OA21950A

OA21950A - Multispecific antigen binding molecules that bind CD38 and 4-1BB, and uses thereof.

Info

Publication number: OA21950A
Application number: OA1202400433
Authority: OA
Inventors: Robert Babb; David Dilillo; Aynur Hermann; Kara Olson; Yang Shen; Erica ULMANN
Original assignee: Regeneron Pharmaceuticals, Inc
Priority date: 2022-05-18
Filing date: 2023-05-17
Publication date: 2025-07-14

Abstract

CD38 is expressed on malignant plasma cells. 4lBB is a costimulatory molecule required for T-cell activation and survival. Provided herein are novel multispecific antigen binding molecules (MABMs) that bind to both CD38 and 4lBB, for example, to provide "signal 2" in order to enhance the activation of T cells in the presence of a "signal 1", provided by a Tumor-associated antigen (TAA) x CD3 bispecific antibody or an allogeneic response 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 tumors expressing CD38. The multispecific antigen binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced CD38-targeted immune response is desired and/or therapeutically beneficial. For example, the multispecific antigen binding molecules of the invention are useful for the treatment of various cancers, including multiple myeloma, lymphoma, and leukemia.

Description

MULTISPECIFIC ANTIGEN BINDING MOLECULES THAT BIND CD38 AND 4-1BB, AND USES THEREOF

FIELD OF THE INVENTION

[0001] The présent invention relates to multispecific antigen binding molécules, which are spécifie for CD38 and 4-1 BB, and methods of use thereof.

SEQUENCE LISTING

[0002] An official copy of the sequence listing is submitted concurrently with the spécification electronically via Patent Center. The contents of the electronic sequence listing (10927W001_Sequence_Listing_ST26.xml; Size: 151,552 bytes; and Date of Création: May 17, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

[0003] Multiple Myeloma (MM) is the second most common blood cancer after non-Hodgkin lymphoma, with a prevalence of -120,000, and roughly 30,000 new cases and 13,000 deaths i 15 each year in the US. MM is characterized by a clonal expansion of malignant plasma cells which secrete cytokines in an unregulated manner. The production of cytokines, especially IL-6, causes localized organ and tissue damage responsible for many of the symptoms associated with myeloma. Subjects with MM suffer from bone pain and osteoporosis, anémia, impaired kidney function and kidney failure, bacterial infections, and neurological impairments. MM is rarely curable with a médian life expectancy of 4-5 years. While progress has been made in treating MM, new thérapies hâve disproportionately benefited younger patients. Prognosis of relapsed MM patients is poor, and novel therapeutic approaches are urgently needed.

[0004] 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, 25 and dendrîtic cells. CD38 functions as an ectoenzyme involved in the metabolism of extracellular nicotinamide adenine dinucleotide (NAD⁺) and cytoplasmic nicotinamide adenine dinucleotide phosphate (NADP) (Howard, et al. Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38. Science (1993) 262:1056—9), resulting in the production of Ca²⁺-mobilizing compounds, such as cyclic adenosine diphosphate (ADP) ribose, ADP ribose 30 (ADPR) and nicotinic acid adenine dinucleotide phosphate. Calcium régulation results in the activation of signaling pathways that control a wide range of physiological functions, including lymphocyte prolifération, insulin release by the pancréas, cardiac muscle contraction, neutrophil chemotaxis and T cell activation. CD38 enzymatic activities regulate NAD* levels and improve the function of protéasome inhibitors (Cagnetta, et al. Intracellular NAD(+) déplétion 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 molécule

I i i

adenosine (ADO), facilitating the escape of tumor cells from the control of the immune System (Chillemi et al. Rôles and modalities of ectonucleotidases in remodeling the multiple myeloma niche. Front Immunol. (2017) 8.305). CD38 appears to contribute to the proliférative 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 thought to deplete CD38+ immunosuppressive cells, such as myeloid-derived suppressor cells, regulatory T cells, and regulatory B cells, leading to increased anti-tumor activity of immune effector cells. Daratumumab, an anti-CD38 antigen binding molécule, has been approved for multiple myeloma patients who are refractory to conventional therapy.

[0005] T cell activation involves co-stimulation via the TNF-receptor superfamily and is key to survival, acquisition of effector fonctions, and memory différentiation. 4-1BB (Tnfrsf9), also known as CD137, is a surface glycoprotein and member of the TNF-receptor superfamily. Receptor expression is induced by lymphocyte activation following TCR-mediated priming, but its levels can be augmented by CD28 co-stimulation. Exposure to ligand or agonist monoclonal antibodies on CD8⁺ T cells costimulates 4-1 BB, contributing to the clonal expansion, survival, and development of T cells, induced prolifération in peripheral monocytes, activation of NFkappaB, enhanced T cell apoptosis induced by TCR/CD3 triggered activation, memory génération, and régulation of CD28 co-stimulation to promote Th1 cell responses. Urelumab (BMS-663513), a fully human lgG4 monoclonal antibody, was the first anti-4-1BB therapeutic to enter clinical trials. Clinical development halted when liver toxicity associated with the antibody was revealed. Utomilumab (PF-05082566) is a humanized lgG2 monoclonal antibody that activâtes 4-1 BB while blocking binding to endogenous 4-1BBL. Thus, a need exists in the art for alternative approaches to treating cancer.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention relates, in part, to multispecific antigen binding molécules that bind CD38 and 4-1 BB and their use in treating various diseases, including cancer.

[0007] The multispecific antigen binding molécules can be used alone or in combination with other agents for treating cancers that express CD38.

[0008] The multispecific antigen binding molécules provided herein comprise two antigen binding arms, A1 and A2. The A1 arm binds specifically to CD38. The A2 arm comprises a first antigen-binding domain (R1) and a second antigen-binding domain (R2) and A2 binds specifically to 4-1 BB. R1 is linked to R2 via a linker, forming stacked antigen-binding domains on the A2 arm. The combination ofthe A1 arm and the stacked A2 arm is termed a 1+2 format. The antigen-binding domains of A2 may be contained in Fabs. See Figure 1. In some aspects, 35 R1 and R2 bind different 4-1 BB epitopes. In some aspects, R1 and R2 bind the same 4-1 BB epitopes. In some aspects, the amino acid sequence of R1 and R2 heavy chain variable régions are identical or substantially similar, i.e., less than 5, or less than 4, or less than 3, amino acid i

différences in a heavy chain variable région or a heavy chain complementarity determining région, or 2 or 1 amino acid différences in a heavy chain variable région or a heavy chain complementarity determining région. In some aspects, the R1 and R2 heavy chain variable régions are different, i.e., hâve different antigen binding sequences. In some embodiments, R1 is comprised in a first Fab (Fab2) and R2 is comprised in a second Fab (Fab3). The Fab2 and Fab3 of the anti-CD38xanti-4-1 BB 1+2 construct are connected via a linker from the N-terminus of the VH-2 4-1 BB “IN” Fab2 to the C-terminus of the CH 1-3 “OUT” Fab3.

Multispecific Antigen Binding Molécules Comprising Anti-CD38 and Anti-4-1BB Antigen 10 Binding Domains

[0009] The présent disclosure provides multispecific antigen binding molécules that bind CD38 and 4-1 BB. Such multispecific antigen binding molécules are also referred to herein as antiCD38/anti-4-1BB multispecific antigen binding molécules. The CD38 antigen binding arm A1 comprises one antigen binding domain. The 4-1 BB antigen binding arm A2 comprises two antigen binding domains, R1 and R2. The R1 is referred to herein as the 4-1 BB “IN” binding domain, and the R2 is referred to herein as the 4-1 BB “OUT” domain. The multispecific antigen binding molécules having the stacked antigen-binding domains are referred to herein as antiCD38/anti-4-1 BB 1+2 multispecific antigen binding molécules, or anti-CD38xanti-4-1BB 1+2 multispecific antigen binding molécules, etc.

[0010] The anti-CD38 portion of the anti-CD38/anti-4-1BB multispecific molécule is useful for targeting tumor cells that express CD38 (e.g., plasma cells), and the anti-4-1BB portion of the multispecific molécule is useful for providing co-stimulation of T cells activated by cognate MHC peptide or tumor targeted CD3 multispecific antigen binding molécules. The simultaneous binding of CD38 on a tumor cell and 4-1 BB on a T-cell facilitâtes directed killing (cell lysis) of the targeted tumor cell by the activated T-cel). The anti-CD38/anti-4-1 BB 1+2 multispecific molécules provided herein are therefore useful, inter alia, for treating diseases and disorders related to or caused by CD38-expressing tumors (e.g., lymphomas, leukemias, multiple myeloma, hepatocellular carcinoma, non-smalî cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer).

[0011] The multispecific antigen binding molécules provided herein comprise a first antigen binding arm A1 comprising an antigen binding domain, that specifically binds human CD38, and a second antigen binding arm A2 comprising two antigen binding domains, R1 and R2, that specifically bind 4-1 BB. The present disclosure includes anti-CD38/anti-4-1 BB 1+2 multispecific molécules (e.g., multispecific antigen binding molécules) wherein each antigen binding domain, comprises a heavy chain variable région (HCVR) paired with a light chain variable région (LCVR). In certain exemplary embodiments of the invention, the anti-CD38 antigen binding domain and the anti-4-1BB antigen binding domains each comprise different, distinct HCVRs paired with a common LCVR or universal LCVR. For example, as illustrated in Example 4 herein, multispecific antigen binding molécules were constructed comprising a first antigen binding domain that specifically binds CD38, wherein the first antigen binding domain comprises an HCVR derived from an anti-CD38 antigen binding molécule; and a second antigen binding domain (R1 ) and a third antigen binding domain (R2) that specifically bind 4-1BB, wherein the second antigen binding domain (R1) and third antigen binding domain (R2) each comprise an HCVR derived from an anti-4-1 BB antigen binding molécule, where each HCVR is paired with a universal light chain LCVR. In such embodiments, the first, second, and third antigen binding domains comprise distinct anti-CD38 and anti-4-1 BB HCVRs, respectively, but share a common light chain LCVR.

[0012] Provided herein are bispecific antigen-binding molécules comprising:

(a) a first antigen-binding arm comprising three CDRs of a heavy chain variable région (HCVR) and three CDRs of a LCVR, wherein the first antigen-binding arm binds specifically to CD38; and (b) a second antigen-binding arm comprising a first antigen-binding région (R1) comprising three CDRs of a HCVR (R1-HCVR) and three CDRs of a LCVR (R1-LCVR); and a second antigen-binding région (R2) comprising three CDRs of a HCVR (R2-HCVR) and three CDRs of a LCVR (R2-LCVR), wherein the second antigen-binding arm binds specifically to 41BB.

[0013] In some aspects, R1 and R2 bind tothe same epitope on 4-1 BB. In some aspects, R1 and R2 bind to different epitopes on 4-1 BB.

[0014] In some aspects, R1 and R2 are connected via a peptide linker. An exemplary peptide linker comprises a peptide sequence of (GGGGS)n, wherein n is 1 to 6.

[0015] In some aspects, the bispecific antigen-binding molécule comprises a first antigenbinding arm, wherein the first antigen-binding arm comprises three CDRs of a HCVR comprising 25 an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40.

[0016] In some aspects, the bispecific antigen-binding molécule comprises a first antigenbinding 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.

[0017] In some aspects, the bispecific antigen-binding molécule comprises a first antigen30 binding arm, wherein the first antigen-binding arm comprises three heavy chain complementarity determining régions (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. [0018] In some aspects, the bispecific antigen-binding molécule comprises a first antigenbinding arm, wherein the first antigen-binding arm comprises three light chain complementarity determining régions (LCDR1-LCDR2-LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24, and 50-52-54, respectively.

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

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

[0021] In some aspects, the bispecific antigen-binding molécule comprises a first antigenbinding arm, wherein the first antigen-binding arm comprises a HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40; and a LCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0022] In some aspects, the bispecific antigen-binding molécule comprises a first antigenbinding arm, wherein the second antigen-binding arm comprises a first antigen-binding région (R1); and a second antigen-binding région (R2).

[0023] In some aspects, R1 comprises three CDRs of a HCVR (R1-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 15 94.

[0024] In some aspects, R1 comprises three CDRs of a LCVR (R1-LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0025] In some aspects, R1 comprises three heavy chain complementarity determining régions (R1-HCDR1-R1-HCDR2-R1-HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16, 34-36-38, 64-66-68, 74-76-78, 88-90-92, and 9698-100, respectively.

[0026] In some aspects, R1 comprises three light chain complementarity determining régions (R1-LCDR1-R1-LCDR2-R1-LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24, and 50-52-54, respectively.

[0027] In some aspects, R1 comprises a HCVR (R1-HCVR) comprising the 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 a LCVR (R 1-LCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0029] In some aspects, R1 comprises a R1-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94; and a R1-LCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0030] In some aspects, R2 comprises three CDRs of a HCVR (R2-HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 35 94.

[0031] In some aspects, R2 comprises three CDRs of a LCVR (R2-LCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0032] In some aspects, R2 comprises three heavy chain complementarity determining régions (R2-HCDR1-R2-HCDR2-R2-HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16, 34-36-38, 64-66-68, 74-76-78, 88-90-92, and 9698-100, respectively.

[0033] In some aspects, R2 comprises three light chain complementarity determining régions (R2-LCDR1-R2-LCDR2-R2-LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24, and 50-52-54, respectively.

[0034] In some aspects, R2 comprises a HCVR (R2-HCVR) comprising the 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 a LCVR (R2-LCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0036] In some aspects, R2 comprises a R2-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94; and a R2-LCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

[0037] In some aspects, the bispecific antigen-binding molécule comprises:

(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 (b) a second antigen-binding arm comprising:

²⁰ (O a first antigen-binding région (R1) comprising three CDRs of R1-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 62 and 72; and three CDRs of R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding région (R2) comprising three CDRs of R2-HCVR ²⁵ comprising the amino acid sequence selected from the group consisting of SEQ ID NOs:

62, 84 and 94; and three CDRs of R2-LCVR comprising the amino acid sequence of SEQ ID No: 48.

[0038] In some aspects, the bispecific antigen-binding molécule comprises:

(a) a first antigen-binding arm comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 42-4446-50-52-54, respectively; and (b) a second antigen-binding arm comprising:

(î) a first antigen-binding région (R1) comprising R1-HCDR1-R1-HCDR2-R1-HCDR3-R1LCDR1-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 74-76-7850-52-54, respectively; and (ii) a second antigen-binding région (R2) comprising R2-HCDR1-R2-HCDR2-R2HCDR3-R2-LCDR1-R2-LCDR2-R2-LCDR3 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.

[0039] In some aspects, the bispecific antigen-binding molécule comprises:

(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 (b) a second antigen-binding arm comprising:

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 62 and 72; and R1LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ü) a second antigen-binding région (R2) comprising R2-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 62, 86 and 94; and R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48.

[0040] in some aspects, the bispecific antigen-binding molécule comprises:

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence of SEQ ID NOs: 32; and R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding région (R2) comprising R2-HCVR comprising the amino acid sequence of SEQ ID NO: 86; and R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48.

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

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence of SEQ ID NOs: 72; and R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ü) θ second antigen-binding région (R2) comprising R2-HCVR comprising the amino acid sequence of SEQ ID NO: 94; and R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48.

[0042] In some aspects, the bispecific antigen-binding molécule comprises:

(a) a 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) second antigen-binding arm comprises:

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence of SEQ ID NOs: 62, and R1-LCVR comprising the amino acid sequence of

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

[0043] In some aspects, the bispecific antigen-binding molécule is a bispecific antibody.

[0044] In some aspects, the bispecific antigen-binding molécule is a bispecific antibody comprising a heavy chain constant région of lgG1 or lgG4 isotype.

[0045] In some aspects, 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 R210 HCVR of the second antigen-binding arm, wherein the second heavy chain comprises the mutations H435R and Y436F (EU numbering).

[0046] In some aspects, the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen-binding arm paired with a light chain comprising the LCVR of the first antigen-binding arm, wherein the first heavy chain comprises the amino acid sequence of SEQ 15 ID NO: 58 and the light chain comprises the amino acid sequence of SEQ ID NO: 60.

[0047] In some aspects, the bispecific antibody comprises a second heavy chain comprising R1-HCVR and R2-HCVR of the second antigen-binding arm 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 selected from the group consisting of SEQ ID NO:

56, 70, 80, 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.

[0048] In some aspects, the bispecific antibody comprises:

(a) a first antigen-binding arm that specifically binds human CD38 comprising a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and (b) a second antigen-binding arm that specifically binds human 4-1BB comprising a heavy chain comprising the sequence of SEQ ID NO: 56, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60.

[0049] In some aspects, the bispecific antibody comprises:

(a) a first antigen-binding arm that specifically binds human CD38 comprising a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and (b) a second antigen-binding arm that specifically binds human 4-1 BB comprising a 35 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

[0050] In some aspects, the bispecific antibody comprises:

(a) a first antigen-binding arm that specifically binds human CD38 comprising a heavy Chain comprising the sequence of SEQ ID NO: 58, and a light Chain comprising the sequence of SEQ ID NO: 60; and (b) a second antigen-binding arm that specifically binds human 4-1BB comprising a heavy chain comprising the sequence of SEQ ID NO: 80, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60.

[0051] In some aspects, the bispecific antibody comprises:

(a) a first antigen-binding arm that specifically binds human CD38 comprising a heavy 10 chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and (b) a second antigen-binding arm that specifically binds human 4-1 BB 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 15 NO: 60.

[0052] In some aspects, the bispecific antibody comprises:

(a) a first antigen-binding arm that specifically binds human CD38 comprising a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and (b) a second antigen-binding arm that specifically binds human 4-1 BB 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.

[0053] Provided herein is a bispecific antigen-binding molécule comprising a first antigen 25 binding arm that binds specifically to CD38 and a second antigen-binding arm that binds specifically to 4-1 BB, 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 LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (b) the second antigen-binding arm comprises:

(i) a first antigen-binding région (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 région (R2) comprising three CDRs of a HCVR (R2-HCVR) 35 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.

[0054] In some aspects, the bispecific antigen-binding molécule is a bispecific antibody.

[0055] 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.

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

(a) a first antigen-binding arm that specifically binds human CD38 comprising a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and (b) a second antigen-binding arm that specifically binds human 4-1 BB comprising:

(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.

[0057] In some embodiments, the multispecific antigen binding molécule inhibits the prolifération of CD38+ tumor cells selected from the group consisting of myeloma cells, leukemia cells, lymphoma cells, hepatocellular carcinoma cells, non-small cell lung cancer cells, melanoma cells, pancreatic ductal adenocarcinoma cells, glioma cells, or breast cancer cells. [0058] In another aspect, pharmaceutical compositions comprising the multispecific antigen binding molécule and a pharmaceutically acceptable carrier or diluent are provided. In a related aspect, the invention features a composition which is a combination of an anti-CD38/anti-4-1 BB 1 + 2 multispecific antigen binding molécule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-CD38/anti-4-1BB 1 + 2 multispecific antigen binding molécule.

[0059] In another aspect, nucleic acid molécules comprising a nucléotide sequence encoding the any one of the A1, or A2 are provided. In some aspects, nucleic acid molécules comprising a nucléotide sequence encoding the any one of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, heavy chain, and/or light chain are provided. In some embodiments, the nucleic acid molécule comprises one or more nucléotide sequences set forth in Tables 2, 4, 6, 8, or 10 are provided. The nucleic acid molécules comprising the nucleic acid sequences can be in any functional combination or arrangement thereof.

[0060] In some aspects, provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain variable région (HCVR) of a multispecific antigen binding molécule antigen binding arm A1, wherein A1 binds to CD38, and wherein (a) the HCVR comprises three heavy chain complementarity determining régions (HCDR1-HCDR2HCDR3) comprising the amino acid sequence of SEQ ID NOs: 42, 44, and 46, respectively or (b) the HCVR comprises an amino acid sequence of SEQ ID NO: 40.

[0061] In some aspects, provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain of a multispecific antigen binding molécule antigen binding arm A1, wherein A1 binds to CD38, and wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 58.

[0062] In some aspects, provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding heavy chain variable régions (HCVR) of a multispecific antigen binding molécule antigen binding arm A2, 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 complementarity determining régions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequence of SEQ ID NOs: 34, 36, and 38, and the R2-HCVR comprises three heavy chain complementarity determining régions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequence of SEQ ID NOs: 88, 90, and 92, or (b) the R1-HCVR comprises an amino acid sequence of SEQ ID NO: 32 and R2HCVR comprises an amino acid sequence of SEQ ID NO: 86.

[0063] Provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain of a multispecific antigen binding molécule antigen binding arm A2, wherein A2 binds to 4-1 BB, and wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 56.

[0064] Also provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a first heavy chain variable région (HCVR) and a second HCVR of a multispecific antigen binding molécule antigen binding arm A2, wherein A2 binds to 4-1 BB, and wherein (a) the first HCVR comprises three heavy chain complementarity determining régions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequence of SEQ ID NOs: 64, 66, and 68, and the second HCVR comprises three heavy chain complementarity determining régions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequence of SEQ ID NOs: 64, 66, and 68, or (b) the first HCVR comprises an amino acid sequence of SEQ ID NO: 62 and the second HCVR comprises an amino acid sequence of SEQ ID NO: 62.

[0065] Also provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain of a multispecific antigen binding molécule antigen binding arm A2, 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.

[0066] Provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a first heavy chain variable région (HCVR) and a second HCVR of a multispecific antigen binding molécule antigen binding arm A2, wherein A2 binds to 4-1 BB, and wherein (a) the first HCVR comprises three heavy chain complementarity detemnining régions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequence of SEQ ID NOs: 74, 76, and 78, and the second HCVR comprises three heavy chain complementarity determining régions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequence of SEQ ID NOs: 96, 98, and 100, or (b) the first HCVR comprises an amino acid sequence of SEQ ID NO: 72 and the second HCVR comprises an amino acid sequence of SEQ ID NO: 94.

[0067] Provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain of a multispecific antigen binding molécule antigen binding arm A2, wherein A2 binds to 4-1 BB, and wherein the heavy chain comprises an amino acid sequence of 15 SEQ ID NO: 80.

[0068] Also provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a light chain variable région (LCVR) of a multispecific antigen binding molécule, wherein (a) the LCVR comprises three heavy chain complementarity determining régions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequence of SEQ ID NOs:

50, 52, and 54, or (b) the LCVR comprises an amino acid sequence of SEQ ID NO: 48.

[0069] And provided herein is an isolated nucleic acid molécule comprising a nucleic acid sequence encoding a light chain of a multispecific antigen binding molécule, wherein the light chain comprises an amino acid sequence of SEQ ID NO: 60.

