cytokines are given as the average maximum response detected in the dose range tested. Results are provided in Tables 15 and 16.

[0420] HEK293 / hCD20 and HEK293 / hCD20 / hCD38

[0421] With matching isotype IgG4 P-PVA IgG4 P In the presence of the target and "signal 1" provided by REGN2281, 4-1BB antibody treatment resulted in higher IL-2 and IFNγ responses compared to the 1+1CD38x4-1BB (REGN7150) and bivalent 4-1BB (REGN4249) antibodies, or bispecific antibody controls (REGN7540, REGN1945, and REGN13168, respectively). Notably, the 1+2 format of CD38x4-1BB resulted in higher maximum cytokine release and greater potency compared to the 1+1CD38x4-1BB (REGN7150) and bivalent 4-1BB (REGN4249) antibodies, with similar cytokine release levels and potency observed regardless of the connection between the 2 tandem 4-1BB binding Fab domains. In the absence of the CD38 target, only the bivalent 4-1BB (REGN4249) showed a dose-dependent increase in cytokine release.

[0422] MOLP8

[0423] In the presence of allogeneic MOLP8 cells and in the absence of CD3 bispecific antibody stimulation, the 1+2CD38x4-1BB antibodies REGN7647, REGN9682, and REGN9686 mediated dose-dependent increases in IL-2 and IFNγ. In contrast, the 1+1CD38x4-1BB antibody REGN7150 and the bivalent 4-1BB antibody REGN4249 resulted in a slight dose-dependent increase in IL-2, but not IFNγ. No dose-dependent increase in IL-2 or IFNγ was observed for both isotype and non-targeting 1+2 4-1BB control antibodies. For all CD38-targeted 4-1BB antibodies and for REGN4249, the addition of a fixed amount of REGN5458 (BCMAxCD3) resulted in a dose-dependent increase in IL-2 and IFNγ under conditions using MOLP8 cells and primary human T cells. As previously described, the 1+2 CD38x4-1BB antibody exhibited greater potency and maximal cytokine increases compared to REGN7150 and REGN4249, with the linkage format between the 4-1BB binding Fabs having little effect on potency or maximal cytokine release. Isotype controls and non-TAAx4-1BB 1+2 control antibodies did not result in dose-dependent cytokine release.

[0424] NALM-6

[0425] In the presence of allogeneic NALM-6 cells and in the absence of CD3 bispecific antibody stimulation, all CD38 targeting 4-1BB antibodies (REGN7647, REGN9682, REGN9686 and REGN7150) and bivalent 4-1BB antibodies (REGN4249) will lead to a dose-dependent increase in IFNγ and IL-2. Compared with 1+1CD38x4-1BB antibody REGN7150 and bivalent 4-1BB antibody REGN4249, 1+2CD38x4-1BB antibodies REGN7647, REGN9682 and REGN9686 show greater efficacy and maximum cytokine release. As previously described, the performance of all 1+2CD38x4-1BB multispecific antigen binding molecules is similar, regardless of the linker between 4-1BB binding Fab. Isotype control and non-TAAx4-1BB 1+2 control antibodies do not lead to dose-dependent cytokine release.

[0426]

[0427] Example 9: Characterization of CD38x4-1BB (1+2) multispecific antigen binding molecules in engineered reporter gene assays using HEK293 / hCD20 / hCD38, HEK293 / hCD20, MOLP8, OPM2 and HEK293 / NFkB-Luc / h4-1BB cells

[0428] The ability of CD38x4-1BB multispecific antigen binding molecules to specifically activate 4-1BB receptors in the presence of target cells expressing CD38 was measured in an engineered reporter gene assay. In this assay, engineered HEK293 cells express reporter gene luciferase under the control of transcription factor NF-KB (NFKB-Luc) and co-stimulatory receptor 4-1BB (HEK293 / NFkB-Luc / h4-1BB). The target cells used in this assay are engineered to express HEK293 cells or endogenous CD38 cell lines, i.e. OPM2 and MOLP8, of CD20 alone or in combination with CD38. The ability of 4-1BB arms to stimulate 4-1BB activity was assessed by combining reporter cells with target cells and titration of CD38x4-1BB 1+2 antibodies. 4-1BB activation results in NFKB-driven luciferase production, which is then measured by luminescent readings. In these assays, the effects of different types of connections between 2 series 4-1BB targeting domains were also evaluated.

[0429] Multispecific antigen-binding molecules:

[0430] The multispecific antigen-binding molecules and controls tested in this experiment are shown in Table 17.

[0431] Table 17: CD38x4-1BB binding molecules and controls

[0432] MABM Identifier describe REGN7647 <![CDATA[CD38x4-1BB(1+2),(hIgG4 P-PVA )]]> REGN7650 <![CDATA[CD38x4-1BB(1+2),(hIgG4 P-PVA )]]> REGN9682 <![CDATA[CD38x4-1BB(1+2),(G4S)3(hIgG4 P-PVA )]]> REGN9686 <![CDATA[CD38x4-1BB(1+2),(G4S)2(hIgG4 P-PVA )]]> REGN4249 Comparison 1 REGN7540 <![CDATA[Isotype control (hIgG4 P-PVA )]]> REGN1945 <![CDATA[Isotype control (hIgG4 P )]]>

[0433] One day before the experiment, HEK293 reporter cells were split to 5 x 10 in DMEM + 10% FBS + P / S / G + 500 μg / ml G418 growth medium. 5 cells / mL.