[0070] Provided herein is an expression vector or a set of expression vectors comprising one more nucleic acid molécules of any one of the nucleic acids described above. Also provided is a host cell comprising one or more expression vectors provided herein. In some 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. colî) cell. Further provided are compositions comprising one or more nucleic acid molécules described herein.

[0071] In some embodiments, methods are provided for producing a multispecific antigen binding molécule. As provided herein, the method comprises growing a host cell under conditions permitting production of the multispecific antigen binding molécule, wherein the host cell comprises a nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain variable région (HCVR) of a multispecific antigen binding molécule antigen binding arm

A1, a nucleic acid molécule comprising a nucleic acid sequence encoding heavy chain variable régions (HCVRs) of a multispecific antigen binding molécule antigen binding arm A2, and/or a nucleic acid molécule comprising a nucleic acid sequence encoding a common light chain variable région (LCVR). In some aspects, each nucleic acid molécule is in the same expression vector. In some aspects, one or more of the nucleic acid molécules are in different expression vectors. In some aspects, the host cell comprises a nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain of the multispecific antigen binding molécule antigen binding arm A1, a nucleic acid molécule encoding a heavy chain of the multispecific antigen binding molécule antigen binding arm A2, and/or a nucleic acid molécule comprising a nucleic acid sequence encoding a common light chain. In some aspects, each nucleic acid molécule is in the same expression vector. In some aspects, one or more of the nucleic acid molécules are in different expression vectors.

[0072] Provided herein are methods of inhibiting growth of a plasma cell tumor in a subject, comprising administering any one or more of the multispecific antigen binding molécules described herein, or pharmaceutical compositions provided herein, to the subject. In some aspects, the plasma cell tumor is multiple myeloma.

[0073] Also provided herein is the use of a multispecific antigen binding molécule described herein, or pharmaceutical composition provided herein, in the manufacture of a médicament for inhibiting growth of a plasma cell tumor in a subject. The multispecific antigen binding molécule or pharmaceutical composition can be administered to the subject. In some aspects, the plasma cell tumor is multiple myeloma.

[0074] Provided herein are methods of inhibiting growth of a tumor in a subject, the method comprising administering any one or more of the multispecific antigen binding molécules described herein, or pharmaceutical compositions provided herein, 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.

[0075] Also provided herein is the use of a multispecific antigen binding molécule described herein, or pharmaceutical composition provided herein, in the manufacture of a médicament for inhibiting growth of a tumor in a subject. The multispecific antigen binding molécule 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.

[0076] Provided herein is a method of treating a patient suffering from multiple myeloma, or from another BCMA-expressing B cell malignancy comprising administering a multispecific antigen binding molécule provided herein, or a pharmaceutical composition provided herein, to the subject. In some aspects, the BCMA-expressing B cell malignancy is selected from the group consisting of Waidenstrôm's macroglobulinemia, Burkitt's lymphoma, Diffuse Large B-Cell lymphoma, Non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, follicular lymphoma, μ 21950 mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin’s lymphoma.

[0077] Also provided herein is the use of a multispecific antigen binding molécule described herein, or pharmaceutical composition provided herein, in the manufacture of a médicament for treating a patient suffering from multiple myeioma, or from another BCMA-expressing B cell malignancy. The multispecific antigen binding molécule or pharmaceutical composition can be administered to the subject.

[0078] Provided herein is a method of treating a patient suffering from a CD38+ tumor and/or a BCMA-expressing tumor. The method comprises administering a multispecific antigen binding molécule described herein, or a pharmaceutical composition described herein, to the subject in combination with an anti-PD-1 antibody or antigen binding fragment thereof. In some aspects, the anti-PD-1 antibody or antigen binding fragment is an anti-PD-1 antibody, for example, cemiplimab (bsAb2810).

[0079] Also provided herein is the use of a multispecific antigen binding molécule described herein, or pharmaceutical composition provided herein, in the manufacture of a médicament for treating a patient suffering from a CD38+ tumor and/or a BCMA-expressing tumor.

[0080] In some embodiments, the methods or uses provided herein further comprise administering a second therapeutic agent or therapeutic regimen. In some aspects, the second therapeutic is an antibody that binds plasma cell tumors. In some aspects, the second therapeutic is an anti-BCMA/anti-CD3 bispecific antigen binding molécule. In some aspects, the second therapeutic is an anti-CD20/anti-CD3 bispecific antigen binding molécule. In some aspects, the second therapeutic is an anti-CD28/anti-4-1BB bispecific antigen binding molécule. In some aspects, the second therapeutic agent or therapeutic regimen comprises a chemotherapeutic drug, DNA alkylators, immunomodulators, protéasome inhibitors, histone deacetylase inhibitors, radiotherapy, a stem cell transplant, an oncolytic virus, a cancer vaccine, an immunocytokine, a CAR-T cell, a different bispecific antibody that interacts with a different tumor cell surface antigen and a T cell or immune cell antigen, an antibody drug conjugale, a bispecific antibody conjugated to an anti-tumor agent, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 checkpoint inhibitor, a CD22 inhibitor, a BCMA inhibitor, a CD28 agonist, a CD20 inhibitor, or combinations thereof.

[0081] Further provided are uses of a multispecific antigen binding molécule provided herein, or a pharmaceutical composition provided herein, in the treatment of a disease or disorder associated with expression of CD38, CD20, and/or BCMA. In some aspects, the disease or disorder is cancer. In some aspects, the cancer is multiple myeioma. In some aspects, the multispecific antigen binding molécule or pharmaceutical composition is for use in combination with an anti-PD-1 antibody or antigen binding fragment thereof. The multispecific antigen binding molécule, or pharmaceutical composition comprising the multispecific antigen binding molécule, is injected intravenously, intramuscularly or subcutaneously.

[0082] Provided herein are anti-CD38/anti-4-1 BB 1+2 multispecific antigen binding molécules having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or an antigen binding molécule lacking a fucose moiety présent on the oligosaccharide chain, for example, to increase antigen binding molécule dépendent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complément dépendent cytotoxicity (CDC).

[0083] In yet another aspect, provided herein are therapeutic methods for targeting/killing tumor cells expressing CD38 using an anti-CD38/anti-4-1 BB multispecific antigen binding molécule of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-CD38/anti-4-1 BB multispecific antigen binding molécule provided herein to a subject in need thereof.

[0084] The présent disclosure also includes the use of an anti-CD38/anti-4-1BB multispecific antigen binding molécule provided herein in the manufacture of a médicament for the treatment of a disease or disorder related to or caused by CD38 expression.

[0085] Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0086] Figure 1 is a schematic depicting the structure of an exemplary antr-CD38 x anti-41BB 1+2 multispecific antigen binding molécule. The A1 antigen binding arm comprises Fab1, which is spécifie for CD38, while the A2 antigen binding arm comprises two Fabs, Fab2 and Fab3, each of which are spécifie to 4-1 BB. The Fab2 and Fab3 of the anti-CD38xanti-4-1BB 1+2 construct are connected via a linker from the N-terminus of the VH-2 4-1 BB “IN” Fab2 to the C-terminus of the CH1-3 “OUT Fab3. The Fab1, Fab2, and Fab3 light chains comprise VL-1, VL-2, and VL-3 universal light chains, respectively.

[0087] Figure 2A compares percent activation of 4-1 BB in the Jurkat reporter assay by multispecific antigen binding molécules in various formats, including identical split 4-1 BB Fabs, identical stacked 4-1 BB Fabs, and mixed stacked 4-1 BB Fabs. Figure 2B illustrâtes the various constructs tested in the screen, and depicts the target dépendent bioassay.

[0088] Figure 3 illustrâtes in vivo tumor burden over time after administration of human multiple myeloma tumor cells to immunodeficient NOD.Cg-Prkdc^scldll2rg^tm1W|,/SzJ (NSG) mice intraperitoneally injected with 4x10⁶ human peripheral blood mononuclear cells (PBMC).

[0089] Figure 4A illustrâtes tumor burden over time in the mice treated with PBS relative to mice that received no tumor cells; Figure 4B illustrâtes tumor burden over time in the mice treated with CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg) relative to mice that received no tumor cells; Figure 4C illustrâtes tumor burden over time in the mice treated with CD3-binding control bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg) relative to mice that received no tumor cells; Figure 4D illustrâtes tumor burden over time in the mice treated with BCMAxCD3 bsAb (0.4mg/kg) + 4-1BB-binding control bsAb (4mg/kg) relative to mice that received no tumor cells; Figure 4E illustrâtes tumor burden over time in the mice treated with BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg) relative to mice that received no tumor cells.

DETAILED DESCRIPTION

[0090] Before the présent invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the présent invention will be limited only by the appended daims.

[0091] Unless defined otherwise, ail technical and scientific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0092] As used herein, the term about, when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and ail values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0093] Although any methods and materials similar or équivalent to those described herein can be used in the practice or testing of the présent invention, the preferred methods and materials are now described. Ail patents, applications and non-patent publications mentioned in this spécification are incorporated herein by reference in their entireties.

Définitions

[0094] The expression “4-1 BB”, as used herein, refers to a receptorfor4-1BBL. Crosslinking of 4-1 BB by 4-1BBL enhances T cell activation. Human 4-1 BB (or CD137) having UniProt accession numberQ07011 comprises the amino acid sequence as setforth in SEQ ID NO: 101; amino acid residues 1-23 are the signal peptide. Residues 24-186 make up the extracellular domain of the receptor.

Human 4-1 BB (Immunogen) amino acid sequence

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNN RNQICSPCPPNSFSSAGGQR TCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQ ELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDWCGPSPADLSPGAS SVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSWKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 101)

[0095] AH référencés to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression 4-1 BB means

I I human 4-1 BB unless specified as being from a non-human species, e.g., mouse 4-1 BB, monkey 4-1 BB, etc.

[0096] As used herein, an antigen binding molécule that binds4-1BB or an anti-4-1BB antigen binding molécule includes antigen binding molécules that specifically recognize 4-1 BB 5 expressed on the surface of a cell. The anti-4-1 BB antigen binding molécules provided herein comprise VRs and CDRs as disclosed herein. In certain embodiments, the antigen-binding molécules are antibodies. In certain embodiments, the antigen-binding molécules are bispecific antibodies.

[0097] The expression “CD38,” as used herein, also known as cyclic ADP ribose hydrolase, refers to a glycoprotein expressed on malignant plasma cells. CD38 plays a central rôle in regulating intracellular calcium levels. The protein has an N-terminal cytoplasmic tail, a single membrane-spanning domain, and a C-terminal extracellular région with four N-glycosylation sites. The term CD38 as used herein, refers to the human CD38 protein unless specified as being from a non-human species (e.g., mouse CD38, monkey CD38, etc.). The human CD38 protein has the amino acid sequence shown in SEQ ID NO: 102 (Human CD38 extracellular domain (V43-l300).mFc), and/or having the amino acid sequence as set forth in NCBI accession No. NP_001766.2 or NM_001775.3.

Human CD38 extracellular domain (V43-l300).mFc (Immunogen) amino acid

[0098] VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPC

NITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEF NTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDWHVMLNGSRSKIFDKNSTFGSV EVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKN PEDSSCTSEIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCWVDVSEDD

PDVQISWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIE

RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEP VLDSDGSYFMYSKLRVEKKNVWERNSYSCSWHEGLHNHHTTKSFSRTPGK* (SEQ ID NO: 102) mFc sequence underlined

[0099] As used herein, an antigen binding molécule that binds CD38 or an anti-CD38 antigen binding molécule includes antigen binding molécules that specifically recognize CD38.

[0100] The term antigen binding molécule includes multispecific antigen binding molécules,

e.g., anti-CD38 x anti-4-1 BB 1+2 multispecific antigen binding molécules. The anti-CD38 antigen binding molécules provided herein comprise VRs and CDRs as disclosed herein. In certain embodiments, the antigen-binding molécules are antibodies. In certain embodiments, the antigen-binding molécules are bispecific antibodies.

[0101] The term antigen binding molécule, as used herein, means any antigen binding molécule or molecular complex comprising at least one complementarity determining région (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD38 or 4-1 BB). The term antigen binding molécule includes immunoglobulin molécules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term antigen binding molécule includes immunoglobulin molécules comprising two antigen binding arms, A1 and A2. The term “antigen binding molécule” also includes immunoglobulin molécules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each 5 antigen binding arm comprises a heavy chain, which in turn comprises at least one heavy chain variable région (abbreviated herein as HCVR or VH-1, VH-2, or VH-3) and a heavy chain constant région (CH1-1, CH1-2, and CH1-3). The heavy chain constant région also comprises CH2 and CH3. Each light chain comprises a light chain variable région (abbreviated herein as LCVR; on the A1 arm, VL-1; on the A2 arm, VL-2 and VL-3) and a light chain constant région (on the A1 arm, CL-1, and on the A2 arm, CL-2 and CL-3). The VH and VL régions can be further subdivided into régions of hypervahabilîty, termed complementarity determining régions (CDRs), interspersed with régions that are more conserved, termed framework régions (FR).

Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-CD38 antigen binding arm or anti-4-1 BB antigen binding arm (or antigen binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0102] The terms antigen binding portion of an antigen binding molécule, antigen binding fragment thereof' of an antigen binding molécule, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. An antigen binding fragment of an antigen binding molécule may be derived, e.g., from full antigen binding molécule molécules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antigen binding molécule variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antigen binding molécule libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0103] An anti-CD38 x anti-4-1 BB 1+2 antigen binding molécule will typically comprise at least three variable domains. A variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable région may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding molécule may contain a monomeric VH or VL domain.

[0104] In certain embodiments, an antigen binding molécule 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 within a multispecific antigen binding molécule ofthe présent invention include: (i) Vh1-Ch1-1; (ii) V_H2-C_H1-2; (iti) V_h3-Ch1-3; (iv) V_h1-Ch1-Ch2; (v) V_h1-C_h1-C_h2-Ch3; (vi) V_h2-Ch1-2-C_h2; (vii) V_h2-C_h1-2-Ch2-C_h3; (viii) V_H3C_h1-3; (ix) V_h3-Ch1-3-V_h2-Ch1-2; (x) V_h3-C_h1-3-Vh2-C_h1-2-Ch2; (xi) V_h3-Ch1-3-Vh2-Ch1-2-C_h2C_h3; (xii) V_H-C_L; (xiü) V_L-1-C_L1; (xiv) V_L-2-C_L2; (xv) V_l-3-C_l3; (xvi) V_L-3-C_L3-C_H1-3; (xvii) V_L-2-C_L2-C_h1-2; (xviii) V_L1- C_l1-C_h1-Ch2-C_h3; (xix) V_L-2-C_L2-C_H1-2; (xx) V_L-2-C_L2-C_H1-2-C_H2; (xxi) V_L2-C_l2-Ch1-2-Ch2-Ch3; (xxii) Vl-3-C_l3-Ch1-3-V_h2-Ch1-2-Ch2; (xxiii) V_l-3-C_l3-Ch1-3-V_h2-Ch1-2Ch2-Ch3; and (xxiv) V_l-Cl. In any configuration of variable and constant domains, încluding any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker région. A hinge région may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which resuit in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molécule.

[0105] lllustratively, Figure 1 shows an A1 antigen binding arm that comprises Fab1, which is spécifie for CD38, and an A2 antigen binding arm that comprises two Fabs, Fab2 and Fab3, each of which are spécifie to 4-1BB. The Fab1, Fab2, and Fab3 light chains can be universal light chains. In this example, the V_L-1 is linked to the C_L-1, which is linked to the C_H1 -1 on the A1 arm. On the A2 arm, the Vl-3 is linked to the Cl-3, which is linked to the Ch 1-3. Likewise, the Vl-2 is linked to the Cl-2, which is linked to the C_H1-2. The Fab2 and Fab3 of the antiCD38xanti-4-1BB 1+2 construct are connected via a linker from the N-terminus ofthe Vh-2 41BB ΊΝ” Fab2 tothe C-terminus ofthe C_H1-3 OUT” Fab3.

[0106] The antigen binding molécules of the présent invention may function through complement-dependent cytotoxicity (CDC) or antigen binding molecule-dependent ceîlmediated cytotoxicity (ADCC). Complement-dependent cytotoxicity (CDC) refers to lysis of antigen-expressing cells by an antigen binding molécule of the invention in the presence of complément. Antigen binding molecule-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antigen binding molécule on a target ceîl and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Patent Nos 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant région of an antigen binding molécule is important in the ability of an antigen binding molécule to fix complément and médiate cell-dependent cytotoxicity. Thus, the isotype of an antigen binding molécule may be selected on the basis of whether it is désirable for the antigen binding molécule to médiate cytotoxicity.

[0107] In certain embodiments ofthe invention, the anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molécules provided herein are human antigen binding molécules. The term human antigen binding molécule, as used herein, is intended to include antigen binding molécules having variable and constant régions derived from human germline immunoglobulin sequences. The human antigen binding molécules ofthe invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term human antigen binding molécule, as used herein, is not intended to include antigen binding molécules in which CDR sequences derived from the germline of another mammalian species, such as a mouse, hâve 10 been grafted onto human framework sequences.

[0108] The multispecific antigen binding molécules provided herein may, in some embodiments, be recombinant human antigen binding molécules. The term 'Tecombinant human antigen binding molécule, as used herein, is intended to include ail human antigen binding molécules that are prepared, expressed, created or isolated by recombinant means, such as antigen binding molécules expressed using a recombinant expression vector transfected into a host cell (described further below), antigen binding molécules isolated from a recombinant, combinatorial human antigen binding molécule library (described further below), antigen binding molécules isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antigen binding molécules prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antigen binding molécules hâve variable and constant régions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antigen binding molécules are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the Vh and Vl régions ofthe recombinant antigen binding molécules are sequences that, while derived from and related to human germline V_H and V_L sequences, may not naturally exist within the human antigen binding molécule germline répertoire in vivo.

[0109] Human antigen binding molécules can exist in two forms that are associated with hinge 30 heterogeneity. In one form, an immunoglobulin molécule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molécule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antigen binding molécule). These forms hâve been extremely difficult to separate, even after affinity purification.

[0110] The frequency of appearance ofthe second form in various intact IgG isotypes is due to, but not limited to, structural différences associated with the hinge région isotype of the antigen binding molécule. A single amino acid substitution in the hinge région ofthe human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human lgG1 hinge. The instant invention encompasses antigen binding molécules having one or more mutations in the hinge, Ch2 or Ch3 région which may be désirable, for example, in production, to improve the yield of the desired antigen binding molécule form.

[0111] The multispecific antigen binding molécules of the invention may be isolated antigen binding molécules. An isolated multispecific antigen binding molécule, as used herein, means an antigen binding molécule that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antigen binding molécule that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antigen binding molécule naturally exists or is naturally produced, is an isolated antigen binding molécule for purposes of the présent invention. An isolated antigen binding molécule also includes an antigen binding molécule in situ within a recombinant cell. Isolated antigen binding molécules are antigen binding molécules that hâve been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antigen binding molécule may be substantially free of other cellular material and/or Chemicals.

[0112] The anti-CD38 x anti-4-1BB 1+2 antigen binding molécules disclosed herein may comprise one or more amino acid substitutions, insertions and/or délétions in the framework and/or CDRs of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antigen binding molécules were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antigen binding molécule sequence databases. The présent disclosure includes multispecific antigen binding molécules, or antigen binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDRs are mutated relative to the sequences provided herein. In some aspects, the mutation is made to reflect the corresponding residue(s) of the germline sequence from which the antigen binding molécule was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations). A person of ordinary skill in the art, starting with the heavy and light chain variable région sequences disclosed herein, can easily produce numerous antigen binding molécules and which comprise one or more mutations such as individual germline mutations or combinations thereof. In certain embodiments, ail of the framework and/or CDR residues within the V_H and/or Vl domains are mutated back to the residues found in the original germline sequence from which the antigen binding molécule was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within

CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence {i.e., a germline sequence that is different from the germline sequence from which the antigen binding molécule was originally derived).

[0113] Furthermore, the multispecific antigen binding molécules of the présent invention may contain any combination of two or more mutations within the framework and/or CDR régions, e.g., wherein certain individual residues are mutated relative to the sequences provided herein, or are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen binding molécules and that contain one or more mutations can be easily tested for one or more desired property 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 molécules and obtained in this general manner are encompassed within the présent invention.

[0114] Provided herein are anti-CD38 x anti-4-1 BB antigen binding molécules comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more substitutions. In some aspects, the substitutions are conservative amino acid substitutions. For example, the présent disclosure includes anti-CD38 x anti-4-1 BB antigen binding molécules having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Tables 1, 3, 5, or 7 herein.

[0115] The term epitope refers to an antigenic déterminant that interacts with a spécifie antigen binding site in the variable région of an antigen binding molécule known as a paratope. A single antigen may hâve more than one epitope. Thus, different antigen binding molécules may bind to different areas on an antigen and may hâve different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0116] The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucléotide insertions or délétions with another nucleic acid (or its complementary strand), there is nucléotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucléotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molécule having substantiai identity to a reference nucleic acid molécule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molécule.

[0117] As applied to polypeptides, the temn substantiai similarity or substantially similar means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Contemplated herein are amino acid substitutions that will not substantially change the functional properties of the multispecific antigen binding molécule. In some aspects, residue positions which are not identical differ by conservative amino acid substitutions. A conservative amino acid substitution is one 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). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that hâve side chains with similar Chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine;

(3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and méthionine. Exemplary conservative amino acids substitution groups are: valine-leucine25 isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Altematively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A moderately conservative replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0118] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, délétions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to détermine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisme or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version

6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the régions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisme is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. 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 herein incorporated by reference.

Binding Properties of the Antigen binding molécules

[0119] As used herein, the term binding in the context of the binding of an antigen binding molécule, immunoglobulin, antigen binding molecule-binding fragment, or Fc-containing protein to either, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antigen binding molecule-antigen interaction.

[0120] For instance, binding affinity typically corresponds to a K_D value of about 10’⁷ M or less, such as about 10'⁸ M or less, such as about 10⁹ M or less when determined by, for instance, surface plasmon résonance (SPR) technology in a BIAcore instrument using the antigen as the ligand and the antigen binding molécule, Ig, antigen binding molecule-binding fragment, or Fccontaining protein as the analyte (or antiligand). Cell-based binding strategies, such as fluorescent-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand compétition 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).

[0121] Accordingly, the multispecific antigen binding molécules or antigen binding protein of the invention binds to the predetermined antigen or cell surface molécule (receptor) having an affinity corresponding to a Ko value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the présent disclosure, the affinity of an antigen binding molécule corresponding to a Kd value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non-detectable binding, however such an antigen binding molécule may be paired with a second antigen binding arm for the production of a multispecific antigen binding molécule of the invention.