[0434] On the day of the experiment, adherent HEK293 reporter cells 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 plated at 5 x 10 3 A final concentration of 10 cells / well was added to the wells of a 96-well white microtiter plate and then 1x 10 4 Target cells HEK293 / hCD20 or HEK293 / hCD20 / hCD38 were added at a final concentration of 10 cells / well, or 2.5 x 10 4 Add MOLP8 and OPM2 target cells at a final concentration of 10 cells / well.

[0435] CD38x4-1BB 1+2 and control antibodies were titrated in a 1:3, 10-point serial dilution ranging from 3.0 pM to 20 nM final concentration, with the last point containing no antibody included as a control. After addition of the antibodies, the 96-well white microtiter plates were incubated at 37°C / 5% CO2 for 5 hours, and then an equal volume of ONE-Glo TM (Promega) reagent was used to lyse cells and detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multi-label microplate reader Envision (PerkinElmer). The EC of the antibody was determined according to a 4-parameter logistic equation on a 10-point dose-response curve (the 10th point did not contain antibody) using GraphPad Prism software. 50 The results are provided in Table 18.

[0436] HEK293 / hCD20 and HEK293 / hCD20 / hCD38

[0437] In the presence of HEK293 / hCD20 / hCD38 target cells, CD38x4-1BB 1+2 bispecific antibodies (REGN7647, REGN9682, REGN9686 and REGN7650) resulted in similar increases in luciferase activity regardless of the connection 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, whereas the isotype control antibody did not.

[0438] In the presence of CD38-deficient HEK293 / hCD20 target cells, no response was seen using the CD38x4-1BB bispecific antibody or using an isotype control. Only the anti-4-1BB bivalent antibody (REGN4249) resulted in a dose-dependent increase in luciferase activity.

[0439] MOLP8 and OPM2 cells

[0440] In the presence of MOLP8 or OPM2 target cells, CD38x4-1BB 1+2 (REGN7647, REGN7650, REGN9682 and REGN9686) antibodies and bivalent anti-4-1BB antibodies (REGN4249) resulted in a dose-dependent increase in luciferase activity, with different linker length 1+2CD38x4-1BB variants resulting in similar activity. Isotype control antibodies did not result in any signal.

[0441] Table 18: Maximum luciferase activity and potency values ​​of antibodies

[0442]

[0443] Abbreviations: ND: not determined, as no concentration-dependent increase was observed; NC: not calculated, as the data did not fit the 4-parameter logistic equation.

[0444] Example 10: Characterization of CD38x4-1BB 1+2 bispecific antibody in T cell allogeneic cemiplimab combination assay using NALM-6, NALM-6 / PDL1 and human primary T cells

[0445] Correct T cell activation requires two signals, i.e., "signal 1" and "signal 2". "Signal 1" is induced by the binding of the major histocompatibility complex (MHC) molecules of the T cell receptor (TCR) on the T cell to the peptide on the antigen presenting cell (APC). "Signal 2" is provided by engaging the co-stimulatory receptors on the T cell. One such co-stimulatory 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. After antigen or mitogen-induced activation, the expression of 4-1BB receptors on the T cell surface is induced. The activation of 4-1BB occurs by engaging with the 4-1BBL presented on the APC. Therefore, the activation of 4-1BB signaling provides a targeted method for enhancing existing TCR signaling.

[0446] The CD38x4-1BB(1+2) bispecific antibody was designed by + Target cells and 4-1BB receptor-positive T cells are bridged to simulate the natural ligand of 4-1BB to provide "signal 2", so as to enhance T cell activation in the presence of "signal 1" provided by tumor-associated antigen (TAA) x CD3 bispecific antibodies or allogeneic reactions provided by APC. However, T cell activation can be inhibited by connecting the programmed cell death protein 1 receptor (PD-1) on T cells to its ligand PD-L1 on APC. The connected PD-1 leads to the recruitment of phosphatases to CD28 and TCR complexes (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 aprimary target for PD-1-mediated inhibition. Science. 2017, 355 (6332): 1428-33), which in turn counteracts TCR signaling and 4-1BB stimulation. Therefore, blocking the PD-1 / PD-L1 interaction using the PD-1 antagonist cemiplimab combined with a CD38x4-1BB bispecific antibody may enhance T cell function.

[0447] Multispecific antigen-binding molecules:

[0448] The multispecific antigen-binding molecules and controls tested in this experiment are shown in Table 19.