[0122] The term Kd (M) refers to the dissociation equilibrium constant of a particular antigen binding molecule-antigen interaction, or the dissociation equilibrium constant of an antigen binding molécule or antigen binding molecule-binding fragment binding to an antigen. There is an inverse relationship between Kd and binding affinity, therefore the smaller the Kd value, the higher, i.e., stronger, the affinity. Thus, the terms “higher affinity or “stronger affinity relate to a higher ability to form an interaction and therefore a smaller K_D value, and conversely the terms “lower affinity” or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger K_D value. In some circumstances, a higher binding affinity (or Kd) of a particular molécule (e.g. antigen binding molécule) to its interactive partner molécule (e.g. antigen X) compared to the binding affinity of the molécule (e.g. antigen binding molécule) to another interactive partner molécule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger Kd value (lower, or weaker, affinity) by the smaller Ko (higher, or stronger, affinity), for example 5 expressed as 5-fold or 10-fold greater binding affinity, as the case may be.

[0123] The term kd (sec -1 or 1/s) refers to the dissociation rate constant of a particular antigen binding molecule-antigen interaction, or the dissociation rate constant of an antigen binding molécule or antigen binding molecule-binding fragment. Said value is also referred to as the k_Off value.

[0124] The term k_a (M-1 x sec-1 or 1/M/s) refers to the association rate constant of a particular antigen binding molecule-antigen interaction, or the association rate constant of an antigen binding molécule or antigen binding molecule-binding fragment.

[0125] The term Ka (M-1 or 1/M) refers to the association equilibrium constant of a particular antigen binding molecule-antigen interaction, or the association equilibrium constant of an antigen binding molécule or antigen binding molecule-binding fragment. The association equilibrium constant is obtained by dividing the k_a by the kd.

[0126] The term “EC50” or “EC50” refers to the half maximal effective concentration, which includes the concentration of an antigen binding molécule which induces a response halfway between the baseline and maximum after a specified exposure time. The EC50 essentially represents the concentration of an antigen binding molécule where 50% of its maximal effect is observed. In certain embodiments, the EC50 value equals the concentration of an antigen binding molécule of the invention that gives half-maximal binding to cells expressing 4-1BB or tumor-associated antigen (e.g., CD38), as determined by e.g., a FACS binding assay. Thus, reduced or weaker binding is observed with an increased EC₅o, or half maximal effective concentration value.

[0127] In one embodiment, decreased binding can be defined as an increased EC50 antigen binding molécule concentration which enables binding to the half-maximal amount of target cells.

[0128] In another embodiment, the EC50 value represents the concentration of an antigen binding molécule of the invention that elicits half-maximal déplétion of target cells by T cell cytotoxic activity. Thus, increased cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed with a decreased EC50, or half maximal effective concentration value.

Multispecific Antigen binding Molécules

[0129] The antigen binding molécules of the présent invention bind both CD38 and 4-1 BB.

Multispecific antigen binding molécules may be spécifie for different epitopes of one target polypeptide or may contain antigen binding domains spécifie for more than one target polypeptide. See, e.g., Tutt et a!., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends

Biotechnol. 22:238-244.

[0130] According to certain exemplary embodiments, the present invention includes multispecific antigen binding molécules that specifically bind 4-1BB and CD38. Such molécules may be referred to herein as, e.g., “anti-CD38 x anti-4-1BB 1+2” or anti-CD38/anti-4-1 BB 1+2, or anti-CD38x4-1 BB 1 +2 or CD38x4-1 BB 1 +2 multispecific molécules, or other similar terminology.

[0131] The present disclosure includes multispecific antigen binding molécules wherein one arm A2 of the immunoglobulin has two antigen-binding domains which bind human 4-1 BB, a 41BB ΊΝ domain and a 4-1 BB OUT” domain, and the other arm A1 of the immunoglobulin is spécifie for binding human CD38. The 4-1 BB-binding arm can comprise any of the HCVR/LCVR or CDR amino acid sequences as setforth in Table 3 (anti-4-1BB “IN”) or Table 5 (anti-4-1BB “OUT”) herein, in a stacked format.

[0132] In certain embodiments, the 4-1 BB-binding arm binds to human 4-1 BB and facilitâtes human T cell activation. In certain embodiments, the 4-1 BB-binding arm binds to human 4-1 BB and induces human T cell activation. In other embodiments, the 4-1 BB-binding arm binds to human 4-1 BB and induces tumor-associated antigen-expressing cell killing in the context of a multispecific or multispecific antigen binding molécule. The CD38-binding arm can comprise any of the HCVR/LCVR or CDR amino acid sequences as setforth in Table 1 herein.

[0133] As used herein, the expression antigen binding molécule means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity determining région (CDR) that alone, or in combination with one or more additional CDRs and/or framework régions (FRs), specifically binds to a particular antigen. In certain embodiments, an antigen binding molécule is an antigen binding molécule or a fragment of an antigen binding molécule, as those terms are defined elsewhere herein.

[0134] As used herein, the expression multispecific antigen binding molécule 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 the multispecific antigen binding molécule comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen. In the context of the present invention, the first antigen binding arm A1 specifically binds a first antigen (e.g., CD38), and the second antigen binding arm A2 specifically binds a second and third antigen (e.g., 41 BB), distinct from the first antigen.

[0135] The multispecific antigen binding molécules discussed herein can comprise a human IgG heavy chain constant région. In some cases, the human IgG heavy chain constant région is isotype lgG1. In some cases, the human IgG heavy chain constant région is isotype lgG4. In various embodiments, the multispecific antigen binding molécule comprises a chimeric hinge that reduces Fcy receptor binding relative to a wild-type hinge of the same isotype.

[0136] The first antigen binding arm A1 and the second antigen binding arm A2 may be directly or indirectiy connected to one another to form a multispecific antigen binding molécule of the présent invention. Alternatively, the first antigen binding arm A1 and the second antigen binding arm A2 may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitâtes the association between the two antigen binding domains, thereby forming a multispecific antigen binding molécule. As used herein, a multimerizing domain is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin Ch3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a Ch2-Ch3 domain), e.g., an Fc domain of an IgG selected from the isotypes lgG1, lgG2, lgG3, and lgG4, as well as any allotype within each isotype group.

[0137] Multispecific antigen binding molécules of the présent invention will typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antigen binding molécule heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., lgG1/lgG1, lgG2/lgG2, lgG4/lgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgG1/lgG2, lgG1/lgG4, lgG2/lgG4, etc.

[0138] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteinecontaining peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0139] Any multispecific antigen binding molécule format ortechnology may be used to make the multispecific antigen binding molécules of the présent invention. For example, an antigen binding molécule or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by Chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as another antigen binding molécule or antigen binding molécule fragment having a second antigen binding specificity to produce a multispecific antigen binding molécule. Spécifie exemplary multispecific formats that can be used in the context of the présent invention include, without limitation, e.g., scFv-based ordiabody multispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-intoholes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab² multispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats).

[0140] In the context of multispecific antigen binding molécules of the présent invention, the multimerizing domains, e.g., Fc domains, may comprise one or more amino acid changes (e.g., insertions, délétions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, the invention includes multispecific antigen binding molécules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the multispecific antigen binding molécule comprises a modification in a Ch2 or a Ch3 région, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Nonlimiting examples of such Fc modifications include, e.g., a modification 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 a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and

256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and

M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).

[0141] The present disclosure also includes multispecific antigen binding molécules comprising a first Ch3 domain and a second Ig Ch3 domain, wherein the first and second Ig Ch3 domains differfrom one another by at least one amino acid, and wherein at least one amino acid différence reduces binding of the multispecific antigen binding molécule to Protein A as compared to a bi-specific antigen binding molécule lacking the amino acid différence. In one embodiment, the first Ig C_h3 domain binds Protein A and the second Ig Ch3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C_h3 may further comprise a Y96F modification (by IMGT; Y436F by EU). The second Ch3 may further comprise a L105P modification (by IMGT, L455P by EU) See, for example, US Patent No. 8,586,713. Further modifications that may be found within the second C_H3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of lgG1 antigen binding molécules; N44S, K52N, and V82I (IMGT; N384S, K392N, and

V422I by EU) in the case of lgG2 antigen binding molécules; 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 lgG4 antigen binding molécules.

[0142] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or ail of a C_H2 sequence derived from a human IgG 1, human lgG2 or human lgG4 Ch2 région, and part or ail of a C_H3 sequence derived from a human lgG1, human lgG2 or human lgG4. A chimeric Fc domain can also contain a chimeric hinge région. For example, a chimeric hinge may comprise an upper hinge sequence, derived from a human lgG1, a human lgG2 or a human lgG4 hinge région, combined with a lower hinge sequence, derived from a human lgG1, a human lgG2 or a human lgG4 hinge région. A particular example of a chimeric Fc domain that can be included in any of the antigen binding molécules set forth herein comprises, from N- to C-terminus: [JgG4 C_H1] - [lgG4 upper hinge] - [lgG2 lower hinge] - [lgG4

CH2] - [lgG4 CH3], Another example of a chimeric Fc domain that can be included tn any of the antigen binding molécules set forth herein comprises, from N- to C-terminus: [IgG 1 Ch1] - [IgG 1 upper hinge] - [lgG2 lower hinge] - [lgG4 CH2] - ]lgG1 CH3], These and other examples of chimeric Fc domains that can be included in any of the antigen binding molécules of the présent invention are described in US Patent No. 9,359,437, which is herein incorporated in its entirety.

Chimeric Fc domains having these general structural arrangements, and variants thereof, can hâve altered Fc receptor binding, which in turn affects Fc effector function.

Sequence Variants

[0143] The antigen binding molécules and multispecific antigen binding molécules of the present invention may comprise one or more amino acid substitutions, insertions and/or délétions in the framework and/or CDR régions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antigen binding molécule sequence databases. The antigen binding molécules of the present invention may comprise antigen binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR régions are mutated to the corresponding residue(s) of the germline sequence from which the antigen binding molécule was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations). A person of ordinary skill in the art, starting with the heavy and light chain variable région sequences disclosed herein, can easily produce numerous antigen binding molécules and which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, ail of the framework and/or CDR residues within the V_H and/or V_L domains are mutated back to the residues found in the original germline sequence from which the antigen binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (Le., a germline sequence that is different from the germline sequence from which the antigen binding domain was originally derived).

[0144] Furthermore, the antigen binding domains may contain any combination of two or more germline mutations within the framework and/or CDR régions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen 5 binding domains that contain one or more germline mutations can be easily tested for one or more desired property 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 molécules comprising one or more antigen binding domains obtained in this general manner are encompassed within the présent 10 invention.

pH-Dependent Binding

[0145] The présent invention includes anti-CD38 x anti-4-1BB multispecific antigen binding molécules, with pH-dependent binding characteristics. For example, an anti-CD38 antigen binding arm of the présent invention may exhibit reduced binding to CD38 at acidic pH as compared to neutral pH. Alternatively, anti-CD38 antigen binding arms ofthe invention may exhibit enhanced binding to CD38 at acidic pH as compared to neutral pH. The expression acidic pH includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5,9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the expression neutral pH means a pH of 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.

[0146] In certain instances, reduced binding ... at acidic pH as compared to neutral pH is expressed in terms of a ratio of the Kd value of the antigen binding molécule binding to its antigen at acidic pH to the K_D value of the antigen binding molécule binding to its antigen at neutral pH (or vice versa). For example, an antigen binding molécule or may be regarded as exhibiting reduced binding to CD38 at acidic pH as compared to neutral pH for purposes of the présent invention if the antigen binding molécule or exhibits an acidic/neutral K_D ratio of about

3.0 or greater. In certain exemplary embodiments, the acidic/neutral K_D ratio for an antigen binding molécule or of the présent invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0.

25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

[0147] Antigen binding molécules with pH-dependent binding characteristics may be obtained, e.g., by screening a population of antigen binding molécules for reduced (or enhanced) binding to a particular antigen at acidic pH as compared to neutral pH. Additionally, modifications of the antigen binding domain at the amino acid level may yield antigen binding molécules with pH35 dépendent characteristics. For example, by substituting one or more amino acids of an antigen binding domain (e.g., within a CDR) with a histidine residue, an antigen binding molécule with reduced antigen binding at acidic pH relative to neutral pH may be obtained.

Antigen Binding Molécules Comprising Fc Variants

[0148] According to certain embodiments of the présent invention, anti-CD38 x anti-4-1BB multispecific antigen binding molécules, are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antigen binding molécule binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the présent invention includes antigen binding molécules comprising a mutation in the C_H2 or a C_H3 région of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may resuit in an increase in sérum half-life of the antigen binding molécule when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification 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 a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). [0149] For example, the présent disclosure includes anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molécules, comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). Ail possible combinations of the foregoing Fc domain mutations, and other mutations within the antigen binding molécule variable domains disclosed herein, are contemplated within the scope of the présent invention.

Biological Characteristics of the Antigen binding molécules and Multispecific Antigen binding Molécules

[0150] Provided herein are anti-CD38 x anti-4-1BB multispecific antigen binding molécules that bind CD38 expressed on MOLP8 cells. As shown in Example 6, dose dépendent binding of the CD38x4-1BB 1+2 (REGN7633, REGN7647 and REGN7650) and 1+1 (REGN7150) bispecific antibodies was observed in the presence of MOLP8 cells, with Max gMFI ranging from 8.8x10⁴ to 1.4x10⁵ and ECsoS ranging from 4.23x10^-9 M to 9.27x10 ® M.

[0151] Provided herein are anti-CD38 x anti-4-1BB multispecific antigen binding molécules that bind 4-1BB expressed on HEK293 cells engineered to express 4-1 BB. Dose dépendent 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

Max gMFI ranging from 7.4x10⁵ to 2.4x10⁶ and ECsoS ranging from 1.47x10'³ M to 9.97x10¹⁰ M.

[0152] CD38x4-1BB (1+1 and 1+2) multispecific antigen binding molécules mimicthe natural ligand of 4-1BB by bridging CD38+ target cells with 4-1 BB receptor positive T cells. In doing so, the constructs provide “signal 2” and enhance the activation of T cells in the presence of a “signal Γ provided by a Tumor-associated antigen (TAA) x CD3 bispecific antibody or an allogeneic response provided by the APC. As shown in Example 7, the multispecific antigen binding molécules provided herein, in the presence of target and “signal 1” (provided by REGN1979), led to higher maximum IL-2 response and greater potency than matched isotype Controls in a T cell activation assay. As shown in Example 8, the multispecific antigen binding molécules had dose dépendent increase in IL-2 and IFNy and greater potency even when the linker length between the Fab2 and Fab3 was varied.

[0153] According to certain embodiments, multispecific antigen binding molécules provided herein activate 4-1 BB receptor and stimulate 4-1 BB activity in presence of target cells expressing CD38 as demonstrated in an engineered reporter assay. As shown in Example 9, 41BB activation was achieved using constructs with different linker lengths.

[0154] In certain embodiments, the multispecific antigen binding molécules provided herein cause dose dépendent increases in IL-2 and IFNy release. As shown in Example 10, in the presence of allogeneic NALM-6 cells or NALM-6 cells engineered to express PD-L1, CD38x41BB 1+2 antibody treatment (REGN7633, REGN7647 and REGN7650), led to dose dépendent increases in IL-2 and IFNy release and greater potency.

[0155] According to certain embodiments, treatment with the multispecific antigen-binding molécules when administered in combination with BCMAxCD3 bispecific antibodies, in vivo, results in a more potent, anti-tumor efficacy that is superior to either treatment alone. In Example 11, immunodeficient NOD.Cg-Prkdc^scidH2rg’^m1v'^fll/SzJ (NSG) mice intraperitoneally injected with 4x10⁶ human peripheral blood mononuclear cells (PBMC) were administered human multiple myeloma cells. After receiving tumor cells, the mice were treated with CD3binding control bispecific Ab or a BCMAxCD3 (REGN5458) bsAb at 0.4 mg/kg, in combination with a 4-1 BB-binding control bispecific Ab (1+2 format) or a CD38x4-1 BB (1+2 format; REGN9686) at 4 mg/kg. The treatment combinations were administered twice more on days 7 and 14, for a total of three doses. Combination treatment with BCMAxCD3 bsAb plus CD38x41BB 1+2 bsAb demonstrated more potent, combinatorial anti-tumor efficacy superior to either therapy alone.

Epitope Mapping and Related Technologies

[0156] The epitope on CD38 and/or 4-1 BB to which the antigen binding molécules of the présent invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a respective CD38 or 4-1 BB protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of CD38 or 4-1 BB.

[0157] The term epitope, as used herein, refers to an antigenic déterminant that interacts with a spécifie antigen binding site in the variable région of an antigen binding molécule p.m. known as a paratope. A single antigen may hâve more than one epitope. Thus, different antigen binding molécules may bind to different areas on an antigen and may hâve different biological

S effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0158] Various techniques known to persons of ordinary skill in the art can be used to détermine whether an antigen binding domain of an antigen binding molécule interacts with one or more amino acids within a polypeptide or protein. Exemplary techniques include, e.g., routine cross-blocking assay such as that described in Antigen binding molécules, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutational analysis, 15 peptide blots 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 employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen binding domain of an antigen binding molécule interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antigen binding molécule to the deuterium-labeled protein. Next, the protein/antigen binding molécule complex is transferred to water to allow hydrogen-deuterium exchange to occur at ail residues except for the residues protected by the antigen binding molécule (which remain deuterium-labeled). After 25 dissociation of the antigen binding molécule, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the spécifie amino acids with which the antigen binding molécule interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen/antigen binding molécule complex may also be used for epitope mapping purposes.

[0159] Provided herein are anti-CD38 antigen binding arms A1 that bind to the same epitope as any of the spécifie exemplary antigen binding arms described herein {e.g., antigen binding molécules comprising any of the amino acid sequences as setforth in Table 1 herein). Likewise, the présent invention also includes anti-CD38 antigen binding arms A1 that compete for binding 35 to CD38 with any of the spécifie exemplary antigen binding arms described herein {e.g., antigen binding molécules comprising any of the amino acid sequences as set forth in Table 1 herein). [0160] Provided herein are anti-4-1 BB antigen binding arms A2 comprising a first antigenbindîng domain (R1) and a second antigen-binding domain (R2), where either R1 or R2 bind to the same epitope as any of the spécifie exemplary antigen binding domains described herein (e.g., antigen binding arms comprising any of the amino acid sequences as set forth in Table 3 or Table 5 herein). Likewise, the présent invention also includes anti-4-1 BB antigen binding molécules that compete for binding to 4-1 BB with any of the spécifie exemplary antigen binding domains described herein (e.g., antigen binding arms comprising any of the amino acid sequences as set forth in Table 3 or Table 5 herein).

[0161] Likewise, provided herein are multispecific antigen binding molécules comprising a first antigen binding arm that specifically binds human CD38 (Fab1 ), and a second antigen binding arm that specifically binds human 4-1 BB (Fab 2 and Fab 3), wherein the first antigen binding domain competes for binding to CD38 with any of the spécifie exemplary CD38-specific antigen binding arms described herein, and/or wherein the second antigen binding arm competes for binding to 4-1 BB with any of the spécifie exemplary 4-1BB-specific antigen binding Fabs described herein.

[0162] One can easily détermine whether a particular antigen binding molécule (e.g., multispecific 1+2 antigen binding molécule) or antigen binding fragment thereof binds to the same epitope as, or competes for binding with, a reference antigen binding molécule of the présent invention by using routine methods known in the art. For example, to détermine if a test antigen binding molécule binds to the same epitope on CD38 (or 4-1 BB) as a reference multispecific antigen binding molécule of the présent invention, the reference multispecific molécule is first allowed to bind to a CD38 protein (or4-1BB protein). Next, the ability of a test antigen binding molécule to bind to the CD38 (or4-1BB) molécule is assessed. If the test antigen binding molécule is able to bind to CD38 (or 4-1 BB) following saturation binding with the reference multispecific antigen binding molécule, it can be concluded that the test antigen binding molécule binds to a different epitope of CD38 (or 4-1 BB) than the reference multispecific antigen binding molécule. On the other hand, if the test antigen binding molécule is not able to bind to the CD38 (or 4-1 BB) molécule following saturation binding with the reference multispecific antigen binding molécule, then the test antigen binding molécule may bind to the same epitope of CD38 (or 4-1 BB) as the epitope bound by the reference multispecific antigen binding molécule of the invention. Additional routine expérimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antigen binding molécule is in fact due to binding to the same epitope as the reference multispecific antigen binding molécule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antigen binding molecule-binding assay available in the art. In accordance with certain embodiments of the présent invention, two antigen binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antigen binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a compétitive binding assay (see, e.g., Junghans et aL, Cancer Res. 1990:50:1495-1502). Alternatively, two antigen binding proteins are deemed to bind to the same epitope if essentially ail amino acid mutations in the antigen that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other. Two antigen binding proteins are deemed to hâve overlapping epitopes if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other.

[0163] To détermine if an antigen binding molécule or antigen binding domain thereof competes for binding with a reference antigen binding molécule, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen binding molécule is allowed to bind to a CD38 protein (or 4-1 BB protein) under saturating conditions followed by assessment of binding of the test antigen binding molécule to the CD38 (or 4-1 BB) molécule. In a second orientation, the test antigen binding moiecule is allowed to bind to a CD38 (or 4-1 BB) moiecule under saturating conditions followed by assessment of binding of the reference antigen binding moiecule to the CD38 (or4-1BB) moiecule. If, in both orientations, only the first (saturating) antigen binding moiecule is capable of binding to the CD38 (or4-1BB) moiecule, then it is concluded that the test antigen binding moiecule and the reference antigen binding moiecule compete for binding to CD38 (or 4-1 BB). As will be appreciated by a person of ordinary skill in the art, an antigen binding moiecule that competes for binding with a reference antigen binding moiecule may not necessarily bind to the same epitope as the reference antigen binding moiecule, but may sterically biock binding of the reference antigen binding moiecule by binding an overlapping or adjacent epitope.