[0449] Table 19: CD38x4-1BB binding molecules and controls

[0450] MABM Identifier describe REGN7633 <![CDATA[CD38x4-1BB 1+2,hIgG4 P-PVA ]]> REGN7647 <![CDATA[CD38x4-1BB 1+2,hIgG4 P-PVA ]]> REGN7650 <![CDATA[CD38x4-1BB 1+2,hIgG4 P-PVA ]]> REGN4249 Comparison 1 REGN2810 <![CDATA[PD-1 antibody (cemiplimab), hIgG4 P > REGN7540 <![CDATA[Isotype control, hIgG4 P-PVA > REGN1945 <![CDATA[Isotype control, hIgG4 P >

[0451] In CD38 cells engineered to express PD-L1 (NALM6 / hPD-L1) + In the presence of human acute lymphoblastic leukemia cancer cell lines, CD38x4-1BB 1+2 multispecific antigen binding molecules were evaluated for their ability to activate human primary T cells by engaging CD38 and 4-1BB receptors to deliver "signal 2" (as determined by IL-2 and IFNγ release). NALM6 cells provided allogeneic TCR responses sufficient to act as "signal 1". The addition of a fixed concentration of PD-1 antagonist antibody cimiprilimumab was evaluated in the presence of titration of CD38x4-1BB 1+2 or control antibodies.

[0452] Human primary CD3 + Isolation of T cells:

[0453] Human peripheral blood mononuclear cells (PBMCs) were isolated and frozen from a healthy donor leukocyte pack (donor 555192) from Precision for Medicine using the EasySep™ Direct Human PBMC Isolation Kit following the manufacturer's recommended protocol. CD3 + T cells. EasySepTM human CD3 T cells from StemCell Technologies were used. + T cell isolation kit and follow the manufacturer's instructions to enrich donor PBMC for CD3 + T cells.

[0454] IL-2 and IFNγ release assays:

[0455] Resuspend the enriched CD3 + T cells were 1×10 5 NALM6 cells engineered to express hPD-L1 or NALM-6 cells were added to a 96-well round-bottom plate at a concentration of 5x10 cells / well. 4 The final concentration of cells / well was added to CD3 +T cells. Subsequently, REGN7633, REGN7647, REGN7650, REGN4249 and REGN7540 were titrated from 0.76pM to 50nM at a 1:4 dilution and added to the wells. The end point of the 10-point dilution did not contain the titrated antibody. After adding the titrated antibody, a constant 20nM of Cimiprili monoclonal antibody or its matching isotype control (REGN1945) was added to the wells. The plate was incubated at 37°C, 5% CO2 for 72 hours, and 5 μL of supernatant was used to measure IL-2 or IFNγ. According to the manufacturer's protocol, the AlphaLisa kit from PerkinElmer was used to determine the amount of cytokines in the assay supernatant. Cytokine measurements were obtained on the Perkin Elmer multilabel microplate reader Envision and reported in pg / mL. All serial dilutions were tested in triplicate.

[0456] result

[0457] Using GraphPad Prism TM The software determines the EC of the antibody based on a four-parameter logistic equation on a 10-point dose-response curve. 50 Values. Maximum cytokines are given as the average maximum response detected in the dose range tested. Results are provided in Tables 20 and 21.

[0458] In the presence of allogeneic NALM6 cells or NALM6 cells engineered to express PD-L1, CD38x4-1BB 1+2 antibody treatment (REGN7633, REGN7647 and REGN7650) resulted in a dose-dependent increase in IL-2 and IFNγ release compared to the matched isotype control (REGN7540). Compared with NALM-6 (not expressing PD-L1), the maximum IL-2 and IFNγ release was lower under the condition of using NALM6 / PD-L1 cells. Compared with the matching isotype control REGN1945 of Cimiprilimab, Cimiprilimab increased the maximum cytokine release in the presence of NALM6 cells expressing PD-L1. It is worth noting that the 1+2 form of the CD38x4-1BB antibody resulted in higher maximum cytokine release and greater efficacy compared to the bivalent 4-1BB (REGN4249) antibody.

[0459] Table 20: Maximum IL-2 release and potency values

[0460]

[0461]

[0462] Abbreviations: ND: not determined; NC: not calculated because data did not fit the 4-parameter logistic equation.

[0463] Table 21: Maximum IFNγ release and potency values

[0464]

[0465] Abbreviations: ND: not determined; NC: not calculated because data did not fit the 4-parameter logistic equation.

[0466] Example 11: In vivo anti-tumor efficacy of the combination of CD38x4-1BB bispecific antibody and BCMAxCD3 bispecific antibody.

[0467] The multispecific antigen-binding molecules and controls tested in this experiment are shown in Table 22.

[0468] Table 22: CD38x4-1BB binding molecules and controls

[0469] MABM Identifier describe REGN5458 BCMAxCD3 bsAB REGN13168 <![CDATA[Non-TAAx4-1BB(1+2) control, (hIgG4 P-PVA )]]> REGN9686 <![CDATA[CD38x4-1BB(1+2),(G4S)2(hIgG4 P-PVA )]]>

[0470] To determine the in vivo antitumor efficacy of the CD38x4-1BB 1+2 bispecific antibody (bsAb) in combination with the BCMAxCD3 bsAb, a xenograft study was performed. scid Iq tm1Wjl / SzJ(NSG) mice were injected intraperitoneally with 4x10 6 Human peripheral blood mononuclear cells (PBMCs). On day 0, mice were intravenously administered 2×10 6 BCMA + CD38 +MOLP-8 human multiple myeloma tumor cells, which are engineered to also express firefly luciferase (MOLP-8-luciferase cells). Then 0.4mg / kg of control bispecific Ab or BCMAxCD3 bispecific antibody (REGN5458; U.S. Patent Application Publication 20200024356) binding CD3 was immediately administered to mice (n=4-5 per group), with 4mg / kg of control bispecific Ab (1+2 form) or CD38x4-1BB bispecific antibody (1+2 form; REGN9686) binding 4-1BB. On the 7th and 14th days, these antibodies were administered to mice twice more, for a total of three doses. Tumor growth within 53 days was assessed by measuring tumor bioluminescence (BLI) of anesthetized animals. As a positive control, a group of mice (n=5) were given only MOLP-8-luciferase cells and PBMCs, but no antibodies (PBS treatment group). To measure background BLI levels, one group of mice (n=5) was untreated and received no tumor, PBMCs, or antibodies (no tumor group).