Préparation of Antigen binding Domains and Construction of Multispecific Molécules [0164] Antigen binding domains spécifie for particular antigens can be prepared by any antigen binding moiecule generating technology known in the art. Once obtained, different antigen binding domains provided herein, spécifie for two different antigens (e.g., CD38 and 41BB), can be appropriately arranged relative to one another to produce a multispecific antigen binding moiecule of the présent invention using routine methods. (A discussion of exemplary multispecific antigen binding moiecule formats that can be used to construct the multispecific antigen binding molécules of the present invention is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen binding molécules of the invention are derived from chimeric, humanized orfully human antigen binding molécules. Methods for making such antigen binding molécules are welî known in the art. For example, one or more of the heavy and/or light chains of the multispecific antigen binding molécules of the present invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antigen binding moiecule generating technology), high affinity chimeric antigen binding molécules to a particular antigen (e.g., CD38 or4-1BB) are initially isolated having a human variable région and a mouse constant région. The antigen binding molécules are characterized and selected for désirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant régions are replaced with a desired human constant région to generate fully human heavy and/or light chains that can be incorporated into the multispecific antigen binding molécules of the présent invention.

[0165] Genetically engineered animais may be used to make human multispecific antigen binding molécules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains 10 encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human antigen binding molécules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable région gene segments. (See, e.g., US 2011/0195454). Fully 15 human refers to an antigen binding molécule, or antigen binding fragment thereof, or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antigen binding molécule or antigen binding fragment thereof, or immunoglobulin domain thereof. In some instances, the fully human sequence is derived from a protein endogenous to a human. In 20 other instances, the fully human protein or protein sequence comprises a chîmeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chîmeric proteins or chîmeric sequences are generally designed to minimize the création of immunogenic epitopes in the junctions of component sequences, e.g., compared to any wild-type human immunoglobulin régions or domains.

Bioequivalents

[0166] Provided herein are antigen binding molécules having amino acid sequences that vary from those of the exemplary molécules disclosed herein but that retain the ability to bind CD38 and/or 4-1 BB. Such variant molécules may comprise one or more additions, délétions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity 30 that is essentially équivalent to that of the described multispecific antigen binding molécules.

[0167] Antigen binding molécules that are bioequivalent to any of the exemplary antigen binding molécules set forth herein are phamnaceutical équivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant différence when administered at the same molar dose under similar experimental conditions, either single dose 35 or multiple doses. Some antigen binding proterns will be considered équivalents or pharmaceutical alternatives if they are équivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such différences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0168] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful différences in their safety, purity, and potency.

[0169] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0170] In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0171] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antigen binding molécule or its métabolites is measured in blood, plasma, sérum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably prédictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antigen binding molécule (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen binding protein.

[0172] Bioequivalent variants of the exemplary multispecific antigen binding molécules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internai residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexte, bioequivalent antigen binding proteins may include variants of the exemplary multispecific antigen binding molécules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the molécules, e.g., mutations which eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

[0173] According to certain embodiments of the invention, antigen binding molécules are provided which bind to human 4-1 BB but not to 4-1 BB from other species. Also provided are antigen binding molécules which bind to human CD38, but not to CD38 from other species. The present invention also includes antigen binding molécules that bind to human 4-1 BB and to CD38 from one or more non-human species; and/or antigen binding molécules that bind to human 4-1 BB and to 4-1 BB from one or more non-human species.

[0174] According to certain exemplary embodiments of the invention, antigen binding molécules are provided which bind to human CD38 and/or human 4-1 BB and may 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, rhésus or chimpanzee CD38 and/or 4-1 BB. For example, in particular exemplary embodiments of the disclosed herein, multispecific antigen binding molécules are provided comprising a first antigen binding arm that binds human CD38 or cynomolgus CD38, and a second antigen binding arm comprising a first antigen-binding domain and a second antigen-binding domain wherein the second antigenbinding arm specifically binds human 4-1 BB, or multispecific antigen binding molécules comprising a second antigen binding arm comprising a first and second antigen-binding domains that bind human 4-1 BB and/or cynomolgus 4-1 BB, and a first antigen binding arm that specifically binds human CD38.

Therapeutic Formulation and Administration

[0175] The présent invention provides pharmaceutical compositions comprising the multispecific antigen binding molécules disclosed herein. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolérance, and the like. A multitude of appropriate formulations can be found in the formulary known to ail pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugales, anhydrous absorption pastes, oil-in-water and water-in-oil émulsions, émulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. Compendium of excipients for parentéral formulations PDA (1998) J Pharm Sci Technol 52:238-311.

[0176] The dose of multispecific antigen binding molécule administered to a patient may vary depending upon the âge and the size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When a multispecific antigen binding molécule of the présent invention is used for therapeutic purposes in an adult patient, it may be advantageous to intravenously administer the multispecific antigen binding molécule of the présent invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.

Effective dosages and schedules for administering a multispecific antigen binding molécule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut.

Res. 8:1351).

[0177] Various delivery Systems are known and can be used to administer the pharmaceutical 5 composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (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, épidural, and oral routes. The composition may be administered by any convenient 10 route, for example by infusion or bolus injection, by absorption through épithélial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0178] A pharmaceutical composition of the present invention can be delivered 15 subcutaneously, intramuscularly, or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once ail of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device cornes prefilled with the pharmaceutical composition held in a réservoir within the device. Once 25 the réservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0179] Numerous reusable pen and autoinjector delivery devices hâve applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 30 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co„ Indianapolis, IN),

NOVOPEN™ I, Il and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having 35 applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the

HIIMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.

[0180] In certain situations, the pharmaceutical composition can be delivered in a controlled release System. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used;

see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Près., Boca Raton, Florida. In yet another embodiment, a controlled release System can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (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 by Langer, 1990, Science

249:1527-1533.

[0181] The injectable préparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable préparations may be prepared by methods publicly known. For example, the injectable préparations may be prepared, e.g., by dissolving, suspending or emulsifying the antigen binding molécule or its sait 15 described above in a stérile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonie solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., éthanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 20 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably fi lied in an appropriate ampoule.

[0182] Advantageously, the pharmaceutical compositions for oral or parentéral use described 25 above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingrédients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antigen binding molécule contained is generally about 1 to about 1000 mg per dosage form in a unit dose, especially in the form of injection, it is preferred that the aforesaid antigen binding molécule is contained in 30 about 1 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. In some embodiments, the unit dosage can be as much as about 750 mg, 800 mg, 900 mg, or 1000 mg.

Therapeutic Uses of the Antigen binding Molécules

[0183] The présent invention includes methods comprising administering to a subject in need thereof a therapeutic composition a multispecific antigen binding molécule that specifically binds 35 CD38 and 4-1 BB. The therapeutic composition can comprise any of the multispecific antigen binding molécules as disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression a subject in need thereof' means a human or non-human animal that exhibits one or more symptoms or indicia of cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned herein below), or who otherwise would benefit from an inhibition or réduction in CD38 activity or a déplétion of CD38+ cells (e.g., multiple myeloma cells).

[0184] The multispecific antigen binding molécules of the invention (and therapeutic compositions comprising the same) are useful, inter alia, fortreating any disease ordisorder in which stimulation, activation and/or targeting of an immune response would be bénéficiai. In particular, the anti-CD38 x anti-4-1BB 1+2 multispecific antigen binding molécules of the present invention may be used for the treatment, prévention and/or amelioration of any disease 10 or disorder associated with or mediated by CD38 and/or BCMA expression or activity or the prolifération of CD38+ and /or BCMA+ cells. The mechanism of action by which the therapeutic methods of the invention are achieved include killîng of the cells expressing CD38 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing CD38 which can be inhibited or killed using the multispecific antigen binding molécules of the invention include, for example, multiple myeloma cells.

[0185] The multispecific antigen binding molécules of the present disclosure may be used to treat a disease or disorder associated with CD38 expression including, e.g., multiple myeloma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic 20 ductal adenocarcinoma, glioma, or breast cancer, or another cancer characterized in part by having CD38+ cells.

[0186] According to certain embodiments, the anti-CD38 x anti-4-1 BB 1 +2 antigen binding molécules are useful for inhibiting growth of a plasma cell tumor in a subject. In some aspects, the plasma cell tumor is multiple myeloma.

[0187] The multispecific antigen binding molécules of the present disclosure may be used to inhibit growth of a tumor 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.

[0188] According to certain embodiments, the anti-CD38 x anti-4-1 BB 1+2 antigen binding molécules are useful for treating tumor cells expressing, for example, BCMA or CD20. The antigen binding molécules provided herein may also be used to treat a disease or disorder associated with BCMA expression including, e.g., a cancer including multiple myeloma or other B-cell or plasma cell cancers, such as Waldenstrôm’s macroglobulinemia, Burkitt 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, the anti-CD38 x anti-4-1 BB antigen binding molécules are useful for treating a patient afflicted with multiple myeloma. According to other related embodiments ofthe invention, methods are provided comprising administering an anti-CD38 x anti-4-1 BB multispecific antigen binding molécule provided herein in combination with an anti-BCMA antigen binding molécule, or an anti-BCMA x anti-CD3 multispecific antigen binding molécule, or an anti-CD20 x anti-CD3 multispecific antigen binding molécule, or an anti-CD28 x anti-4-1 BB multispecific antigen binding molécule as disclosed herein to a patient who is afflicted with cancer cells expressing BCMA or CD20. Analytic/diagnostic methods known in the art, such as tumor scanning, etc., may be used to ascertain whether a patient harbors multiple myeloma or another B-cell lineage cancer.

[0189] In some embodiments, an anti-CD38 x anti-4-1 BB multispecific antigen binding molécule provided herein can be administered in combination with second therapeutic agent or therapeutic regimen comprising a chemotherapeutic drug, DNA alkylators, immunomodulators, protéasome inhibitors, histone deacetylase inhibitors, radiotherapy, a stem cell transplant, a different bispecific antibody that interacts with a different tumor cell surface antigen and a T cell 15 or immune cell antigen, an antibody drug conjugate, a bispecific antibody conjugated to an antitumor agent, a PD-1 inhibitor (such as an anti-PD-1 antibody, e.g., cemiplimab), a PD-L1 inhibitor, a CTLA-4 checkpoint inhibitor, or combinations thereof.

[0190] The present invention also includes methods for treating residual cancer in a subject. As used herein, the term residual cancer means the existence or persistence of one or more 20 cancerous cells in a subject following treatment with an anti-cancer therapy.

[0191] According to certain aspects, the present invention provides methods for treating a disease or disorder associated with CD38 expression (e.g., multiple myeloma) comprising administering one or more of the anti-CD38 x anti-4-1 BB 1 +2 antigen binding molécules described herein to a subject after the subject has been determined to hâve multiple myeloma.

For example, the present invention includes methods for treating multiple myeloma comprising administering an anti-CD38 x anti-4-1 BB multispecific antigen binding molécule to a patient 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.

I

Combination Thérapies and Formulations

[0192] The present invention provides methods which comprise administering a pharmaceutical composition comprising any ofthe exemplary antigen binding molécules and multispecific antigen binding molécules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be therapeutically combined with or administered in combination with an antigen binding molécule ofthe present invention include, e.g., an anti-tumor agent (e.g., chemotherapeutic agents including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or any others known to be effective in treating a plasma cell tumor in a subject.). In some embodiments, the second therapeutic agent comprises steroids. In some embodiments, the second therapeutic agent comprises targeted thérapies including thalidomide, lenalidomide, 5 and bortezomib, which are thérapies approved to treat newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of a second therapeutic agent effective for treating récurrent myeloma.

[0193] In some embodiments, the second therapeutic is an anti-BCMAxCD3 bispecific antigen binding molécule. Illustrative anti-BCMAxCD3 bispecific antigen binding molécules are 10 disclosed in U S. 2020/0024356 incorporated by reference herein. An exemplary antiBCMAxCD3 bispecific antigen binding molécule, as disclosed in U.S. 2020/0024356, is REGN5458. In some embodiments, the second therapeutic is an anti-CD20xCD3 bispecific antigen binding molécule. Illustrative anti-CD20xCD3 bispecific antigen binding molécules are disclosed in U S. Patent No 9,657,102, incorporated by reference herein. An exemplary anti15 CD20xCD3 bispecific antigen binding molécule is REGN1979 (U.S. Patent 9,657,102).

[0194] In certain embodiments the second therapeutic agent is a regimen comprising radiotherapy or a stem cell transplant. In certain embodiments, the second therapeutic agent may be an immunomodulatory agent. In certain embodiments, the second therapeutic agent may be a protéasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib 20 (Ninlaro). In certain embodiments the second therapeutic agent may be a histone deacetylase inhibitor such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may be a monoclonal antibody, an antibody drug conjugate, a multispecific/bispecific/monospecific antigen binding molécule conjugated to an anti-tumor agent, a checkpoint inhibitor, an oncolytic virus, a cancer vaccine, a CAR-T cell, or combinations thereof. Other agents that may be beneficially administered in combination with the antigen binding molécules of the invention include cytokine inhibitors, including smallmolecule cytokine inhibitors and antigen binding molécules 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, 1L-13, IL-17, IL-18, orto their respective receptors. The pharmaceutical compositions of the présent invention (e.g., pharmaceutical compositions comprising an anti-CD38 x anti-4-1BB multispecific antigen binding molécule as disclosed herein) may also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from a monoclonal antigen binding molécule other than those described herein, which may interact with a different antigen on the plasma cell surface, a multispecific antigen binding molécule, which has one arm that binds to an antigen on the tumor cell surface and the other arm binds to an antigen on a T cell, an antibody drug conjugate, a bispecific antibody conjugated with an anti-tumor agent, a checkpoint inhibitor, for example, one thattargets, PD-1 orCTLA-4, or combinations thereof. In certain embodiments, the checkpoint inhibitors may be selected from PD-1 inhibitors, such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab (REGN2810; Libtayo). In certain embodiments, the checkpoint inhibitors may be selected from PD-L1 inhibitors, such as atezolizumab (Tecentriq), avelumab (Bavencio), or Durvalumab (Imfinzi)). In certain embodiments, the checkpoint inhibitors may be selected from CTLA-4 inhibitors, such as ipilimumab (Yervoy). Other combinations that may be 5 used in conjunction with an antigen binding molécule of the invention are described above.

[0195] The présent invention also includes therapeutic combinations comprising any of the antigen binding molécules mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-a, PDGFR-β, F0LH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any ofthe 10 aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molécule, a ribozyme, an siRNA, a peptibody, a nanobody or an antigen binding molécule fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molécules, such as diabodies, triabodies, tetrabodies, minibodies and minimal récognition units). The antigen binding molécules of the invention may also be administered 15 and/or co-formulated in combination with antivirals, antibiotics, analgésies, corticosteroids and/or NSAIDs. The antigen binding molécules ofthe invention may also be administered as part of a treatment regimen that also includes radiation treatment and/or conventional chemotherapy.

[0196] The additional therapeutically active component(s) may be administered just prior to, 20 concurrent with, or shortly after the administration of an antigen binding molécule of the présent invention; (for purposes of the présent disclosure, such administration regimens are considered the administration of an antigen binding molécule in combination with an additional therapeutically active component).

[0197] The présent invention includes pharmaceutîcal compositions in which an antigen 25 binding molécule of the présent invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

Administration Regimens

[0198] According to certain embodiments of the présent invention, multiple doses of an antigen binding molécule (e.g., a multispecific antigen binding molécule that specifically binds 30 CD38 and 4-1 BB) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of a multispecific antigen binding molécule of the invention. As used herein, sequentially administering means that each dose of an antigen binding molécule is administered to the subject at a different point in time, e.g., on different days separated by a 35 predetermined interval (e.g., hours, days, weeks or months). The présent invention includes methods which comprise sequentially administering to the patient a single initial dose of an antigen binding molécule, followed by one or more secondary doses of the antigen binding

I molécule, and optionally followed by one or more tertiary doses of the antigen binding molécule.

[0199] The ternis initial dose, secondary doses, and tertiary doses, refer to the temporal sequence of administration of the antigen binding molécule of the invention. Thus, the initial dose is the dose which is administered at the beginning of the treatment regimen (also referred 5 to as the baseline dose); the secondary doses are the doses which are administered after the initial dose; and the tertiary doses are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may ail contain the same amount of the antigen binding molécule, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antigen binding molécule 10 contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses followed by subséquent doses that are administered on a less frequent basis (e.g., maintenance doses).

[0200] In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, T/₂, 2, 2½. 3, 3½. 4, 4½. 5, 5½. 6, 6½. 7, 7½. 8, 8½. 9, 97₂, 10, 10½. 11, 11½. 12, 12½. 13, 13½. 14, 14½. 15, 15½. 16, 16½. 17, 17½. 18, 18½. 19, 19½. 20, 20½. 21, 21½. 22, 22½. 23, 23½. 24, 24½. 25, 25½. 26, 26½. or more) weeks after the immediately preceding dose. The phrase the immediately preceding dose, as used herein, 20 means, in a sequence of multiple administrations, the dose of antigen binding molécule which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0201] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an antigen binding molécule (e.g., a 25 multispecific 1+2 antigen binding molécule that specifically binds CD38 and 4-1 BB). For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) 30 tertiary doses are administered to the patient.

[0202] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose 35 may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration i

may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

Diagnostic Uses of the Antigen binding molécules

[0203] The anti-CD38 antigen binding molécules disclosed herein may be used to detect and/or measure CD38, or CD38-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-CD38 antigen binding molécule, or fragment thereof, may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, underexpression, lack of expression, etc.) of CD38. Exemplary diagnostic assays for CD38 may comprise, e.g., contacting a sample, obtained from a patient, with an anti-CD38 antigen binding molécule disclosed herein, wherein the anti-CD38 antigen binding molécule is labeled with a détectable label or reporter molécule. Alternatively, an unlabeled anti-CD38 antigen binding molécule can be used in diagnostic applications in combination with a secondary antigen binding molécule which is itself detectably labeled. The détectable label or reporter molécule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵l; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-CD38 antigen binding molécules of the invention includes ⁸⁹Zrlabeled, such as ⁸⁹Zr-desferrioxamine—labeled, antigen binding molécules for the purpose of noninvasive identification and tracking of tumor cells in a subject (e.g., positron émission 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.) Spécifie 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).

[0204] Samples that can be used in CD38 diagnostic assays according to the présent invention include any tissue or fluid sample obtainable from a patient which contains détectable quantities of CD38 protein, or fragments thereof, under normal or pathological conditions.

Generally, levels of CD38 in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease or condition associated with abnormal CD38 levels or activity) will be measured to initially establish a baseline, or standard, level of CD38. This baseline level of

CD38 can then be compared against the levels of CD38 measured in samples obtained from individuals suspected of having a CD38 related disease (e.g., a tumor containing CD38expressing cells) or condition.

Devices

[0205] The présent invention also provides a vessel (e.g., a vial or chromatography column) or injection device (e.g., syringe, pre-filled syringe or autoinjector) comprising a multispecific antigen binding molécule (e.g., pharmaceutical formulation thereof) set forth herein. The vessel or injection device may be packaged into a kit.

[0206] An injection device is a device that introduces a substance into the body of a subject (e.g., a human) via a parentéral route, e.g., intraocular, intravitreal, intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical formulation, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the antigen binding molécule or fragment or a pharmaceutical formulation thereof), a needle for piecing skin, blood vessels or other tissue for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore and into the body of the subject.

[0207] A pharmaceutical composition provided herein can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the présent invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once ail of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device cornes prefilled with the pharmaceutical composition held in a réservoir within the device. Once the réservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0208] Numerous reusable pen and autoinjector delivery devices hâve applications in the subcutaneous delivery of a pharmaceutical composition of the présent invention. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MiX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind), NOVOPEN™ I, Il and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the présent invention include, but are not limited tothe SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif ), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park III.), to name only a few.

[0209] Provided herein are methods for administering a multispecific antigen binding molécule of the présent disclosure comprising introducing e.g., injecting, the molécule into the body of the subject, e.g., with an injection device.

Expression Methods

[0210] Provided herein are recombinant methods for making a multispecific antigen binding moiecule of the present invention, or an immunoglobulin chain thereof, comprising (i) introducing, into a host cell, one or more polynucleotides encoding light and/or heavy immunoglobulin chains of such a multispecific antigen binding moiecule, for example, wherein the one or more polynucleotides is comprised in one or more vectors; and/or intégrâtes into the host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., mammalian, fungal, Chinese hamster ovary (CHO), Pichia or Pichia pastoris) under conditions favorable to expression of the polynucleotide and, (iii) optionally, isolating the multispecific antigen binding moiecule or immunoglobulin chain from the host cell and/or medium in which the host cell is grown. The product of such a method also forms part of the present disclosure along with a pharmaceutical composition thereof.

[0211] In some aspects, step (i) comprises cloning the individual A1 heavy chain, the A2 heavy chain, and the universal light chain into separate expression vectors. For example, CD38binding heavy chain variable régions (HCVR) (VH-1) can be cloned into a heavy chain expression plasmid (CH1-1_CH2_CH3). 4-1BB-binding heavy chain variable régions (HCVR) (VH-3) fused to a CH1 domain (CH1-3) with linkers of various length (linker) followed by another 4-1 BB-binding heavy chain variable régions (HCVR) (VH-2) can be cloned into a heavy chain expression plasmid (CH1-2_CH2_CH3(*)) containing the mutations H435R, and Y436F, (EU numbering) (US 8,586,713). Along with a universal light chain containing plasmid, the expression plasmids can be transfected into a host cell, such as a CHO cell. The host cells can then produce multispecific antigen binding molécules described herein.

[0212] In an embodiment, a method for making a multispecific antigen binding moiecule includes a method of purifying the moiecule, e.g., by column chromatography, précipitation and/or filtration. The product of such a method also forms part of the present disclosure along with a pharmaceutical composition thereof.

[0213] Host cells comprising a multispecific antigen binding moiecule of the present disclosure and/or a polynucleotide encoding immunoglobulin chains of such a moiecule (e.g., in a vector) are also part of the present invention. Host cells include, for exampie, mammalian cells such as Chinese hamster ovary (CHO) cells and fungal cells such as Pichia cells (e.g., P. pastoris).

EXAMPLES

[0214] The following examples are put forth so as to provide those of ordinary skill in the art with a complété disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts hâve been made to ensure accuracy with respect to numbers used (e.g., amounts, température, etc.) but some experimental errors and déviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, température is in degrees Centigrade, and pressure is at or near atmospheric.

[0215] Antigen binding molécules used as Controls in Examples 6 through 10 include:

• A CD20xCD3 bispecific antîbody (REGN1979) (US 10,550,193) used as signal 1 along with a matched isotype controi (REGN7540). Both REGN1979 and REGN7540 comprise hlgG4^{P PVA} isotype (US 9,359,437).

• A second CD20xCD3 bispecific antîbody (REGN2281) used as signal 1. REGN2281 comprises variable régions identical to REGN1979 with a hlgG4 isotype having S108P substitution (hlgG4^p) · BCMAxCD3 (REGN5458), also used as signal 1 (US 11,384,153).