[0471] These studies demonstrated that while BCMAxCD3 bispecific antibody monotherapy exhibited only modest anti-tumor efficacy and CD38x4-1BB 1+2 bispecific antibody monotherapy exhibited little / no anti-tumor activity, combination treatment with BCMAxCD3 bispecific antibody plus CD38x4-1BB 1+2 bispecific antibody resulted in combined anti-tumor efficacy that was superior to either therapy alone.

[0472] Xenograft tumor implantation and measurements

[0473] On day -11, immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) mice were injected intraperitoneally with 4x10 6 Human peripheral blood mononuclear cells (PBMCs). On day 0, mice were intravenously administered 2×10 6 BCMA + CD38 +MOLP-8 human multiple myeloma tumor cells, which are engineered to also express firefly luciferase (MOLP-8-luciferase cells). Then 0.4mg / kg of control bispecific antibodies or BCMAxCD3 bispecific antibodies (REGN5458) binding to CD3 were immediately administered to mice (n=4-5 per group), with 4mg / kg of 4-1BB binding control bispecific antibodies (1+2 form) or CD38x4-1BB bispecific antibodies (1+2 form; REGN9686) in combination. On the 7th and 14th days, these Abs were administered to mice twice more, for a total of three doses. Tumor growth within 53 days was assessed by measuring the tumor bioluminescence (BLI) of anesthetized animals. As a positive control, a group of mice (n=5) were given only MOLP-8-luciferase cells and PBMCs, but no antibodies (PBS treatment group). In order to measure background BLI levels, a group of mice (n=5) were untreated and did not receive tumors, PBMCs or antibodies (no tumor group).

[0474] Measurement of xenograft tumor growth

[0475] BLI imaging was used to measure tumor burden. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after injection, mice were imaged by BLI using the Xenogen IVIS system under isoflurane anesthesia. Image acquisition was performed with a field of view at D, an object height of 1.5 cm, and a medium frame and level, with automatic exposure time determined by the Living Image software. BLI signals were extracted using the Living Image software: a region of interest was drawn around each tumor mass, and photon intensity was recorded as total flux (photons / second – p / s).

[0476] result :

[0477] Tables 23-32 provide the results for the effect of treatment combinations on tumor burden and subject survival 6 days, 10 days, 13 days, 17 days, 20 days, 24 days, 27 days, 31 days, 34 days and 38 days after administration of human multiple myeloma tumor cells. Figure 3 is a graphical representation of the data shown in the table over a 38-day period. Figure 4A Shown is the tumor burden over time in mice treated with PBS relative to mice that did not receive tumor cells; Figure 4B Shown is the tumor burden over time in mice treated with a control bsAb binding CD3 (0.4 mg / kg) + a control bsAb binding 4-1BB (4 mg / kg) relative to mice that did not receive tumor cells; Figure 4CShown is the tumor burden over time in mice treated with a control CD3-binding bsAb (0.4 mg / kg) + CD38x4-1BB (4 mg / kg) relative to mice that did not receive tumor cells; Figure 4D Shown is the tumor burden over time in mice treated with BCMAxCD3 bsAb (0.4 mg / kg) + a control bsAb that binds 4-1BB (4 mg / kg) relative to mice that did not receive tumor cells; Figure 4E Shown is the tumor burden over time in mice treated with BCMAxCD3 bsAb (0.4 mg / kg) + CD38x4-1BB (4 mg / kg) relative to mice that did not receive tumor cells.

[0478] BCMAxCD3 monotherapy: The BCMAxCD3 bsAb (REGN5458) plus a control bsAb that binds 4-1BB provided some anti-tumor efficacy, with reduced mean BLI readings compared to mice receiving a control bsAb that binds CD3 plus a control bsAb that binds 4-1BB, with p<0.0001 on day 24 and p=0.0015 on day 26 (two-way ANOVA).

[0479] CD38x4-1BB monotherapy: Treatment with a control bsAb binding CD3 plus a CD38x4-1BB 1+2 bsAb (REGN9686) did not significantly reduce mean BLI readings compared to mice receiving a control bsAb binding CD3 plus a control bsAb binding 4-1BB.

[0480] BCMAxCD3+CD38x4-1BB 1+2: The combination of BCMAxCD3 bsAb (REGN5458) plus CD38x4-1BB 1+2 bsAb (REGN9686) resulted in lower mean BLI readings than those receiving BCMAxCD3 bsAb plus a control bsAb that binds 4-1BB (p<0.0001 at day 38, two-way ANOVA).

[0481] Thus, these studies demonstrate that while BCMAxCD3 bsAb monotherapy exhibited only modest antitumor efficacy and CD38x4-1BB 1+2bsAb monotherapy exhibited little / no antitumor activity, combination therapy with BCMAxCD3bsAb plus CD38x4-1BB 1+2bsAb resulted in combined antitumor efficacy superior to that of either therapy alone.

[0482] Table 23: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 6

[0483]

[0484] Table 24: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 10

[0485]

[0486] Table 25: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 13

[0487]

[0488]

[0489] Table 26: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 17

[0490]

[0491] Table 27: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 20

[0492]

[0493]

[0494] Table 28: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 24

[0495]

[0496] Table 29: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 27

[0497]

[0498] Table 30: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 31

[0499]

[0500]

[0501] Table 31: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 34

[0502]

[0503] Table 32: Anti-tumor efficacy obtained by combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb – Day 38

[0504]

[0505]

[0506] Example 12: Biacore binding kinetics of anti-CD38x4-1BB antigen binding molecules.

[0507] The binding kinetics of the anti-CD38x4-1BB antibodies were determined by antibody capture format Biacore binding kinetics of the anti-CD38x4-1BB 1+2 antibodies binding to monomeric and dimeric human 4-1BB reagents and antigen capture format Biacore binding kinetics of the anti-CD38x4-1BB 1+2 antibodies binding to dimeric human 4-1BB reagents. Both experiments were performed at 25°C.

[0508] Antibody capture format method:

[0509] 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 murine Fc tag (h4-1BB.mFc, REGN3585) binding to purified anti-CD38x4-1BB 1+2 antibodies was determined using a real-time surface plasmon resonance biosensor using a Biacore 8k instrument. DValues). The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567). All Biacore binding studies were performed in a buffer consisting of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v surfactant P20 (HBS-EP operating buffer). Different concentrations of h4-1BB.mmH (REGN3584) prepared in HBS-EP operating buffer (ranging from 100nM to 6.25nM, 4-fold serial dilutions) or different concentrations of h4-1BB.mFc (REGN3585) prepared in HBS-EP operating buffer (ranging from 100nM to 1.56nM, 4-fold serial dilutions) were injected onto the captured anti-CD38x4-1BB 1+2 antibodies at a flow rate of 30μL / min. The antibody-reagent binding was monitored for 5 minutes, while the dissociation in the HBS-EP operating buffer was monitored for 10 minutes. At the end of each cycle, the anti-CD38x4-1BB 1+2 antibody capture surface was regenerated using a 10 second injection of 20 mM phosphoric acid. All binding kinetic experiments were performed at 25°C. The results are presented in Tables 33 and 34.

[0510] Antigen capture format method:

[0511] The equilibrium dissociation constant (K) for binding of human 4-1BB expressed with a C-terminal murine Fc tag (h4-1BB.mFc, REGN3585) to purified anti-CD38x4-1BB 1+2 antibodies was determined using a Biacore 4000 instrument using a real-time surface plasmon resonance biosensor. D Value). 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 studies were performed in a buffer consisting of 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v surfactant P20 (HBS-EP operating buffer). Different concentrations of CD38x4-1BB 1+2 constructs (ranging from 100nM to 6.25nM, 4-fold serial dilutions) prepared in HBS-EP operating buffer were injected onto the h4-1BB.mFc (REGN3585) capture surface at a flow rate of 30 μL / min. Antibody-reagent binding was monitored for 5 minutes, while dissociation in HBS-EP operating 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.

[0512] Two forms of data analysis:

[0513] The kinetic binding rate constants (k) were determined by fitting the real-time sensorgrams to a 1:1 binding model using Cytiva Insight curve fitting software. a ) and the dissociation rate constant (k d Calculate the association and dissociation equilibrium constant (K) from the kinetic rate constant D ) and dissociation half-life (t1 / 2) are:

[0514]

[0515] Table 33: Kinetic and equilibrium binding parameters of monomeric human 4-1BB to surface captured anti-CD38x4-1BB 1+2 antibodies at 25°C

[0516]

[0517] *Dose-dependent binding of h4-1bb.mmH was observed

[0518] **IC: Observed binding does not fit the binding simulation model and binding kinetic parameters were not determined under the current experimental conditions.

[0519] Table 34: Kinetic and equilibrium binding parameters of dimeric human 4-1BB to surface captured anti-CD38x4-1BB 1+2 antibodies at 25°C

[0520]

[0521]

[0522] Table 35: Kinetic and equilibrium binding parameters of anti-CD38x4-1BB 1+2 antibodies to surface captured dimeric human 4-1BB at 25°C

[0523]

[0524] The scope of the present invention is not limited by the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A bispecific antigen-binding molecule, comprising: (a) a first antigen-binding arm comprising three complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) and three CDRs of a LCVR, wherein the first antigen-binding arm specifically binds to CD38; and (b) a second antigen-binding arm comprising a first antigen-binding region (R1) comprising three CDRs of a HCVR (R1-HCVR) and three CDRs of a LCVR (R1-LCVR); and a second antigen-binding region (R2) comprising three CDRs of a HCVR (R2-HCVR) and three CDRs of a LCVR (R2-LCVR), wherein the second antigen-binding arm specifically binds to 4-1BB.