• Comparator 1: 4-1 BB bivalent agonist (REGN4249) comprising the variable régions of antîbody “10C7” (US 7,288,638).

• Anti-PD-1 antîbody cemiplimab (REGN2810; LIBTAYO®) (US 9,987,500).

• isotype controi to 4-1 BB bivalent agonist (hlgG4P) (REGN1945); REGN1945 was also 15 used as the isotype controi for cemiplimab.

• Bispecific 4-1 BB x non-TAA isotype controi (REGN13168).

[0216] Two signais, “signal 1 & “signal 2”, are required for properT cell activation. “Signal 1” is induced by binding of the T cell receptor (TCR) on T cells to peptide-bound major histocompatibility complex (MHC) molécules on antigen presenting cells (APCs). “Signal 2 is provided by engaging co-stimulatory receptors on T cells. One such costimulatory receptor is 41BB receptor, which is an inducible type I membrane protein and memberof the tumor necrosis factor receptor (TNFR) superfamily. Expression of 4-1 BB receptor is induced on the surface of T-cells after antigen- or mitogen-induced activation. The activation of 4-1BB occurs via engagement with 4-1BBL, présent on APCs. Therefore, activation of 4-1 BB signaling provides a targeted approach to enhance existing TCR signaling.

Cell Line Descriptions

[0217] The characteristics of the cell lines used in several of the following examples are provided below:

NALM6 (ACL14036)

[0218] 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 are maintained in RPMI 1640 + 10% FBS + P/S/G; 37°C 5% CO₂[0219] Staining to confirm expression

NALM6/hPD-L1 (ACL16389)

[0220] NALM6 cells that were genetically engineered to stably express human PD-L1 (amino acids M1-T290 of accession number NP_054862.1). Cells are maintained in RPMI 1640 + 10% FBS + P/S/G + 1ug/ml Puro; 37°C 5% CO₂.

[0221] Virus Transfection and Transduction

[0222] Staining to confirm expression

HEK293 parental (ACL2397)

[0223] A human embryonic kidney cell line isolated from a fétus [HEK-293 (ATCC, #CRL1573)]. Referred to as HEK293 parental cell line. HEK293 cells are maintained in DMEM + 10% FBS + P/S/G.

HEK293/hCD20 (ACL14268)

[0224] A cell line made by stably transducing HEK293 (HZ) cells with human CD20 (Uniprot accession #: P11836, amino acids M1 to P297). Engineered line is maintained in DME + 10% FBS + penicillin/streptomycin/glutamine (P/S/G) + 100 pg/mL hygromycin @ 5% CO₂.

[0225] Virus Transfection/Production

[0226] Cell Line Transduction

[0227] Staining to confirm expression

HEK293/hCD20/hCD38 (ACL14270)

[0228] A cell line made by stably transducing HEK293 (HZ) cells with human CD20 (Uniprot accession #: P11836, amino acids M1 to P297) and human CD38 (Uniprot accession #: P28907, amino acids M1 to I300). Engineered line is maintained in DME + 10% FBS + penicillin/streptomycin/glutamine (P/S/G) + 500 pg/mL G418 + 100 pg/mL hygromycin @ 5% CO₂.

[0229] Virus Transfection/Production

[0230] Cell Line Transduction

[0231] Staining to confirm expression

HEK293/h4-1BB (ACL7888)

[0232] A cell line made by stably transducing HEK293 (HZ) cells with human TNFRSF9 (4-1 BB) (Accession #: NM_001561, amino acids M1 to L255). Engineered line is maintained in DME + 10% FBS + penicillin/streptomycin/glutamine (P/S/G) + 500 pg/mL G418 @ 5% CO₂.

[0233] Virus Transfection/Production

[0234] Cell Line Transduction

[0235] Staining to confirm expression

HEK293/NFkB-Luc (ACL5021)

[0236] A cell line made by transfecting a Firefly Luciferase-IRES-GFP gene driven by five copies of NF-κΒ response element located upstream of the minimal TATA promoter. Positive cell line sélection was performed by flow cytometry and a single clone, D9, was isolated.

[0237] Plasmid Génération.

[0238] Staining to confirm expression.

HEK293/NFkB-Luc/h4-1 BB ( ACL11090)

[0239] A cell line made by stably transducing HEK293/NFkB-Luc cells with human TNFRSF9 (4-1 BB) (Accession #: NM_001561, amino acids M1 to L255). Engineered line is maintained in DME + 10% FBS + penicillin/streptomycin/glutamine (P/S/G) + 500 pg/mL G418 @ 5% CO2. [0240] Virus Transfection/Production

[0241] Cell Line Transduction

[0242] Staining to confirm expression

MOLP8 (ACL14198)

[0243] A human multiple myeloma line established from the peripheral blood of a 52-year-old Japanese man with multiple myeloma in 2002. Acquired through DSMZ #: ACC 569.

[0244] Staining to confirm expression

OPM2 (ACL14164)

[0245] Established from the peripheral blood of a 56-year-old woman with multiple myeloma in leukemic phase in 1982. Acquired through DSMZ #: ACC50.

[0246] Staining to confirm expression

Example 1. Génération and Screening of Anti-CD38 Antibodies and Anti-4-1 BB Antibodies

[024η Anti-CD38 antibodies were obtained by immunizing a genetically engineered mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable régions with cells expressing CD38 or with DNA encoding CD38. The antibody immune response was monitored by a CD38-specific immunoassay. When a desired immune response was achieved splénocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce CD38-specific antibodies. Using this technique several anti-CD38 chimeric antibodies {i.e., antibodies possessing human variable domains and mouse constant domains) were obtained. In addition, several fully human anti-CD38 antibodies were generated from directly isolating antigen-positive B cells without fusion to myeloma cells, as described in US 2007/0280945A1.

[0248] Likewise, anti-4-1 BB antibodies were obtained by immunizing a genetically engineered mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable régions with cells expressing 4-1 BB or with DNA encoding 4-1 BB. The antibody immune response was monitored by a 4-1BB-specific immunoassay. When a desired immune response was achieved splénocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce 4-1BB-specific antibodies. Using this technique several anti-4-1 BB chimeric antibodies {i.e., antibodies possessing human variable domains and mouse constant domains) were obtained. In addition, several fully human anti-4-1 BB antibodies were generated from directly isolating antigen-positive B cells without fusion to myeloma cells, as described in US 2007/0280945A1.

[0249] The antibodies were characterîzed and selected for désirable characteristics, including affinity, selectivity, etc. If necessary, mouse constant régions were replaced with a desired human constant région, for example wild-type or modified IgG 1 or lgG4 constant région, to generate a fully human anti-CD38 antigen binding molécule or fully human anti-4-1BB antigen binding molécule. While the constant région selected may vary according to spécifie use, high affinity antigen binding and target specificity characteristics résidé in the variable région.

Example 2. Heavy and Light Chain Variable Région Amino Acid and Nucleic Acid Sequences of anti-CD38 Binding Arm.

[0250] Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable régions and CDRs of selected anti-CD38 antigen binding arms of the invention. The corresponding nucleic acid sequence identifiers are set forth in Table 2.

Table 1 Anti-CD38 Amino Acid Sequence Identifiers
Ab Désignation	HCVR (VH-1)	HCDR1	HCDR2	HCDR3	LCVR (VL-1)	LCDR1	LCDR2	LCDR3
mAb26871P2	2	4	6	8	18 (3-20GL)	20	22 GAS	24
mAb26812P2	40	42	44	46	48 (1-39GL)	50	52 AAS	54

Table 2 Anti-CD38 Nucleic Acid Sequence Identifiers
Ab Désignation	HCVR (VH-1)	HCDR1	HCDR2	HCDR3	LCVR (VL-1)	LCD RI	LCDR2	LCDR3
mAb26871P2	1	3	5	7	17 (3-20GL)	19	21 ggggcaagt	23
mAb26812P2	39	41	43	45	47 (1-39GL)	49	51 gctgcatcc	53

[0251] The anti-CD38 binding arms may comprise variable domain and CDR sequences as set forth in Table 1 and a human Fc domain of isotype lgG4, lgG1, etc. For certain applications or experiments the Fc domain may be a mouse Fc domain. As will be appreciated by a person of ordinary skill in the art, an antigen binding arm having a particular Fc isotype can be converted to an antigen binding arm with a different Fc isotype (e.g., an antigen binding molécule with a mouse lgG4 Fc can be converted to an antigen binding molécule with a human lgG1, etc.), but in any event, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Table 1 - will remain the same, and the binding properties are expected to be identical or substantialiy similar regardless of the nature of the Fc domain.

Example 3: Heavy and Light Chain Variable Région Amino Acid and Nucleic Acid Sequences of anti-4-1BB Binding Arm.

[0252] Table 3 sets forth the amino acid sequence identifiers of the heavy and light chain variable régions and CDRs of selected anti-4-1BB binding arms of the bispecific antibodies. The corresponding nucleic acid sequence identifiers are set forth in Table 4.

Table 3 Anti-4-lbb Amino Acid Sequence Identifiers

Ab Désignation	HCVR (VH-2)	HCDR1	HCDR2	HCDR3	LCVR (VL-2)	LCDR1	LCDR2	LCDR3
mAb25921P	10	12	14	16	18	20	22	24
mAb25889P2	32	34	36	38	48	50	52	54
mAb35333P2	62	64	66	68	48	50	52	54
mAb25898P2	72	74	76	78	48	50	52	54
mAb35388P2	86	88	90	92	48	50	52	54
mAb35321P2	94	96	98	100	48	50	52	54

Table 4 Anti-4-lbb Nucleic Acid Sequence Identifiers

Ab Désignation	HCVR (VH-2)	HCDR1	HCDR2	HCDR3	LCVR (VL-2)	LCDR1	LCDR2	LCDR3
mAb25921P	9	11	13	15	17	19	21	23
mAb25889P2	31	33	35	37	47	49	51	53
mAb35333P2	61	63	65	67	47	49	51	53
mAb25898P2	71	73	75	77	47	49	51	53
mAb35388P2	85	87	89	91	47	49	51	53
mAb35321P2	93	95	97	99	47	49	51	53

[0253] The anti-4-1 BB antigen binding arms may comprise variable domain and CDR sequences as set forth in Table 3 and a human Fc domain of isotype lgG4, lgG1, etc. For certain applications or experiments the Fc domain may be a mouse Fc domain. As will be appreciated by a person of ordinary skill in the art, an antigen binding molécule having a particular Fc isotype can be converted to an antigen binding molécule with a different Fc isotype (e.g., an antigen binding molécule with a mouse lgG4 Fc can be converted to an antigen binding molécule with a human lgG1, etc.), but in any event, the variable domains (including the

CDRs) - which are indicated by the numerical identifiers shown in Table 3 - will remain the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

Example 4: Génération of Multispecific Antigen Binding Molécules that Bind CD38 and 420 1BB

[0254] The multispecific antigen binding molécules that bind CD38 and 4-1 BB are also referred to herein as “anti-CD38 x anti-4-1 BB 1 + 2” or “anti-CD38 x anti-4-1 BB multispecific molécules”, or “anti-CD38/anti-4-1BB 1 + 2”, or “CD38x4-1 BB multispecific molécules. The antiCD38 portion of the anti-CD38 x anti-4-1 BB multispecific molécule is useful for targeting tumor 25 cells that express CD38, and the anti-4-1 BB portion of the multispecific molécule is useful for activating T-cells.

[0255] Various 4-1 BB Fabs (Fab3; heavy chain variable régions (HCVR) with heavy chain CH1 domain and light chain) binding to 4-1 BB epitope 1 (ep1) or epitope 2 (ep2) were fused to the N-terminus of a 4-1 BB VH domain from an existing IgG-like bispecific molécule targeting both 4-1 BB and CD38.

[0256] DNA fragments encoding (i) various 4-1 BB heavy chain variable régions (HCVR) (ii) heavy chain CH1 domain followed by linkers of varied lengths for connecting the heavy chain CH1 domain to a second 4-1 BB heavy chain variable régions (HCVR) (iii) CD38 heavy chain variable régions (HCVR) were synthesized by Integrated DNA Technologies, Inc. (San Diego,

California).

[0257] Mammalian expression vectors for individual heavy chains were created by InFusion Cloning (Takara Bio USA Inc.) following protocols provided by Takara Bio USA Inc. CD38 heavy chain variable régions (HCVR) (VH-1) were cloned into a heavy chain expression plasmid (CH11_CH2_CH3). 4-1 BB heavy chain variable régions (HCVR) (VH-3) fused to a CH1 domain (CH 1-3) with linkers of various length (linker) followed by another 4-1 BB heavy chain variable régions (HCVR) (VH-2) were cloned into a heavy chain expression plasmid (CH12_CH2_CH3(*)) containing the star mutation (H435R, Y436F, EU numbering).

[0258] Recombinant CD38 x 4-1 BB x 4-1 BB 1 +2 N-Fab MBMs were produced in CHO cells after transfection with 3 expression piasmids (i) CD38 heavy chain plasmid (ii) 4-1 BB + 4-1 BB 20 heavy chain star plasmid (iii) a universal light chain containing plasmid. Stably transfected CHO cell pools were isolated after sélection with 400 pg/ml hygromycin for 12 days. The CHO cell pools were used to produce the CD38 x 4-1 BB x 4-1 BB 1+2 N-Fab MBMs which were subsequently purified as described previously (Sci Rep. 2015 Dec 11; 5:17943).

[0259] A summary of the component parts of the antigen binding domains of selected multispecific antigen binding molécules made in accordance with this Example is set forth in Table 5. The respective nucleic acid sequence identifiers of the component parts are provided in Table 6. Tables 7 and 8 provide the component parts, polypeptide sequences and nucleic acid sequences, respectively, for the control bispecific antigen binding molécule.

Table 5 Component Parts of Selected 1+2 Formatted Antibodies-Amino Acid Sequence Identiflers
	CD38 arm (Fabl)	4-lBB-IN arm (Fab2)	4-1B B OUT arm (Fab3)	LCVR	Full Length Heavy and Light Chain
REGN No.	Ab Désignation	VH -1	CDR 1	CDR 2	CDR 3	Ab Désignation	VH2	CDR 1	CDR 2	CDR 3	Ab Désignation	VH3	CDR 1	CDR 2	CDR 3	Ab Désignation	Vk	CDR 1	CDR 2	CDR 3	HC4-1BB	HCCD38	LC ULC 139GL
7633	26812P2	40	42	44	46	25889P2	32	34	36	38	35388P2	86	88	90	92	1-39(PP) GL	48	50	52	54	56	58	60
7647	26812P2	40	42	44	46	35333P2	62	64	66	68	35333P2	62	64	66	68	1-39(PP) GL	48	50	52	54	70	58	60
76S0	26812P2	40	42	44	46	25898P2	72	74	76	78	35321P2	94	96	98	100	1-39(PP) GL	48	50	52	54	80	58	60
9682	26812P2	40	42	44	46	35333P2	62	64	66	68	35333P2	62	64	66	68	1-39(PP) GL	48	50	52	54	82	58	60
9686	26812P2	40	42	44	46	35333P2	62	64	66	68	35333P2	62	64	66	68	1-39(PP) GL	48	50	52	54	84	58	60

un un

Table 6 Component Parts of Selected 1+2 Formatted Antlbodies-Nucleic Acid Sequence Identifier;

	CD38 arm (Fabl)	4-1ΒΒ-ΊΝ arm (Fab2)	4-1BB OUT arm (Fab3)	LCVR	Full Length Heavy and Light Chain
REGN No.	Ab Désignation	VH -1	CDR 1	CDR 2	CDR3	Ab Désignation	VH -2	CDR 1	CDR 2	CDR 3	Ab Désignation	VH3	CDR 1	CDR 2	CDR 3	Ab Désignation	Vk	CDR 1	CDR 2	CDR 3	HC4-1BB	HCCD 38	LC ULC 139GL
7633	26812P2	39	41	43	45	25889P2	31	33	35	37	35388P2	85	87	89	91	1-39(PP) GL	47	49	51	53	55	57	59
7647	26812P2	39	41	43	45	35333P2	61	63	65	67	35333P2	61	63	65	67	1-39(PP)GL	47	49	51	53	69	57	59
7650	26812P2	39	41	43	45	25898P2	71	73	75	77	35321P2	93	95	97	99	1-39(PP) GL	47	49	51	53	79	57	59
9682	26812P2	39	41	43	45	35333P2	61	63	65	67	35333P2	61	63	65	67	1-39(PP) GL	47	49	51	53	81	57	59
9686	26812P2	39	41	43	45	35333P2	61	63	65	67	35333P2	61	63	65	67	1-39(PP) GL	47	49	51	53	83	57	59

Table 7 Component Parts of 1 +1 Formatted Control Antibody- Amino Acid Sequence Identiffers

CD38 arm

4-1ΒΒ-ΊΝ (Fab2)

4-lBB-0UT (Fab3)

LCVR

Full Lenf

jth HC and LC

REGN No.

Ab Désignation

VH -1

CDR 1

CDR 2

CDR3

Ab Désignation

VH -2

CDR 1

CDR 2

CDR 3

Ab Désignation

VH- 3

CDR 1

CDR 2

CDR 3

Ab Désignation

Vk

CDR 1

CDR 2

CDR 3

HC

LC

7150

26871P2

2

4

6

8

NA

25921P

10

12

14

16

3-20 G L

18

20

22

24

26

28

30

Table 8 Component Parts of 1 + 1 Formatted Control Antibody- Nucleic Acid Sequence Identifiers

CD38 arm

4-1ΒΒ-ΊΝ (Fab2)

4-lBB-OUT (Fab3)

LCVR

Full Length HC and LC

REGN No.

Ab Désignation

VH -1

CDR 1

CDR 2

CDR3

Ab Désignation

VH -2

CDR 1

CDR 2

CDR 3

Ab Désignation

VH3

CDR 1

CDR 2

CDR 3

Ab Désignation

Vk

CDR 1

CDR 2

CDR 3

HC

LC

7150

26871P2

1

3

5

7

NA

25921P

9

11

13

15

3-20 G L

17

19

21

23

25

27

29

Example 5: Screening of Multispecific Antigen Binding Molécules

[0260] Constructs activating the 4-1 BB signaling pathway were next identified by screening. [0261] Thirty-four 4-1 BB binding antibodies were selected for inclusion in the 1+2 screen based on cell binding to human 4-1 BB, utilization of a common light chain and diversity of CDR3 sequences. For the screen, 4-1 BB variable domains were arrayed in two different 1+2 formats, split 4-1 BB or stacked 4-1 BB. See Figure 2B.

[0262] For the split format, a standard bivalent architecture for 4-1 BB was maintained and tumor targeting was achieved by adding the variable domain and CH1 from a CD38 tumor targeting arm (26812) at the N-terminus of one side ofthe 4-1 BB molécule separated by a G4Sx3 spacer domain.

These constructs were expressed as Knob in Hole (KiH) bispecifics so only one arm of the expressed molécule contained the CD38 targeting motif.

[0263] For the stacked FAB format, tandem 4-1 BB sequences were linked in the same manner as CD38 x 4-1 BB described above, and tumor targeting was achieved by expression as a bispecific molécule with anti-CD38 present on the opposing arm. The thirty-four 4-1 BB variable domains along with one irrelevant control sequence (anti-BetV1) were assembled in al) possible combinations of tandem FABs generating 1225 combinations. This included thirty-four molécules where the two stacked 4-1 BB variable sequences were identical.

[0264] For screening, the 1+2 constructs were transiently expressed in CHO cells and bispecific containing supernatants harvested 4 days later. Supernatants were added to a co-culture of cells containing H EK cells over-expressing hCD38 and Jurkat cells harboring an NFkB luciferase reporter and over-expressing human 4-1 BB. Engagement and activation of 4-1 BB by the test samples was compared to a co-culture of the Jurkat reporter cells with HEK cells expressing the 41BB ligand (100%). Screening results from split FABs showed little to no activity in the Jurkat reporter assay with the highest activity observed to be 3.7%. In contrast, robust activation was observed with the stacked FAB format with over twenty-five percent of the samples tested yielding activities greater than 30%. Activity was observed when the 4-1 BB variable domains within the stacked FAB were identical or unique sequences and activities as high as 66% were observed. Introduction of the irrelevant control FAB at any location within the stacked FAB resulted in a loss of activity. See Figure 2A.

Example 6: Characterization of CD38x4-1BB Multispecific Antigen Binding Molécules Binding Using Flow Cytometry

[0265] Binding of the CD38 arm was tested using MOLP8 cells which endogenously express CD38. Binding ofthe 4-1 BB arm was assessed using HEK293 cells engineered to express h4-1BB (HEK293/h4-1BB). HEK293 cells were used to détermine non-targeted cell binding, as they do not 35 express CD38 nor 4-1 BB.

[0266] The ability ofthe multispecific antigen binding molécules to bind cells was assessed using flow cytometry. Cell lines were chosen to détermine the ability of both the anti-CD38 and anti-4-1 BB arms to bind their targets. In the first experiment test antibodies were incubated with MOLP8 (endogenously express hCD38) and HEK293 (do not express hCD38) cells. In the second experiment test antibodies were incubated with HEK293/h4-1 BB (engineered to express h4-1BB) and HEK293 (do not express h4-1BB) cells. Binding was detected by using a labeled secondary antibody and measuring fluorescence on a flow cytometer.

Multispecific Antigen Binding Molécules:

[0267] Multispecific antigen binding molécules and the Controls tested in these two experiments are as shown in Table 9.

Table 9: CD38x4-1BB Binding Molécules and Controls

MABM Identifier	Description
REGN7633	CD38x4-1BB (1+2), (hlgG4^p-^pvA)
REGN7647	CD38x4-1BB (1+2), (hlgG4^{P PVA})
REGN7650	CD38X4-1BB (1+2), (htgG4^{P PVA})
REGN7150	CD38x4-1BB (1+1), (hlgG4^p-^pvA)
REGN4249	Comparator 1
REGN7540	Isotype control (hlgG4^{P PVA})
REGN1945	Isotype control (hlgG4^p)

Experiment 1 - CD38 Binding:

[0268] HEK293 cells were lifted with trypsin, washed and resuspend in stain buffer (2% FBS in PBS). MOLP8 cells were washed and resuspended in stain buffer. Cells were added to wells of a 96 well V-bottom plate (3x10⁵cells/well). A 1:4, 9-point, dose titration of test antibodies was diluted in stain buffer and added to cells to a final concentration ranging from 610 fM to 10 nM (a “no antibody” control was included as the ninth point (153fM), labeled as “secondary only”). Cells and antibodies were incubated for 30 min on ice and then washed in stain buffer. Cells were resuspended in 2 ug/ml allophycocyanin (APC) conjugated goat-anti human secondary antibody and incubated for 30 min on ice. Cells were then washed and resuspended in viability dye (according to the manufacturer's protocol) and incubated for 30 min on ice. Cells were then washed with stain buffer and resuspended in 2% PFA for 30 min on ice. After washing, cells were filtered and analyzed by flow cytometry to détermine the géométrie mean fluorescence intensity (gMFI), which was subsequently plotted using GraphPad Prism software. EC50 values of the antibodies were determined from a 4-parameter logistic équation over a 9-point dose response curve (încluding secondary only control, representing the ninth point). Results are shown in Table 10.