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

3. The bispecific antigen binding molecule of claim 1, wherein R1 and R2 bind to different epitopes on 4-1BB. 4 . The bispecific antigen binding molecule of claim 1 , wherein R1 and R2 are connected via a peptide linker. 5 . The bispecific antigen binding molecule of claim 4 , wherein the peptide linker comprises a peptide sequence of (GGGGS)n, wherein n is 1 to 6.

6. The bispecific antigen binding molecule of any one of claims 1-5, wherein the first antigen binding arm comprises three CDRs of a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40.

7. The bispecific antigen binding molecule of any one of claims 1-6, wherein the first antigen binding arm comprises three CDRs of a LCVR, and the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

8. The bispecific antigen binding molecule of any one of claims 1 to 7, wherein the first antigen binding arm comprises three heavy chain complementarity determining regions (HCDR1-HCDR2-HCDR3) comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6-8 and 42-44-46, respectively.

9. The bispecific antigen binding molecule of any one of claims 1 to 8, wherein the first antigen binding arm comprises three light chain complementary determining regions (LCDR1-LCDR2-LCDR3) comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54, respectively.

10. The bispecific antigen binding molecule of any one of claims 1-9, 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.

11. The bispecific antigen binding molecule of any one of claims 1-10, wherein the first antigen binding arm comprises a LCVR and the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

12. The bispecific antigen binding molecule of any one of claims 1-11, 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.

13. The bispecific antigen binding molecule of any one of claims 1-12, wherein R1 comprises three CDRs of a HCVR (R1-HCVR), wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94.

14. The bispecific antigen binding molecule of any one of claims 1-13, wherein R1 comprises three CDRs of a LCVR (R1-LCVR), wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

15. The bispecific antigen binding molecule of any one of claims 1-14, wherein R1 comprises three heavy chain complementary determining regions (R1-HCDR1-R1-HCDR2-R1-HCDR3), each of which comprises 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.

16. The bispecific antigen binding molecule of any one of claims 1-15, wherein R1 comprises three light chain complementary determining regions (R1-LCDR1-R1-LCDR2-R1-LCDR3), and the three light chain complementary determining regions respectively comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 20-22-24 and 50-52-54.

17. The bispecific antigen binding molecule of any one of claims 1-16, wherein R1 comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94 (R1-HCVR).

18. The bispecific antigen binding molecule of any one of claims 1-17, wherein R1 comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48 (R1-LCVR).

19. The bispecific antigen binding molecule of any one of claims 1-18, wherein 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.

20. The bispecific antigen binding molecule of any one of claims 1-19, wherein R2 comprises three CDRs of a HCVR (R2-HCVR), wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94.

21. The bispecific antigen binding molecule of any one of claims 1-20, wherein R2 comprises three CDRs of a LCVR (R2-LCVR), wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

22. The bispecific antigen binding molecule of any one of claims 1-21, wherein R2 comprises three heavy chain complementarity determining regions (R2-HCDR1-R2-HCDR2-R2-HCDR3), each of which comprises 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.

23. The bispecific antigen binding molecule of any one of claims 1-22, wherein R2 comprises three light chain complementary determining regions (R2-LCDR1-R2-LCDR2-R2-LCDR3), and the three light chain complementary determining regions respectively comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54.

24. The bispecific antigen binding molecule of any one of claims 1-23, wherein R2 comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94 (R2-HCVR).

25. The bispecific antigen binding molecule of any one of claims 1-24, wherein R2 comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48 (R2-LCVR).

26. The bispecific antigen binding molecule of any one of claims 1-25, wherein 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.

27. The bispecific antigen binding molecule of any one of claims 1 to 26, wherein: (a) the first antigen-binding arm comprises three CDRs of a HCVR comprising the amino acid sequence of SEQ ID NO: 40, and three CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen binding arm comprises: (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 an amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding region (R2) comprising three CDRs of an R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 86 and 94; and three CDRs of an R2-LCVR comprising an amino acid sequence of SEQ ID NO:

48.

28. The bispecific antigen binding molecule of any one of claims 1 to 27, wherein: (a) the first antigen-binding arm comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 respectively comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42-44-46-50-52-54; and (b) the second antigen binding arm comprises: (i) a first antigen-binding region (R1) comprising amino acid sequences 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, respectively. R1-HCDR1-R1-HCDR2-R1-HCDR3-R1-LCDR1-R1-LCDR2-R1-LCDR3; and (ii) a second antigen-binding region (R2) comprising 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, respectively. R2-HCDR1-R2-HCDR2-R2-HCDR3-R2-LCDR1-R2-LCDR2-R2-LCDR3.

29. The bispecific antigen binding molecule of any one of claims 1 to 28, wherein: (a) the first antigen-binding arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 40 and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen binding arm comprises: (i) a first antigen-binding region (R1) comprising an R1-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 62, and 72; and an R1-LCVR comprising an amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding region (R2) comprising an R2-HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:62, 86 and 94; and an R2-LCVR comprising an amino acid sequence of SEQ ID NO:

48.