[0269] Dose dépendent binding of the CD38x4-1 BB 1 +2 (REGN7633, REGN7647 and

REGN7650) and 1+1 (REGN7150) multispecific antigen binding molécules was observed in the presence of MOLP8 cells, which endogenously express CD38. However, no binding was observed for the bivalent anti-4-1BB antibody REGN4249. Nor was binding observed for the isotype control antibodies (REGN7540 and REGN1945).

[0270] No binding of the CD38x4-1 BB 1 +2 antibody REGN7633 or the isotype control antibodies (REGN7540 and REGN1945) was observed in the presence of HEK293 cells, which do not express CD38. Weak binding (>1 OO-fold lower gMFI than on the MOLP8 cells) was observed with the CD38x4-1BB 1+2 (REGN7647 and REGN7650) and 1+1 bispecific (REGN7150) antibodies as well as the bivalent anti-4-1 BB antibody REGN4249.

Table 10: Maximum Binding and EC50 Values of Binding for Antibodies

	MOLP8	HEK293
MAX [gMFI]	EC50 [M]	MAX [gMFI]	EC50 [M]
REGN7633	8.8E+04	NC	7.1E+02	ND
REGN7647	1.2E+05	NC	1.2E+03	2.43E-10
REGN7650	1.4E+05	NC	9.5E+02	ND
REGN7150	2.1E+05	NC	1.1E+03	NC*
REGN4249	5.3E+02	ND	1.4E+03	NC*
REGN7540	4.6E+02	ND	7.4E+02	ND
REGN1945	4.5E+02	ND	7.5E+02	ND

Abbreviations: ND: Not Determined; NC: Not calculated because the data did not fit a 4-parameter logistic équation. *Most likely due to nonspecific binding

Experiment 2 - 4-1BB Binding:

[0271] HEK293 and HEK293/4-1 BB cells were lifted with trypsin, washed and resuspended in stain buffet (2% FBS in PBS) and added to weîls of a 96 well V-bottom plate (3x10⁵ceils/well). A 1:5, 9-point, dose titration of test antibodies was diluted in stain buffer and added to cells to a final concentration ranging from 1.3 pM to 100 nM (a “no antibody” control was included as the ninth point (0.26nM) labeled as “secondary only”). Cells and antibodies were incubated for 30 min on ice and then washed in stain buffer. Cells were resuspended in 2 ug/ml allophycocyanin (APC) conjugated goat-anti human secondary antibody and incubated for 30 min on ice. Cells were then washed and resuspended in viability dye (according to the manufacturées protocol) and incubated for 30 min on ice. Cells were then washed with stain buffer and resuspended in 2% PFA for 30 min on ice. After washing, cells were filtered and analyzed by flow cytometry to détermine the géométrie mean fluorescence intensity (gMFI), which was subsequently plotted using GraphPad Prism software. EC_So values of the antibodies were determined from a 4-parameter logistic équation over a 9-point dose response curve (including secondary only control). Results are shown in Table 11.

[0272] Dose dépendent binding of the CD38x4-1BB 1+2 (REGN7633, REGN7647 and

REGN7650) and 1+1 (REGN7150) multispecific antigen binding molécules was observed in the presence of HEK293/h4-1 BB cells, which were engineered to express h4-1 BB. Binding of the bivalent anti-4-1 BB antibody REGN4249 was also observed. Binding was not observed for the isotype control antibodies (REGN7540 and REGN1945).

[0273] No binding ofthe CD38x4-1BB 1+2 antibody REGN7633 orthe isotype control antibodies (REGN7540 and REGN1945) was observed in the presence of HEK293 cells, which do not express h4-1 BB. Slight binding (more than 1,000-fold lower gMFI than on the HEK293/h4-1 BB cells) was observed with the CD38x4-1BB 1+2 multispecific antigen binding molécules (REGN7647 and REGN7650), the CD38x4-1BB bispecific (REGN7150), and the bivalent anti-4-1 BB antibody REGN4249.

Table 11 : Maximum Binding and EC50 Values of Binding for Antibodies

	HEK293/4-1BB	HEK293
MAX [gMFI]	EC50 [M]	MAX [gMFI]	EC50 [M]
REGN7633	8.9E+05	1.47E-08	1.6E+04	ND
REGN7647	1 4E+06	6.08E-10	1.8E+04	ND
REGN7650	1.0E+06	9.97E-10	2.4E+04	NC*
REGN7150	2.4E+06	2.86E-09	2.5E+04	2.72E-09*
REGN4249	7.4E+05	8.86E-10	1.8E+04	ND
REGN7540	2.5E+04	ND	1.9E+04	ND
REGN1945	1.6E+04	ND	1.4E+04	ND

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.

[0274] Two signais, “signal 1 & “signal 2”, are required for proper T cell activation. Signal 1” is induced by binding ofthe T cell receptor (TCR) on T cells to peptide-bound major histocompatibility complex (MHC) molécules on antigen presenting cells (APCs). Whereas, “signal 2” is provided by engaging co-stimulatory receptors on T cells. One such costimulatory receptor is 4-1 BB receptor, which is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily. Expression of 4-1BB receptor is induced on the surface of T-cells after antigen- or mitogen-induced activation. The activation of 4-1BB occurs via engagement with 41BBL, présent on APCs. Therefore, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0275] CD38x4-1BB (1+1 and 1+2) bispecific antibodies are designed to mimicthe naturel ligand of 4-1 BB, by bridging CD38⁺ target cells with 4-1 BB receptor positive T cells, to provide “signal 2” in order to enhance the activation of T cells in the presence of a signal 1” provided by a Tumor associated antigen (TAA) x CD3 bispecific antibody or an allogeneic response provided by the APC.

Multispecific Antigen Binding Molécules:

[0276] Multispecific antigen binding molécules and the Controls tested in this experiment are as shown in Table 12.

Table 12: CD38x4-1BB Binding Molécules and Controls

MABM Identifier	Description
REGN7633	CD38x4-1BB (1+2), (hlgG4^pPVAj
REGN7647	CD38x4-1BB (1+2), (hlgG4^p‘ PVAJ
REGN7650	CD38x4-1BB (1+2), (hlgG4^pPVAJ
REGN7150	CD38x4-1BB (1+1), (hlgG4^pPVAj
REGN4249	Comparator 1
REGN7540	Isotype control, (hlgG4^p_pVA)
REGN1945	Isotype control, (hlgG4^p)
REGN5458	BCMAxCD3, (hlgG4^PPVA)
REGN1979	CD20xCD3, (hlgG4^PPVA)

[0277] The ability of CD38x4-1 BB multispecific antigen binding molécules to activate human primary T-cells by engaging CD38 and 4-1 BB receptor to deliver “signal 2, as determined by IL-2 release, was evaluated in the presence of a human embryonic kidney cancer cell line engineered to express hCD20 and hCD38 (HEK293/hCD20/hCD38) using REGN1979 (CD20xCD3) to serve as “signal 1.” HEK293 cells expressing only hCD20 were included as a control to measure activity that may occur in the absence of CD38 on APC’s. Additionally, a multiple myeloma cell line that endogenously expresses hCD38, MOLP8, was included in testing CD38x4-1 BB bispecific antibodies. As MOLP8 cells endogenously express BCMA, REGN5458 (BCMAxCD3) was included to serve as signal 1. Of note, unlike HEK293 cells, MOLP8 cells are able to provide détectable allogeneic stimulation of T-cells, serving as “signal 1’, in the absence CD3 stimulation provided by REGN5458.

Isolation of human primary CD3^¥ T cells:

[0278] Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor leukocyte pack from Précision for Medicine (Donor 555105) using density gradient centrifugation. Bhefly, 15ml of Ficoll-Paque PLUS is added to 50ml conical tubes, and subsequently 30ml of blood diluted 1:1 with PBS containing 2% FBS is layered on top. After a 30-minute centrifugation at 400 x g, with the brake off, the buffy coat (containing mononuclear cells) is transferred to a fresh tube, diluted 5x with PBS containing 2% FBS and centrifuged for 8 minutes at 300 x g. Subsequently, CD3⁺ T-cells were isolated from PBMC’s using an EasySepTM Human CD3⁺ T Cell Isolation Kit from StemCell Technologies and following the manufacturées recommended instructions.

IL-2 release assay:

[0279] Enriched CD3* T-cells, resuspended in stimulation media, were added into 96-well round bottom plates at a concentration of 1 x 10⁵ cells/well. Growth-arrested HEK293/hCD20/hCD38 or HEK293/hCD20 were added to CD3⁺ T-cells at a final concentration of 1 x 10“ cells/well. Growtharrested MOLP8 cells were added to CD3⁺ T-cells at a final concentration of 5 x 10⁴ cells/well. Following addition of cells, a constant of 0.1nM REGN1979 or its matched isotype control (REGN7540) was added to wells containing HEK293/hCD20/hCD38 or HEK293/hCD20. A constant of 0.5nM REGN5458 or an isotype control was added to wells containing MOLP8 cells.

Subsequently, CD38x4-1BB (1+1 or 1+2), bivalent 4-1BB (REGN4249), or isotype Controls (REGN7540 or REGN1945) were titrated from 3pM to 200nM in a 1:4 dilution and added to wells. The final point of the 10-point dilution contained no titrated antibody. Plates were incubated for 72 hours at 37°C, 5% CO₂ and 5 μΙ_ total supernatant was used for measuring IL-2. The amount of cytokine in assay supernatant was determined using AlphaLisa kits from PerkinElmer following the manufacturées protocol. The cytokine measurements were acquired on Perkin Elmer’s multilabel plate reader Envision and values were reported as pg/mL. AH serial dilutions were tested in duplicate.

[0280] The ECso values of the antibodies were determined from a four-parameter logistic équation over a 10-point dose-response curve using GraphPad Prism™ software. Maximal IL-2 is given as the mean max response detected within the tested dose range. Results are provided in Table 13.

Results with HEK293/hCD20 & HEK293/hCD20/hCD38 Cell Lines

[0281] In the presence of target and “signal T, provided by REGN1979, 4-1BB antibody treatment led to higher IL-2 response compared to matched isotype Controls (REGN7540 and REGN1945). Of note, 1+2 formats of CD38x4-1BB led to higher maximum IL-2 and greater potency, compared to 1+1 CD38x4-1BB (REGN7150) and bivalent 4-1 BB (REGN4249) antibody. In the absence of CD38 target only bivalent 4-1 BB (REGN4249) exhibited a dose dépendent increase in IL-2 release. In the absence of 'signal Γ none of the antibodies lead to dose dépendent enhancement of IL-2 release.

Results with MOLP8 Cell Line

[0282] In the presence of allogeneic MOLP8 cells, and absence of REGN5458, there was a dose-dependent increase in IL-2 observed for 1+2 CD38x4-1BB antibodies REGN7647 and REGN7650 and to a lesser extent for 1+2 CD38x4-1BB antibody REGN7633 and bivalent 4-1 BB antibody REGN4249. The 1+1 CD38x4-1BB and isotype control antibodies did not exhibit dose dépendent enhancement of IL-2 release. While “signal 1” can be provided by allogeneic MOLP8 cells, the addition of REGN5458, was also evaluated. Under these conditions ail 4-1 BB antibodies led to dose dépendent increases in IL-2 release compared to matched isotype control, with the 1+2 5 CD38x4-1BB antibodies exhibiting the greatest potency and maximum increase in IL-2.

Table 13: Maximum IL-2 release and Potency Values of Antibodies

	HEK293/CD20/CD38 + REGN1979	HEK293/CD20 + REGN1979	HEK293/CD20/CD 38 + IsoC	MOLP-8 + IsoC	MOLP-8 + REGN5458
	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]
REGN7633	1934	8.3E-11	218	2.16E-09	16	ND	177	1.31E-11	534	4.482E-10
R EG N 7647	2847	5.8E-12	199	NC	18	ND	331	2.21E-12	692	2.31 E-11
REGN7650	2721	1.4E-11	184	NC	10	ND	288	NC	553	3.166E-11
REGN7150	1303	1E-10	182	ND	12	ND	163	ND	308	6.338E-08
REGN4249	931	1 4E-09	729	1.42E-09	15	ND	174	NC	183	5.883E-10
REGN7540	176	ND	195	ND	9	ND	170	ND	156	ND
REGN1945	173	ND	208	ND	10	ND	155	ND	164	ND

Abbreviations: ND: Not Determined; NC: Not calculated because the data did not fit a 4-parameter logistic équation.

Example 8: Characterization of CD38x4-1 BB (1+2) Multispecific Antigen Binding Molécules in T-cell Activation Assays Using HEK293/hCD20/hCD38, HEK293/hCD20, MOLP8, NALM6, and Human Primary T-cells.

[0283] Two signais, “signal 1 & “signal 2”, are required for properT cell activation. “Signal Γ is induced by binding of the T cell receptor (TCR) on T cells to peptide-bound major histocompatibility complex (MHC) molécules on antigen presenting cells (APCs). Signal 2” is provided by engaging co-stimulatory receptors on T cells. One such costimulatory receptor is 4-1 BB receptor, which is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily. Expression of 4-1 BB receptor is induced on the surface of T-cells after antigen- or mitogen-induced activation. The activation of 4-1BB occurs via engagement with 4-1BBL, présent on APCs. Therefore, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0284] CD38x4-1BB (1+1 and 1+2) bispecific antibodies are designed to mimic the natural ligand 30 of 4-1 BB, by bridging CD38⁺ target cells with 4-1 BB receptor positive T cells, to provide “signal 2 in orderto enhance the activation of T cells in the presence of a signal Γ provided by a Tumor associated antigen (TAA) x CD3 bispecific antibody or an allogeneic response provided by the APC.

Multispecific Antigen Binding Molécules:

[0285] Multispecific antigen binding molécules and the Controls tested in this experiment are as 5 shown in Table 14.

Table 14: CD38x4-1BB Binding Molécules and Controls

MABM Identifier	Description, Linker, Isotype
REGN7647	CD38x4-1BB (1+2), (G4S)3v15 (hlgG4^{P PVA})
REGN9682	CD38x4-1BB (1+2), (G4S)3 (hlgG4^{P PVA})
REGN9686	CD38x4-1BB (1+2), (G4S)2 (hlgG4^p'^PVA)
REGN7150	CD38x4-1BB (1+1), (hlgG4^{P PVA})
REGN13168	Non-TAAx4-1BB (1+2) Control, (hlgG4^p-^pVA)
REGN4249	Comparator 1
REGN5458	BCMAxCD3, (hlgG4^{P PVA})
REGN2281	CD20xCD3, (hlgG4^p)
REGN7540	Isotype control, (hlgG4^{p PVA})
REGN1945	Isotype Control, (hlgG4^p)

[0286] The ability of CD38x4-1BB 1+2 multispecific antigen binding molécules, harboring different linkages between 2 tandem 4-1 BB binding Fab domains, to activate human primary T-cells 20 by engaging CD38 and 4-1 BB receptor to deliver “signal 2”, as determined by IL-2 or IFNy release, was evaluated in the presence of a human embryonic kidney cancer cell line engineered to express hCD20 and hCD38 (HEK293/hCD20/hCD38) using REGN2281 (CD20xCD3) to serve as “signal 1.” HEK293 cells expressing only hCD20 were included as a control to measure activity that may occur in the absence of CD38 on APC’s. Additionally, a multiple myeloma cell line that endogenously expresses hCD38, MOLP8, was included in testing CD38x4-1 BB (1 +2) bispecific antibodies. As MOLP8 cells endogenously express BCMA, REGN5458 (BCMAxCD3) was included to serve as “signal 1. Lastly, conditions that included either MOLP8 or NALM-6 (an acute lymphoblastic leukemia cell line that endogenously expresses CD38) as target cells, in the absence of a CD3 bispecific were tested. Of note, unlike HEK293 cells, MOLP8 and NALM-6 cells are able to provide détectable allogeneic stimulation of T-cells, serving as “signal 1’, in the absence CD3 antibody stimulation.

Isolation of human primary CD3⁺ T cells:

[0287] Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor leukocyte pack from Précision for Medicine (Donor 555114) using an EasySep Human T-Cell 35 Isolation kit from StemCell Technologies, following the manufacturées recommendations.

Subsequently, CD3⁺ T-cells were isolated from PBMC’s using an EasySepTM Human CD3⁺ T Cell

Isolation Kit from StemCell Technologies and following the manufacturer’s recommended instructions.

Primary T-cell activation assay:

[0288] Enriched CD3⁺ T-cells, resuspended in stimulation media, were added into 96-well round 5 bottom plates at a concentration of 1 x 10⁵ cells/well. Target cells were added to CD3⁺ T-cells at a final concentration of 1 x 10⁴ cells/well for HEK293/hCD20/hCD38 or HEK293/hCD20 cells or 5 x 10⁴ cells/well for MOLP8 and NALM-6 cells. Following addition of cells, a constant of 0.25nM REGN2281 was added to wells containing HEK293/hCD20/hCD38 or HEK293/hCD20 cells. A constant of 0.5nM REGN5458 or an isotype control was added to wells containing MOLP8 cells. No 10 CD3 bispecific was added to wells containing NALM-6 target cells. Subsequently, CD38x4-1BB (1+1 or 1+2), bivalent 4-1 BB (REGN4249), or isotype Controls (REGN7540 or REGN1945) were titrated from 128fM to 50nM in a 1:5 dilution and added to wells. The final point of the 10-point dilution contained no titrated antibody. Plates were incubated for 72 hours at 37°C, 5% CO? and 5 pL total supernatant was used for measuring IL-2 or IFNy. The amount of cytokine in assay supernatant was determined using AlphaLisa kits from PerkinElmer following the manufacturer’s protocol. The cytokine measurements were acquired on Perkin Elmer’s multilabel plate reader Envision and values were reported as pg/mL. Ail serial dilutions were tested in duplicate.

[0289] The ECso values of the antibodies were determined from a four-parameter logistic équation over a 10-point dose-response curve using GraphPad Prism™ software. Maximal cytokine 20 is given as the mean max response detected within the tested dose range. Results are provided in

Tables 15 and 16.

HEK293/hCD20 & HEK293/hCD20/hCD38

[0290] In the presence of target and “signal Γ, provided by REGN2281,4-1 BB antibody treatment led to higher IL-2 and IFNy response compared to matched isotype lgG4^{P PVA}, lgG4^p, or 25 bispecific antibody Controls (REGN7540, REGN1945 and REGN13168, respectively). Of note, 1+2 formats of CD38x4-1 BB led to higher maximum cytokine release and greater potency, compared to 1+1 CD38x4-1BB (REGN7150) and bivalent 4-1 BB (REGN4249) antibody, with similar level and potency of cytokine release observed regardless of the linkage between the 2 tandem 4-1 BB binding Fab domains. In the absence of CD38 target, only bivalent 4-1 BB (REGN4249) exhibited a 30 dose dépendent increase in cytokine release.

MOLP8

[0291] In the presence of allogeneic MOLP8 cells, and absence of CD3 bispecific antibody stimulation, 1+2 CD38x4-1BB antibodies REGN7647, REGN9682, and REGN9686, mediated a dose-dependent increase in IL-2 and IFNy. In comparison, the 1+1 CD38x4-1BB antibody, 35 REGN7150, and bivalent 4-1 BB antibody, REGN4249, led to minor dose dépendent increases in

IL-2 and not IFNy. No dose dépendent increase was observed for isotype and non-targeted 1+2 41BB control antibodies for either IL-2 or IFNy. Addition of a fixed amount of REGN5458 (BCMAxCD3) in conditions with MOLP8 cells and primary human T-cells resulted in a dose dépendent increase in IL-2 and IFNy for ail CD38-targeted 4-1BB antibodies as well as for

REGN4249. As noted previously, the 1+2 CD38x4-1BB antibodies exhibited greater potency and maximum cytokine increase in comparison to REGN7150 and REGN4249, with the format of linkage between the 4-1BB-binding Fabs having little impact on potency or maximum cytokine release. Isotype control and non-TAAx4-1 BB 1+2 control antibodies did not lead to dose dépendent cytokine release.

NALM-6

[0292] In the presence of allogeneic NALM-6 cells, and absence of CD3 bispecific antibody stimulation, ail CD38-targeted 4-1 BB antibodies (REGN7647, REGN9682, REGN9686, and REGN7150) as well as the bivalent 4-1 BB antibody (REGN4249), led to dose-dependent increases in IFNy and IL-2. The 1+2 CD38x4-1BB antibodies REGN7647, REGN9682, and REGN9686 exhibited greater potency and maximum cytokine release compared to the 1+1 CD38x4-1BB antibody, REGN7150, and bivalent 4-1 BB antibody, REGN4249. As noted previously all 1+2 CD38x4-1 BB multispecific antigen binding molécules performed similarly, regardless of the linker between the 4-1 BB-binding Fabs. Isotype control and non-TAAx4-1BB 1+2 control antibodies did not lead to dose dépendent cytokine release.

Table 15: Maximum IL2 Release and Potency Values

	HEK293/CD20/CD38 + REGN2281	HEK293/CD20 + REGN2281	MOLP-8 + REGN5458	MOLP-8 + IsoC	NA	LM-6
MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50[M]
REGN7647	781	5.35E-11	61	ND	66	2.16E-10	73	NC	192	4.85E-11
REGN9682	912	6.07E-11	52	ND	79	1.33E-10	96	NC	205	7.17E-11
REGN9686	709	4.53E-11	57	ND	62	8.33E-11	90	3.61 E-11	169	7.23E-11
REGN7150	593	NC	63	ND	32	NC	48	NC	122	1.08E-10
REGN13168	41	ND	57	ND	16	ND	28	ND	48	ND
REGN4249	110	NC	126	8.19E-10	19	NC	54	NC	108	NC
REGN7540	37	ND	54	ND	17	ND	21	ND	35	ND
REGN1945	48	ND	63	ND	18	ND	33	ND	41	ND

Abbreviations: NC, not calculated because the data did not fit a 4-parameter logistic équation; ND, not determined because a concentration-dependent lo increase was not observed.