30. The bispecific antigen binding molecule of any one of claims 1 to 29, wherein: (a) the first antigen-binding arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 40 and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen binding arm comprises: (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; and (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.

31. The bispecific antigen binding molecule of any one of claims 1 to 29, wherein: (a) the first antigen-binding arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 40 and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen binding arm comprises: (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; and (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.

32. The bispecific antigen binding molecule of any one of claims 1 to 29, wherein: (a) the first antigen-binding arm comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 40 and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen binding arm comprises: (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.

33. The bispecific antigen binding molecule of any one of claims 1-32, wherein the molecule is a bispecific antibody. The bispecific antibody of claim 33 , wherein the bispecific antibody comprises a heavy chain constant region of the IgG1 or IgG4 isotype.

35. The bispecific antibody of claim 33 or 34, wherein the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen binding arm, and a second heavy chain comprising R1-HCVR and R2-HCVR of the second antigen binding arm, wherein the second heavy chain comprises mutations H435R and Y436F (EU numbering).

36. The bispecific antibody of any one of claims 33-35, comprising a first heavy chain comprising the HCVR comprising the first antigen binding arm, the first heavy chain paired with a light chain comprising the LCVR comprising 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.

37. The bispecific antibody of claim 36, comprising a second heavy chain comprising R1-HCVR and R2-HCVR comprising 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, wherein the second heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 70, 80, 82, and 84; and the 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.

38. The bispecific antibody of any one of claims 33-37, wherein: (a) the first antigen-binding arm that specifically binds to human CD38 comprises a heavy chain containing the sequence of SEQ ID NO: 58, and a light chain containing the sequence of SEQ ID NO: 60; and (b) The second antigen-binding arm that specifically binds to human 4-1BB comprises a heavy chain containing the sequence of SEQ ID NO: 56, a first light chain containing the sequence of SEQ ID NO: 60, and a second light chain containing the sequence of SEQ ID NO:

60.

39. The bispecific antibody of any one of claims 33-37, wherein: (a) the first antigen-binding arm that specifically binds to human CD38 comprises a heavy chain containing the sequence of SEQ ID NO: 58, and a light chain containing the sequence of SEQ ID NO: 60; and (b) The second antigen-binding arm that specifically binds to human 4-1BB comprises a heavy chain containing the sequence of SEQ ID NO: 70, a first light chain containing the sequence of SEQ ID NO: 60, and a second light chain containing the sequence of SEQ ID NO:

60.

40. The bispecific antibody of any one of claims 33-37, wherein: (a) the first antigen-binding arm that specifically binds to human CD38 comprises a heavy chain containing the sequence of SEQ ID NO: 58, and a light chain containing the sequence of SEQ ID NO: 60; and (b) The second antigen-binding arm that specifically binds to human 4-1BB comprises a heavy chain containing the sequence of SEQ ID NO: 80, a first light chain containing the sequence of SEQ ID NO: 60, and a second light chain containing the sequence of SEQ ID NO:

60.

41. The bispecific antibody of any one of claims 33-37, wherein: (a) the first antigen-binding arm that specifically binds to human CD38 comprises a heavy chain containing the sequence of SEQ ID NO: 58, and a light chain containing the sequence of SEQ ID NO: 60; and (b) The second antigen-binding arm that specifically binds to human 4-1BB comprises a heavy chain containing the sequence of SEQ ID NO: 82, a first light chain containing the sequence of SEQ ID NO: 60, and a second light chain containing the sequence of SEQ ID NO:

60.

42. The bispecific antibody of any one of claims 33-37, wherein: (a) the first antigen-binding arm that specifically binds to human CD38 comprises a heavy chain containing the sequence of SEQ ID NO: 58, and a light chain containing the sequence of SEQ ID NO: 60; and (b) The second antigen-binding arm that specifically binds to human 4-1BB comprises a heavy chain containing the sequence of SEQ ID NO: 84, a first light chain containing the sequence of SEQ ID NO: 60, and a second light chain containing the sequence of SEQ ID NO:

60.

43. 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 a HCVR comprising the amino acid sequence of SEQ ID NO: 40, and three CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen binding arm comprises: (i) a first antigen-binding region (R1) comprising three CDRs of a HCVR (R1-HCVR) comprising the amino acid sequence of SEQ ID NO:62, and three CDRs of a LCVR (R1-LCVR) comprising the amino acid sequence of SEQ ID NO:48; and (ii) a second antigen-binding region (R2) comprising three CDRs of a HCVR (R2-HCVR) comprising the amino acid sequence of SEQ ID NO:62, and three CDRs of a LCVR (R2-LCVR) comprising the amino acid sequence of SEQ ID NO:

48.

44. The bispecific antigen binding molecule of claim 43, wherein the molecule is a bispecific antibody.

45. The bispecific antigen binding molecule of claim 44, wherein the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen binding arm, wherein the first heavy chain is 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.

46. ​​The bispecific antigen binding molecule of claim 45, wherein the bispecific antibody comprises a second heavy chain comprising R1-HCVR and R2-HCVR of the second antigen binding arm, wherein the second heavy chain is paired with a first light chain comprising R1-LCVR and a second light chain comprising R2-LCVR, wherein the second heavy chain comprises the amino acid sequence of SEQ ID NOs: 70, 82, and 84; the 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.