Table 16: Maximum IFNy Release and Potency Values

	HEK293/CD20/CD38 + REGN2281	HEK293/CD20 + REGN2281	MOLP-8 + REGN5458	MOLP-8 + IsoC	NALM-6
MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]
REGN7647	6046	6.17E-11	1908	ND	3284	7.07E-11	103	NC	655	7.03E-11
REGN9682	5271	5.26E-11	1573	ND	3406	7.25E-11	161	NC	658	4.19E-11
REGN9686	6823	8.74E-11	1449	ND	3891	1.02E-10	85	NC	651	NC
REGN7150	3951	7.80E-10	1843	ND	1297	4.38E-08	56	NC	620	NC
REGN13168	1805	ND	1638	ND	656	ND	1	ND	102	ND
REGN4249	2474	6.40E-10	2279	4.29E-09	1003	NC	37	NC	400	NC
REGN7540	2080	ND	1455	ND	602	ND	1	ND	130	ND
REGN1945	2086	ND	1664	ND	542	ND	83	NC	117	ND

Abbreviations: NC, not calculated because the data did not fit a 4-parameter logistic équation; ND, not determined because a concentration-dependent increase was not served.

Example 9: Characterization of CD38x4-1BB (1+2) Multispecific Antigen Binding Molécules in an Engineered Reporter Assay Using HEK293/hCD20/hCD38, HEK293/hCD20, MOLP8, OPM2 and HEK293/NFkB-Luc/h4-1BB cells

[0293] The ability of CD38x4-1BB multispecific antigen binding molécules to specifically activate 4-1 BB receptor in the presence of target cells expressing CD38 was measured in an engineered reporter assay. In this assay, engineered HEK293 cells express the reporter gene luciferase under the control of the transcription factor NF-KB (NFKB-Luc) along with the costimulatory receptor 41BB (HEK293/NFkB-Luc/h4-1BB). The target cells used in this assay were HEK293 cells engineered to express CD20 alone or in combination with CD38 or cell lines that endogenously express CD38, namely OPM2 and MOLP8. The ability of 4-1 BB arms to stimulate 4-1 BB activity is assessed by combining reporter cells with target cells and a titration of CD38x4-1BB 1+2 antibody. Activation of 4-1 BB results in NFKB-driven luciferase production, which is then measured via a luminescence readout. In these assays the impact of different types of linkages between the 2 tandem 4-1 BB targeting domains was also evaluated.

Multispecific Antigen Binding Molécules:

[0294] Multispecific antigen binding molécules and the Controls tested in this experiment are as shown in Table 17.

Table 17: CD38x4-1BB Binding Molécules and Controls

MABM Identifier	Description
REGN7647	CD38x4-1BB (1+2), (hlgG4^p-^pvA)
REGN7650	CD38x4-1BB (1+2), (hlgG4^{P PVA})
REGN9682	CD38x4-1BB (1+2), (G4S)3 (hlgG4^{P PVA})
REGN9686	CD38x4-1BB (1+2), (G4S)2 (hlgG4^{P PVA})
REGN4249	Comparator 1
REGN7540	Isotype control (hlgG4^p_pVA)
REGN1945	Isotype control (hlgG4^p)

[0295] One day before the experiment, HEK293 reporter cells were split to 5 x 10⁵ cells/ml in DMEM + 10% FBS + P/S/G + 500 pg/ml G418 growth media.

[0296] On the day of the experiment adhèrent HEK293 reporter and target cells were trypsinized, washed, and resuspended in assay media (DMEM + 10% FBS + P/S/G). The reporter HEK293/NFKB-Luc/h4-1 BB cells were added to the wells of 96-well white microtiter plates at a final concentration of 5 x 10³ cells/well, followed by the addition of target cells, either HEK293/hCD20 or HEK293/hCD20/hCD38, added at a final concentration of 1 x 10⁴ cells/well or MOLP8 and OPM2 target cells added at a final concentration of 2.5 x 10⁴ cells/well.

[0297] CD38x4-1BB 1+2 and contrat 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 antîbody, included as a controi. After addition of antibodies, the 96-well white micratiter plates were incubated at 37'C/5% CO₂ for 5 h followed by the addition of an equal volume of ONE-GIo™ (Promega) reagent to lyse cells and detect luciferase activity. The emitted light was captured in Relative Light Units (RLU) on a multi-label plate reader Envision (PerkinElmer). EC50 values of the antibodies were determined from a 4-parameter logistic équation over a 10-point dose response curve (the 10^th point containing no antîbody) using GraphPad Prism software. Results are provided in Table 18.

HEK293/hCD20 & HEK293/hCD20/hCD38

[0298] In the presence of HEK293/hCD20/hCD38 target cells the CD38x4-1BB 1+2 bispecific antibodies (REGN7647, REGN9682, REGN9686, and REGN7650) led to a similar increase in luciferase activity, regardless of the linkage between the 2 anti-4-1BB binding Fab domains. The anti4-1 BB bivalent antîbody (REGN4249) also led to a dose dépendent increase in luciferase activity, whereas the isotype controi antibodies did not.

[0299] In the presence of HEK293/hCD20 target cells lacking CD38, no response was seen with the CD38x4-1 BB bispecific antibodies nor with the isotype contrais. Only the anti-4-1 BB bivalent antîbody (REGN4249) led to a dose dépendent increase in luciferase activity.

MOLP8 and OPM2 cells

[0300] In the presence of either the MOLP8 or OPM2 target cells the CD38x4-1BB 1+2 (REGN7647, REGN7650, REGN9682 and REGN9686) antibodies as well as the bivalent anti-41 BB antîbody (REGN4249) led to a dose dépendent increase in luciferase activity, with the different linker length 1+2 CD38x4-1BB variants resulting in similar activity. The isotype controi antibodies did not resuit in any signal.

Table 18: Maximum Luciferase Activity and Potency Values of Antibodies

	HEK293/CD20/CD38	HEK293/CD20	OPM2	MOLP-8
MAX [RLU]	EC50 [M]	MAX [RLU]	EC50 [M]	MAX [RLU]	EC50 [M]	MAX [RLU]	EC50 [M]
REGN7647	2.4E+06	NC	1.2E+06	ND	1.9E+06	7.35E-12	2.3E+06	NC
REGN7650	2.3E+06	NC	1.5E+06	NC	2.0E+06	9.89E-11	2.4E+06	NC
REGN9682	2.4E+06	NC	1.0E+06	ND	2.1E+06	2.19E-11	2.2E+06	NC
REGN9686	2.4E+06	NC	1.1E+06	NC	2.1E+06	1.80E-11	2.2E+06	NC
REGN4249	2.8E+06	4.28E-11	3.3E+06	6.82E-11	2.9E+06	5.25E-11	2.7E+06	4.59E-11
REGN7540	9.9E+05	ND	1.2E+06	ND	8.5E+05	ND	7.9E+05	ND
REGN1945	1.0E+06	ND	9.5E+05	ND	7.5E+05	ND	7.8E+05	ND

Abbreviations: ND: Not Determined, because a concentration-dependent increase was not observed; NC: Not calculated because the data did not fit a 4-parameter logistic équation.

Example 10: Characterization of CD38x4-1BB 1+2 bispecific antibodies in T-cell allogeneic

Cemiplimab Combination Assay using NALM-6, NALM-6/PDL1, and Human Primary T-Cells [0301] Two signais, “signal Γ & “signal 2”, are required for proper T cell activation. “Signal Γ is induced by binding ofthe T cell receptor (TCR) on T cells to peptide-bound major histocompatibility complex (MHC) molécules on antigen presenting cells (APCs). “Signal 2” is provided by engaging co-stimulatory receptors on T cells. One such costimulatory receptor is 4-1 BB receptor, which is an inducible type I membrane protein and member ofthe tumor necrosisfactor receptor (TNFR) superfamily. Expression of 4-1 BB receptor is induced on the surface of T-cells after antigen- or mitogen-induced activation. The activation of4-1BB occurs via engagement with 4-1BBL, present on APCs. Therefore, activation of 4-1BB signaling provides a targeted approach to enhance existing TCR signaling.

[0302] CD38x4-1BB (1+2) bispecific antibodies are designed to mimic the natural ligand of 41 BB, by bridging CD38⁺ target cells with 4-1 BB receptor positive T cells, to provide “signal 2” in order to enhance the activation of T cells in the presence of a signal 1 provided by a Tumorassociated antigen (TAA) x CD3 bispecific antibody or an allogeneic response provided by the APC. However, T cell activation can be inhibited by the ligation of programmed cell death protein 1 receptor (PD-1) on T cells to its ligand PD-L1 on APCs. Ligated PD-1 leads to the recruitment of phosphatases to CD28 and the TCR complex (Zou et al. Inhibitory B7-family molécules in the tumor microenvironment. Nature Reviews Immunology 2008, 8:467-477; Francisco et al. 2010, Hui et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017, 355(6332):1428-33), which in turn counteract TCR signaling and 4-1 BB stimulation. Thus, blockade ofthe PD-1/PD-L1 interaction with a PD-1 antagonist, cemiplimab, in combination with CD38x41 BB bispecific antibodies may potentiate T cell function.

Multispecific Antigen Binding Molécules:

[0303] Multispecific antigen binding molécules and the Controls tested in this experiment are as shown in Table 19.

Table 19: CD38x4-1BB Binding Molécules and Controls

MABM Identifier	Description
REGN7633	CD38x4-1BB 1+2, hlgG4^{P PVA}
REGN7647	CD38x4-1BB 1+2, hlgG4^p-^pvA
R EG N 7650	CD38x4-1BB 1+2, hlgG4^p-^pvA
REGN4249	Comparator 1
REGN2810	PD-1 antibody (Cemiplimab), hlgG4^p
REGN7540	Isotype control, hlgG4^{P PVA}
REGN1945	Isotype control, hlgG4^p

[0304] The ability of CD38x4-1BB 1+2 multispecific antigen binding molécules to activate human primary T-cells by engaging CD38 and 4-1 BB to deliver “signal 2”, as determined by IL-2 & IFNy release, was evaluated in the presence of a CD38⁺ human acute lymphoblastic leukemia cancer cell line engineered to express PD-L1 (NALM6/hPD-L1). NALM6 cells provide an allogeneicTCR response sufficient to serve as “signal 1”. The addition of a fixed concentration of the PD-1 antagonist antibody, cemiplimab, was evaluated in the presence of a titration of CD38x4-1BB 1+2 or control antibodies.

Isolation of human primary CD3⁺ T cells:

[0305] Human peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor leukocyte pack from Précision for Medicine (Donor 555192) using the EasySepTM Direct Human PBMC Isolation Kit, following the manufacturers recommended protocol and frozen down. CD3⁺ Tcells were isolated by thawing vials of frozen PBMCs. Donor PBMCs were enriched for CD3⁺ Tcells using an EasySepTM Human CD3⁺ T Cell Isolation Kit from StemCell Technologies and following the manufacturées recommended instructions.

IL-2 & IFNy release assay:

[0306] Enriched CD3⁺ T-cells, resuspended in stimulation media, were added into 96-well round bottom plates at a concentration of 1 x 10⁵ cells/well. NALM6 cells or NALM-6 cells engineered to express hPD-L1, were added to CD3⁺ T-cells at a final concentration of 5 x 10⁴ cells/well.

Subsequently, REGN7633, REGN7647, REGN7650, REGN4249 and REGN7540, were titrated from 0.76pM to 50nM in a 1:4 dilution and added to wells. The final point of the 10-point dilution contained no titrated antibody. Following addition of titrated antibody, a constant 20nM of either cemiplimab or its matched isotype control (REGN1945) was added to wells. Plates were incubated for 72 hours at 37°C, 5% CO2 and 5 μΙ_ from supernatant was used for measuring IL-2 and IFNy. The amount of cytokine in assay supernatant was determined using AlphaLisa kits from PerkinElmer following the manufacturées protocol. The cytokine measurements were acquired on Perkin Elmer’s multilabel plate reader Envision and values were reported as pg/mL. Ail serial dilutions were tested in triplicate.

Results

[0307] The EC50 values of the antibodies were determined from a four-parameter logistic équation over a 10-point dose-response curve using GraphPad Prism™ software. Maximal cytokine is given as the mean max response detected within the tested dose range. Results are provided in Tables 20 and 21.

[0308] 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), in comparison to matched isotype control (REGN7540), led to dose dépendent increases in IL-2 and IFNy release. The maximum IL-2 and IFNy release was lower in conditions with NALM6/PD-L1 cells, compared to

NALM-6 (not expressing PD-L1). In the presence of NALM6 cells expressing PD-L1, cemiplimab increased maximum cytokine release in comparison to the matched isotype control for cemiplimab, REGN1945. Of note, 1+2 formats of CD38x4-1BB antibody led to higher maximum cytokine release and greater potency, compared to bivalent 4-1 BB (REGN4249) antibody.

Table 20: Maximum IL-2 Release and Potency Values

	NALM6 + tgG4P	NALM6 + Cemiplimab	NALM6/PD-L1 + lgG4P	NALM6/PD-L1 + Cemiplimab
IL-2	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]
REGN7633	853	6.22E-10	756	4.54E-10	205	4.13E-10	470	5.20E-10
REGN7647	1117	5.79E-11	1043	4.77E-11	433	1.17E-10	683	9.11E-11
REGN7650	1242	1.79E-10	1359	1.55E-10	326	2.28E-10	665	2.83E-10
REGN7540	125	ND	135	ND	32	ND	102	ND
REGN4249	413	6.03E-10	307	4.132E-10	143	3.96E-10	259	2.43E-10

Abréviations: ND: Not Determined; NC: Not calculated because the data did not fit a 4-parameter logistic équation.

Table 21: Maximum IFNy release and Potency Values

	NALM-6 + IgG4P	NALM-6 + Cemiplimab	NALM-6/PD-L1 + lgG4P	NALM-6/PD-L1 + Cemiplimab
IFNy	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50 [M]	MAX [pg/ml]	EC50[M]	MAX [pg/ml]	EC50 [M]
REGN7633	519	6.37E-10	1095	NC	1	ND	272	1.28E-09
REGN7647	707	8.15E-11	1067	NC	67	NC	557	NC
REGN7650	1008	NC	962	1.56E-10	15	NC	244	NC
REGN7540	86	ND	333	ND	1	ND	14	ND
REGN4249	616	NC	919	NC	7	NC	324	NC

Example 11: In Vivo Anti-tumor Efficacy of CD38x4-1BB Bispecific Antibodies in Combination with a BCMAxCD3 Bispecific Antibody.

[0309] Multispecific antigen binding molécules and the Controls tested in this experiment are as shown in Table 22.

Table 22: CD38x4-1BB Binding Molécules and Controls

MABM Identifier	Description
REGN5458	BCMAxCD3 bsAB
REGN13168	Non-TAAx4-1 BB (1+2) Control, (hlgG4^pPVAJ
REGN9686	CD38X4-1BB (1+2), (G4S)2 (hlgG4^p'^PVA)

[0310] To détermine the in vivo anti-tumor efficacy of CD38x4-1 BB 1 +2 format bispecific antibodies (bsAb) in combination with a BCMAxCD3 bsAb, a xenogenic tumor study was performed. On day -11, immunodeficient NOD.Cg-Prkdc^scldll2rg^lm1wï^|/SzJ (NSG) mice were intraperitoneally injected with 4x10⁶ human peripheral blood mononuclear cells (PBMC) from a normal, healthy donor. On day 0, the mice were intravenously administered 2x10⁶ BCMA*CD38⁺MOLP-8 human multiple myeloma tumor cells that were engineered to also express firefly luciferase (MOLP-8-luciferase cells). The mice (n=4-5 per group) were then immediately administered either a CD3-binding control bispecific Ab or a BCMAxCD3 bispecific antibody (REGN5458; US Patent Application Publication 20200024356) at 0.4 mg/kg, in combination with a 4-1 BB-binding control bispecific Ab (1+2 format) or a CD38x4-1BB bispecific antibody (1+2 format; REGN9686) at 4 mg/kg. The mice were administered these antibodies twice more on days 7 and 14, for a total of three doses. Tumor growth was assessed over 53 days by measuring tumor bioluminescence (BLI) in anesthetized animais. As a positive control, a group of mice (n=5) was given only MOLP-8-luciferase cells and PBMCs, but not antibody (PBS-treated group). In order to measure background BLI levels, a group of mice (n=5) were untreated and did not receive tumors, PBMC, or antibody (No Tumor group).

[0311] These studies demonstrate that while BCMAxCD3 bispecific antibody monotherapy demonstrates only modest anti-tumor efficacy and CD38x4-1BB 1+2 bispecific antibody monotherapy demonstrates little/no anti-tumor activity, combination treatment with BCMAxCD3 bispecific antibody plus CD38x4-1BB 1+2 bispecific antibody results in more potent, combinatorial anti-tumor efficacy that is superior to either therapy alone.

Implantation and measurement of xenogenic tumors

[0312] On day-11, immunodeficient NOD.Cg-Prkdc^scidll2rg^tr,’^1w,!/SzJ (NSG) mice were intraperitoneally injected with 4x10⁶ human peripheral blood mononuclear cells (PBMC) from a normal, healthy donor. On day 0, the mice were intravenously administered 2x10⁶ BCMA⁺CD38⁺

MOLP-8 human multiple myeloma tumor cells that were engineered to also express firefiy luciferase (MOLP-8-luciferase cells). The mice (n=4-5 per group) were then immediately administered either a CD3-binding control bispecific antibody or a BCMAxCD3 bispecific antibody (REGN5458) at 0.4 mg/kg, in combination with a 4-1BB-binding control bispecific antibody (1+2 format) or a CD38x4-1 BB bispecific antibody (1+2 format; REGN9686) at 4 mg/kg. The mice were administered these Abs twice more on days 7 and 14, for a total of three doses. Tumor growth was assessed over 53 days by measuring tumor bioluminescence (BLI) in anesthetized animais. As a positive control, a group of mice (n=5) was given only MOLP-8-luciferase cells and PBMCs, but not antibody (PBS-treated group). In order to measure background BLI levels, a group of mice (n=5) were untreated and did not receive tumors, PBMC, or antibody (No Tumor group).

Measurement ofxenocienic tumor growth

[0313] 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 this injection, BLI imaging of the mice was performed under isoflurane anesthésia using the Xenogen IVIS System. Image acquisition was carried out with the field of view at D, subject height of 1.5 cm, and medium binning level with automatic exposure time determined by the Living Image Software. BLI signais were extracted using Living Image software: régions of interest were drawn around each tumor mass and photon intensities were recorded as total flux (photons/second - p/s).

Results:

[0314] Tables 23-32 provide the results of the treatment combinations on tumor burden and subject survival at 6, 10, 13, 17, 20, 24, 27, 31,34, and 38 days after administration of human multiple myeloma tumor cells. Figure 3 is a graphical représentation of the data shown in the tables over the 38 days. Figure 4A illustrâtes tumor burden over time in the mice treated with PBS relative to mice that received no tumor cells; Figure 4B illustrâtes tumor burden over time in the mice treated with CD3-bindrng control bsAb (0.4mg/kg) + 4-1BB-binding control bsAb (4mg/kg) relative to mice that received no tumor cells; Figure 4C illustrâtes tumor burden over time in the mice treated with CD3binding control bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg) relative to mice that received no tumor cells; Figure 4D illustrâtes tumor burden over time in the mice treated with BCMAxCD3 bsAb (0.4mg/kg) + 4-1BB-binding control bsAb (4mg/kg) relative to mice that received no tumor cells;

Figure 4E illustrâtes tumor burden over time in the mice treated with BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)relative to mice that received no tumor cells.

[0315] BCMAxCD3 monotherapy: BCMAxCD3 bsAb (REGN5458) plus 4-1BB-binding control bsAb provided some anti-tumor efficacy, with mean BLI readings reduced compared to mice receiving CD3-binding control bsAb plus 4-1BB-binding control bsAb p<0.0001 on day 24 and p=0.0015 on day 26 by 2-way ANOVA analysis.

[0316] CD38x4-1 BB monotherapy: Treatment with CD3-binding control bsAb plus CD38x4-1BB 1+2 bsAb (REGN9686) did not significantly reduce mean BLI readings compared to mice receiving CD3-binding control bsAb plus 4-1 BB-binding control bsAb.

[0317] BCMAxCD3 + CD38x4-1BB 1+2: The combination of BCMAxCD3 bsAb (REGN5458) plus CD38x4-1BB 1+2 bsAb (REGN9686) resulted in mean BLI readings that were lowerthan mice receiving BCMAxCD3 bsAb plus 4-1 BB-binding control bsAb (p<0.0001 on day 38 by 2-way ANOVA analysis).

[0318] Thus, these studies demonstrate that while BCMAxCD3 bsAb monotherapy demonstrates only modest anti-tumor efficacy and CD38x4-1 BB 1 +2 bsAb monotherapy demonstrates little/no anti-tumor activity, combination treatment with BCMAxCD3 bsAb plus CD38x4-1BB 1+2 bsAb results in more potent, combinatorial anti-tumor efficacy that is superior to either therapy alone.