47. The bispecific antigen binding molecule of any one of claims 43-46, wherein: (a) the first antigen-binding arm that specifically binds to human CD38 comprises a heavy chain containing the sequence of SEQ ID NO: 58, and a light chain containing the sequence of SEQ ID NO: 60; and (b) the second antigen binding arm that specifically binds to human 4-1BB comprises: (i) 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; (ii) 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) 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.

48. A pharmaceutical composition comprising the bispecific antigen binding molecule of any one of claims 1-47 and a pharmaceutically acceptable carrier.

49. 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 of any one of claims 1-47.

50. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the R1-HCVR and R2-HCVR of the second antigen binding arm of the bispecific antigen binding molecule of any one of claims 1-47.

51. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the LCVR of the bispecific antigen binding molecule of any one of claims 1-47.

52. An expression vector comprising the isolated nucleic acid molecule of any one of claims 49-51.

53. A host cell comprising the expression vector of claim 52.

54. The host cell of claim 53, wherein the host cell is Escherichia coli (E. coli) or a CHO cell.

55. A method for producing a multispecific antigen-binding molecule, the method comprising growing the host cell of claim 53 under conditions that allow 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 second nucleic acid molecule encoding the nucleic acid sequence of the heavy chain variable region (HCVR) of antigen-binding arm A2 of the multispecific antigen-binding molecule, and a third nucleic acid molecule comprising a nucleic acid sequence encoding a common light chain variable region (LCVR).

56. The method of claim 55, wherein the host cell comprises a first nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain of antigen-binding arm A1 of the multispecific antigen-binding molecule, a second nucleic acid molecule encoding the heavy chain of antigen-binding arm A2 of the multispecific antigen-binding molecule, and a third nucleic acid molecule comprising a nucleic acid sequence encoding a common light chain.

57. A method of inhibiting the growth of a plasma cell tumor in a subject, the method comprising administering to the subject the multispecific antigen-binding molecule of any one of claims 1 to 47 or the pharmaceutical composition of claim 48.

58. The method of claim 57, wherein the plasma cell neoplasm is multiple myeloma.

59. A method of inhibiting tumor growth in a subject, the method comprising administering to the subject a multispecific antigen-binding molecule of any one of claims 1 to 47 or a pharmaceutical composition of claim 48, 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 characterized in part by having CD38+ cells.

60. A method of treating a patient suffering from a B-cell malignancy expressing BCMA, the method comprising administering to the subject the multispecific antigen-binding molecule of any one of claims 1 to 47 or the pharmaceutical composition of claim 48.

61. The method of claim 60, wherein the BCMA-expressing B-cell malignancy is selected from the group consisting of Waldenstrom's macroglobulinemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, multiple myeloma, and Hodgkin's lymphoma.

62. The method of any one of claims 57 to 61, further comprising administering a second therapeutic agent or treatment regimen.

63. The method of claim 62, wherein the second therapeutic agent is an antibody that binds to plasma cell neoplasms.

64. The method of claim 62, wherein the second therapeutic agent is an anti-BCMA / anti-CD3 bispecific antigen binding molecule.

65. The method of claim 62, wherein the second therapeutic agent is an anti-CD20 / anti-CD3 bispecific antigen binding molecule.

66. The method of claim 62, wherein the second therapeutic agent is an agonist of CD28 or an agonist of 4-1BB.

67. methods as claimed in claim 62, wherein the second therapeutic agent or treatment regimen comprises a chemotherapeutic drug, 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, antibody drug conjugates, bispecific antibodies conjugated to anti-tumor agents, PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 checkpoint inhibitors, CD28 agonists, 4-1BB agonists, anti-BCMA / anti-CD3 bispecific antigen binding molecules, anti-CD20 / anti-CD3 bispecific antigen binding molecules, cancer vaccines, oncolytic viruses, immunocytokines, CD22 inhibitors, IL4 inhibitors, IL6 inhibitors, T cells (CAR-T cells) comprising chimeric antigen receptors, or a combination thereof.

68. A method of treating a patient having a CD38+ tumor and / or a tumor expressing BCMA, the method comprising administering to the subject a combination of the multispecific antigen-binding molecule of any one of claims 1 to 47 or the pharmaceutical composition of claim 48 and a PD-1 inhibitor.

69. The method of claim 68, 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.

70. The method of claim 69, wherein the anti-PD-1 inhibitor is selected from cemiplizumab, nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab.

71. The method of claim 69, wherein the PD-1 inhibitor is cemiplimab.

72. Use of the multispecific antigen-binding molecule of any one of claims 1 to 47 or the pharmaceutical composition of claim 48 in treating a disease or disorder associated with the expression of CD38, CD20 and / or BCMA.

73. The use of claim 72, wherein the disease or disorder is cancer.

74. The use of claim 73, 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 characterized in part by having CD38+ cells.

75. The use of any one of claims 72 to 74, wherein the multispecific antigen-binding molecule or pharmaceutical composition is used in combination with an anti-PD-1 antibody or antigen-binding fragment thereof.

76. The use of claim 72, wherein the multispecific antigen-binding molecule or pharmaceutical composition is injected intravenously, intramuscularly or subcutaneously.

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