Table 23: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus CD38x4-1 BB 1 +2 bsAb - Day 6

Antibody T reatment	Tumor Burden Mean Total Flux (p/s) on Day 6	Total Flux SEM on Day 6	Number of mice still alive on day 6
PBS vehicle	8.67E+05	4.66E+04	5
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	8.80E+05	4.38E+04	5
CD3-binding control bsAb (0.4mg/kg) + CD38X4-1BB (4mg/kg)	7.81E+05	5.36E+04	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	8.40E+05	4.47E+04	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	7.82E+05	2.34E+04	5
No Tumor (Background BLI)	9.54E+05	3.42E+04	5

Table 24: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus CD38x4-1 BB 1 +2 bsAb - Day 10

Antibody Treatment	Tumor Burden Mean Total Flux (p/s) on Day 10	Total Flux SEM on Day 10	Number of mice still alive on day 10
PBS vehicle	1Ό1Ε+06	4.01 E+04	5
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	8.84E+05	2.98E+04	5
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	7.69E+05	3.04E+04	5

BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	8.96E+05	4.41 E+04	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg)	8.49E+05	4.95E+04	5
No Tumor (Background BLI)	9.41 E+05	3.62E+04	5

Table 25: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus CD38x4-1 BB 1 +2 bsAb - Day 13

Antibody Treatment	Tumor Burden Mean Total Flux (p/s) on Day 13	Total Flux SEM on Day 13	Number of mice still alîve on day 13
P BS vehicle	1.24E+06	1.07E+05	5
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	1.01 E+06	1.60E+05	5
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	7.67E+05	2.92E+04	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	7.20E+05	3.41 E+04	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg)	6.37E+05	2.39E+04	5
No Tumor (Background BLI)	7.88E+05	3.52E+04	5

Table 26: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus CD38x4-1 BB 1 +2 bsAb - Day 17

Antibody Treatment	Tumor Burden Mean Total Flux (p/s) on Day 17	Total Flux SEM on Day 17	Number of mice still alive on day 17
PBS vehicle	6.16E+06	1.51 E+06	5
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	3.81 E+06	1.07E+06	5
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	1.84E+06	3.30E+05	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	6.74E+05	7.70E+04	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	7.69E+05	5.96E+04	5
No Tumor (Background BLI)	7.63E+05	4.76E+04	5

Table 27: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus CD38x4-1BB 1+2 bsAb - Day 20

Antibody Treatment	Tumor Burden Mean Total Flux (p/s) on Day 20	Total Flux SEM on Day 20	Number of mice still alive on day 20
PBS vehicle	1.10E+07	1.70E+06	5
CD3-binding control bsAb (0.4mg/kg) +	1.06E+07	5.11 E+06	5

4-1 BB-binding control bsAb (4mg/kg)
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	3.81E+06	5.67E+05	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	7.88E+05	3.78E+04	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg)	7.36E+05	2.41 E+04	5
No Tumor (Background BLI)	8.52E+05	1.57E+04	5

Table 28: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus

CD38x4-1 BB 1 +2 bsAb - Day 24

Antibody T reatment	Tumor Burden Mean Total Flux {p/s) on Day 24	Total Flux SEM on Day 24	Number of mice still alive on day 24
PBS vehicle	2.44E+07	4.43E+06	5
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	3.60E+07	1.18E+07	5
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1 BB (4mg/kg)	1 82E+07	6.39E+06	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	1.14E+06	4.30E+05	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	7.15E+05	1.19E+04	5
No Tumor (Background BLI)	7.91 E+05	2.05E+04	5

Table 29: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus

CD38x4-1 BB 1 +2 bsAb - Day 27

Antibody Treatment	Tumor Burden Mean Total Flux (p/s) on Day 27	Total Flux SEM on Day 27	Number of mice still alive on day 27
PBS vehicle	4.21 E+07	7.71 E+06	4
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	2.67E+07	7.65E+06	2
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	6.11 E+07	3.02E+07	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	1 47E+06	5.55E+05	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	6.92E+05	5.29E+04	5
No Tumor (Background BLI)	8.85E+05	6.27E+04	5

Table 30: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus CD38x4-1BB 1+2 bsAb - Day 31

Antibody Treatment

Tumor Burden Mean Total Flux (p/s) on

Total Flux SEM on Day 31

Number of mice still alive on day 31

	Day 31
PBS vehicle	No mice alive	N/A	0
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	7.95E+07	0.00E+00	1
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	1.15E+08	5.50E+07	5
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	3.91 E+06	2.35E+06	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38X4-1BB (4mg/kg)	8.08E+05	8.44E+04	5
No Tumor (Background BLI)	8.65E+05	3.41 E+04	5

Table 31: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus

CD38x4-1 BB 1 +2 bsAb - Day 34

Antibody T reatment	Tumor Burden Mean Total Flux (p/s) on Day 34	Total Flux SEM on Day 34	Number of mice still alive on day 34
PBS vehicle	No mice alive	N/A	0
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	No mice alive	N/A	0
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	2.24E+08	1.60E+08	2
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	3.25E+06	1.75E+06	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	9.27E+05	1.26E+05	5
No Tumor (Background BLI)	9.75E+05	2.55E+04	5

Table 32: Anti-Tumor Efficacy Through Combination Treatment with BCMAxCD3 bsAb Plus

CD38x4-1BB 1+2 bsAb - Day 38

Antibody Treatment	Tumor Burden Mean Total Flux (p/s) on Day 38	Total Flux SEM on Day 38	Number of mice still alive on day 38
PBS vehicle	No mice alive	N/A	0
CD3-binding control bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	No mice alive	N/A	0
CD3-binding control bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	No mice alive	N/A	0
BCMAxCD3 bsAb (0.4mg/kg) + 4-1 BB-binding control bsAb (4mg/kg)	2.12E+07	1.11E+07	4
BCMAxCD3 bsAb (0.4mg/kg) + CD38x4-1BB (4mg/kg)	1 79E+06	1.13E+06	4
No Tumor (Background BLI)	8.22E+05	7.30E+04	5

Example 12: Bîacore Binding Kinetics of Anti-CD38x4-1BB Antigen-Binding Molécules.

[0319] Binding kinetics of the anti-CD38x4-1 BB antibodies were determined by antibody capture format Biacore binding kinetics of anti-CD38x4-1BB 1+2 antibodies binding to monomeric and dimeric human 4-1 BB reagents and antigen capture format Biacore binding kinetics of anti CD38x41BB 1+2 antibodies binding to dimeric human 4-1 BB reagent. Both experiments were performed at 25°C.

Antibody Capture Format Method:

[0320] Equilibrium dissociation constants (Kd values) for human 4-1 BB expressed with a C-terminal myc-myc-hexahistidine tag (h4-1BB.mmH, REGN3584) or human 4-1 BB expressed with a Cterminal murine Fc tag (h4-1 BB.mFc, REGN3585) binding to purified anti-CD38x4-1BB 1+2 antibodies were determined using a real-time surface plasmon résonance biosensor using a Biacore 8k instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567). Ail Biacore binding studies were performed in a buffer composed of 0.01 M HEPES pH 7.4, 0.15M NaCI, 3mM EDTA, 0.05% v/v Surfactant P20 (HBS-EP running buffer). Different concentrations of h4-1BB.mmH (REGN3584) prepared in HBS-EP running buffer (ranging from 100 to 6.25nM in 4-fold serial dilutions) or h41 BB.mFc (REGN3585) prepared in HBS-EP running buffer (ranging from 100 to 1,56nM in 4-fold serial dilutions) were injected overthe captured anti-CD38x4-1 BB 1+2 antibodies at a flow rate of 30pL/minute. Antibody-reagent association was monitored for 5 minutes while dissociation in HBSEP running 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 20mM phosphoric acid. AH binding kinetics experiments were performed at 25°C. Results are presented in Tables 33 and 34.

Antigen Capture Format Method:

[0321] Equilibrium dissociation constants (Ko values) for human 4-1 BB expressed with a C-terminal murine Fc tag (h4-1 BB.mFc, REGN3585) binding to purified anti-CD38x4-1 BB 1+2 antibodies were determined using a real-time surface plasmon résonance biosensor using a Biacore 4000 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a polyclonal rabbit anti-mouse Fc antibody (GE, # BR-1008-38). Ail Biacore binding studies were performed in a buffer composed of 0.01 M HEPES pH 7.4, 0.15M NaCI, 3mM EDTA, 0.05% v/v Surfactant P20 (HBS-EP running buffer). Different concentrations of CD38x4-1BB 1+2 constructs prepared in HBSEP running buffer (ranging from 100 to 6.25nM in 4-fold serial dilutions) were injected over the h41 BB.mFc (REGN3585) captured surface at a flow rate of 30pL/minute. Antibody-reagent association was monitored for 5 minutes while dissociation in HBS-EP running buffer was monitored for 10 minutes. At the end of each cycle, the h4-1 BB.mFc capture surface was regenerated using a 40 sec injection of 10mM glycine, pH 1.5. AH binding kinetics experiments were performed at 25°C. Results are presented in Table 35.

Data Analysis of Both Formats:

[0322] Kinetic association (k_a) and dissociation (kd) rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Cytiva Insight curve fitting software. Binding dissociation equilibrium constants (Kd) and dissociative half-lives (1½) were calculated from the kinetic rate constants as:

kd In(2)

[0323] K_o (M) = — , and (min) = ka 60*kd

Table 33: Kinetic and Equilibrium Binding Parameters of Monomeric Human 4-1 BB to Surfacecaptured anti-CD38x4-1BB 1+2 Antibodies at 25°C

REGN#/ Ab PID#	Ab Capture (RU)	lOOnM h4lBB.mmH Bind (RU)	ka (1/Ms)	kd (1/s)	KD (M)	tl/2 (min)
REGN7633	394.6 ± 1.4	24.9*	IC**	IC**	IC**	IC**
REGN7647	506.813.1	86.1	3.24E+05	5.33E-03	1.65E-08	2.2
REGN7650	458.9 ± 0.4	41.9	2.62E+05	5.35E-03	2.04E-08	2.2
REGN9682	607.1 ± 1.1	106.8	3.58E+05	4.94E-03	1.38E-08	2.3
REGN9686	606.9 ±0.5	101.3	3.19E+05	4.74E-03	1.48E-08	2.4
*Dose-dependent binding oi	^: h4-1bb.mmH	was observed

**IC: Observed binding did not fit to the binding simulation model and no binding kinetic parameters were determined under the current experimental conditions.

Table 34: Kinetic and Equilibrium Binding Parameters of Dimeric Human 4-1 BB to Surfacecaptured anti-CD38x4-1BB 1+2 Antibodies at 25°C

REGN#/Ab PID#	Ab Capture (RU)	100nM h4IBB.mFc Bind (RU)	ka(1/Ms)	kd (1/s)	KD (M)	t1/2 (min)
REGN 7633	395.4 ±0.3	82.1	2.74E+05	2.51E04	9.17E- 10	46.0
REGN7647	505.3 ±1.4	223.6	1.95E+05	2.31E-	1.18E-	50.0

				80	04	09
REGN7650	458.3±0.6	152.0	1.02E+05	1.22E- 04	1.20E09	94.9
REGN9682	603.2 1 1.7	283.3	2.06E+05	1.77E04	8.60E- 10	65.3
REGN9686	600.212.4	267.4	1.71E+05	2.05E- 04	1.20E09	56.3

Table 35: Kinetic and Equilibrium Binding Parameters of anti-CD38x4-1 BB 1 +2 Antibodies to Surface-captured Dimeric Human 4-1 BB at 25°C

	REGN #/Ab PID #	Ag+ Capture	100nM Ab Bind (RU)	ka (1/Ms)	kd (1/s)	KD (M)	t1/2 (min)
	REGN7633	132.010.4	285.3	1.86E+05	2.37E03	1.27E08	4.9
	REGN7647	142.2 10.5	205.8	6.26E+05	3.51 E04	5.60E- 10	32.9
10	REGN7650	127.510.8	265.7	2.12E+05	1.69E- 04	7.97E- 10	68.4
	REGN9682	116.810.4	233.7	4.30E+05	3.08E04	7.16E- 10	37.6
	REGN9686	127.410.8	232.3	4.70E+05	3.37E- 04	7. ΠΕΙΟ	34.3

[0324] The present invention is not to be limited in scope by the spécifie embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended daims.

Claims

1. A bispecific antigen-binding molécule comprising:

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

2. The bispecific antigen-binding molécule of claim 1, wherein:

(a) R1 and R2 bind to the same epitope on 4-1 BB; or (b) R1 and R2 bind to different epitopes on 4-1 BB; and/or (c) R1 and R2 are connected via a peptide linker; optionally wherein the peptide linker comprises a peptide sequence of (GGGGS)n, wherein n is 1 to 6.

3. The bispecific antigen-binding molécule of either claim 1 or 2, wherein the first antigen-binding arm comprises:

(a) three CDRs of a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40; and three CDRs of a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48; and/or (b) three heavy chain complementarity determining régions (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, and three light chain complementarity determining régions (LCDR1-LCDR2-LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54, respectively; and/or (c) a HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 40; and a LCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

4. The bispecific antigen-binding molécule of any one of daims 1-3, wherein R1 comprises:

(a) three CDRs of a HCVR (R1-HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94; and three CDRs of a LCVR (R1-LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48; and/or (b) three heavy chain complementarity determining régions (R1-HCDR1-R1-HCDR2-R1-HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16, 34-36-38, 64-66-68, 74-76-78, 88-90-92, and 96-98-100, respectively; and three light chain complementarity determining régions (R1-LCDR1-R1-LCDR2-R1-LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24 and 50-52-54, respectively; and/or (c) a HCVR (R1-HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs; 10, 32, 62, 72, 86 and 94; and a LCVR (R1-LCVR) comprising the amino acid sequence selected from the group consisting of SEQ IDNOs: 18 and 48.

5. The bispecific antigen-binding molécule of any one of claims 1-4, wherein R2 comprises:

(a) three CDRs of a HCVR (R2-HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 32, 62, 72, 86 and 94; and three CDRs of a LCVR (R2-LCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48; and/or (b) three heavy chain complementarity determining régions (R2-HCDR1-R2-HCDR2-R2-HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14-16, 34-36-38, 64-66-68, 74-76-78, 88-90-92, and 96-98-100, respectively; and three light chain complementarity determining régions (R2-LCDR1-R2-LCDR2-R2-LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22-24, and 50-52-54, respectively; and/or (c) a HCVR (R2-HCVR) comprising the amino acid sequence selected from the group consisting of

SEQ ID NOs: 10, 32, 62, 72, 86 and 94; and a LCVR (R2-LCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 48.

6. The bispecific antigen-binding molécule of any one of daims 1-5, 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 the second antigen-binding arm comprises:

(i) a first antigen-binding région (R1) comprising three CDRs of R1-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 62 and 72; and three CDRs of R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding région (R2) comprising three CDRs of R2-HCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 86, and 94; and three CDRs of R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and/or (b) the first antigen-binding arm comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 42-44-46-5052-54, respectively; and the second antigen-binding arm comprises:

(i) a first antigen-binding région (R1) comprising R1-HCDR1-R1-HCDR2-R1-HCDR3-R1LCDR1-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 (ii) a second antigen-binding région (R2) comprising R2-HCDR1-R2-HCDR2-R2-HCDR3R2-LCDR1-R2-LCDR2-R2-LCDR3 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 88-90-92-5052-54, respectively; and/or (c) 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 the second antigen-binding arm comprises:

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

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

7. The bispecific antigen-binding molécule of any one of daims 1-6, wherein:

(a) the first antigen-binding arm comprises a HCVR comprising the amino acid sequence of SEQ ID 10 NO: 40 and a LCVR comprising the amino acid sequence of SEQ ID NO: 48; and the second antigen-binding arm comprises:

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence of SEQ ID NO: 32; and R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and

15 (ii) a second antigen-binding région (R2) comprising R2-HCVR comprising the amino acid sequence of SEQ ID NO: 86; and R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48; or (b) 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

20 the second antigen-binding arm comprises:

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence of SEQ ID NOs: 72; and R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding région (R2) comprising R2-HCVR comprising the amino acid 25 sequence of SEQ ID NO: 94; and R2-LCVR comprising the amino acid sequence of SEQ ID

NO: 48; or (c) 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 the second antigen-binding arm comprises:

(i) a first antigen-binding région (R1) comprising R1-HCVR comprising the amino acid sequence of SEQ ID NOs: 62; and R1-LCVR comprising the amino acid sequence of SEQ ID NO: 48; and (ii) a second antigen-binding région (R2) comprising R2-HCVR comprising the amino acid sequence of SEQ ID NO: 62; and R2-LCVR comprising the amino acid sequence of SEQ ID NO: 48.

8. The bispecific antigen-binding molécule of any one of daims 1-7, wherein the molécule is a bispecific antibody, wherein the bispecific antibody comprises a heavy chain constant région of IgG 1 or lgG4 isotype; optionally wherein the bispecific antibody comprises a first heavy chain comprising the HCVR of the first antigen-binding arm, and a second heavy chain comprising R1HCVR and R2-HCVR of the second antigen-binding arm, wherein the second heavy chain comprises the mutations H435R and Y436F (EU numbering).

9. The bispecific antibody of claim 8 comprising:

(a) a first heavy chain comprising the HCVR of the first antigen-binding arm paired with a light chain comprising the LCVR of the first antigen-binding arm, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 58 and the light chain comprises the amino acid sequence of SEQ ID NO: 60; and (b) R1-HCVR and R2-HCVR of the second antigen-binding arm 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 selected from the group consisting of SEQ ID NO: 56, 70, 80, 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.

10. The bispecific antibody of either claim 8 or 9, wherein:

(a) the first antigen-binding arm that specifically binds human CD38 comprises a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and the second antigen-binding arm that specifically binds human 4-1 BB comprises 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; or (b) the first antigen-binding arm that specifically binds human CD38 comprises a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ

ID NO: 60; and the second antigen-binding arm that specifically binds human 4-1 BB comprises a heavy chain comprising the sequence of SEQ ID NO: 70, a first light chain comprising the sequence of SEQ ID NO: 60, and a second light chain comprising the sequence of SEQ ID NO: 60; or (c) the first antigen-binding arm that specifically binds human CD38 comprises a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and the second antigen-binding arm that specifically binds human 4-1 BB comprises 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; or (d) the first antigen-binding arm that specifically binds human CD38 comprises a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60, and the second antigen-binding arm that specifically binds human 4-1 BB comprises 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 (e) the first antigen-binding arm that specifically binds human CD38 comprises a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and the second antigen-binding arm that specifically binds human 4-1 BB comprises 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.

11. A bispecific antigen-binding molécule comprising a first antigen binding arm that binds specifically to CD38 and a second antigen-binding arm that binds specifically to 4-1 BB, wherein:

(i) a first antigen-binding région (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 région (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 (R2LCVR) comprising the amino acid sequence of SEQ ID NO: 48;

optionally wherein the moiecule is a bispecific antibody; and 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; and 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.

12. The bispecific antigen-binding moiecule of claim 11, wherein:

(a) the first antigen-binding arm that specifically binds human CD38 comprises a heavy chain comprising the sequence of SEQ ID NO: 58, and a light chain comprising the sequence of SEQ ID NO: 60; and (b) the second antigen-binding arm that specifically binds human 4-1 BB 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 ofSEQ IDNO: 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 ofSEQ ID NO: 60.

i

13. A pharmaceutical composition comprising the bispecific antigen-binding molécule of any one of

I daims 1-12, and a pharmaceutically acceptable carrier.

14. An isolated nucleic acid molécule comprising;

(a) a nucleic acid sequence encoding for HCVR of the first antigen-binding arm of the bispecific

5 antigen-binding molécule of any one of daims 1-12;

(b) a nucleic acid sequence encoding for R1-HCVR and R2-HCVR of the second antigen-binding arm of the bispecific antigen-binding molécule of any one of daims 1-12; and/or (c) a nucleic acid sequence encoding for LCVR of the bispecific antigen-binding molécule of any one of daims 1-12.

10

15. An expression vector containing the isolated nucleic acid molécule of claim 14.

16. A host cell containing the expression vedor of claim 15; optionally wherein the host cell is E.coli or CHO cell.

17. A method of producing a multispecific antigen binding molécule, the method comprising growing the host cell of claim 16 under conditions permitting production of the multispecific antigen

15 binding molécule, wherein the host cell comprises:

(a ) a first nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain variable région (HCVR) of a multispecific antigen binding molécule antigen binding arm A1, a second nucleic acid molécule comprising a nucleic acid sequence encoding heavy chain variable régions (HCVRs) of a multispecific antigen binding molécule antigen binding arm A2, and a third

20 nucleic acid molécule comprising a nucleic acid sequence encoding a common light chain variable région (LCVR); or (b ) a first nucleic acid molécule comprising a nucleic acid sequence encoding a heavy chain of the multispecific antigen binding molécule antigen binding arm A1, a second nucleic acid molécule encoding a heavy chain of the multispecific antigen binding molécule antigen binding arm A2, and a 25 third nucleic acid molécule comprising a nucleic acid sequence encoding a common light chain.

18. Use of a multispecific antigen binding molécule of any one of daims 1 through 12, or a pharmaceutical composition of claim 13, in the manufacture of a médicament for inhibiting growth of a plasma cell tumor in a subject; optionally wherein the plasma cell tumor is multiple myeloma.

19. Use of a multispecific antigen binding molécule of any one of daims 1 through 12, or a

30 pharmaceutical composition of claim 13, in the manufacture of a médicament for inhibiting growth of a tumor in a subject, wherein the tumor is selected from the group consisting of multiple myeioma, lymphoma, B-cell leukemia, hepatocellular carcinoma, non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, glioma, or breast cancer, or another cancer charactehzed in part by having CD38+ cells.

20. Use of a multispecific antigen binding molécule of any one of daims 1 through 12, or a pharmaceutical composition of claim 13, in the manufacture of a médicament for treating a patient suffering from a BCMA-expressing B cell malignancy, 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-Hodgkiris lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, multiple myeioma, and Hodgkin’s lymphoma.

21. The use of any one of daims 18 through 20, further comprising administering a second therapeutic agent or therapeutic regimen, wherein the second therapeutic agent or therapeutic regimen is:

(a) an antibody that binds a plasma cell tumor;

(b) an anti-BCMA/anti-CD3 bispecific antigen binding molécule;

(c) an anti-CD20/anti-CD3 bispecific antigen binding molécule;

(d) an agonist of CD28 or an agonist of 4-1BB; or (e) a chemotherapeutic drug, DNA alkylators, immunomodulators, protéasome inhibitors, histone deacetylase inhibitors, radiotherapy, a stem cell transplant, a different bispecific antibody that interacts with a different tumor cell surface antigen and a T cell or immune cell antigen, an antibody drug conjugale, a bispecific antibody conjugated to an anti-tumor agent, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 checkpoint inhibitor, a CD28 agonist, a 4-1 BB agonist, an anti-BCMA/anti-CD3 bispecific antigen binding molécule, an anti-CD20/anti-CD3 bispecific antigen binding molécule, a cancer vaccine, an oncolytic virus, an immunocytokine, a CD22 inhibitor, IL4 inhibitor, IL6 inhibitor, a Tcell comprising a chimeric antigen receptor (CAR-Tcell), or combinations thereof.

22. Use of a multispecific antigen binding molécule of any one of daims 1 through 12, or a pharmaceutical composition of claim 13, in the manufacture of a médicament for treating a patient suffering from a CD38+ tumor and/or a BCMA-expressing tumor in combination with a PD-1 inhibitor, wherein the PD-1 inhibitor is an anti-PD-1 antibody or antigen binding fragment thereof, or an anti-PD-L1 antibody or antigen-binding fragment thereof;

optionally wherein the anti-PD-1 inhibitor is selected from cemiplimab, nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab;

further wherein the PD-1 inhibitor is cemiplimab.

23. Use of a multispecific antigen binding molécule of any one of daims 1 through 12, or a

5 pharmaceutical composition of daim 13, in the manufacture of a médicament for treatment of a disease or disorder assodated with expression of CD38, CD20, and/or BCMA, wherein the disease or disorder is cancer;

optionally wherein the cancer is multiple myeloma, lymphoma, B-cell leukemia, hepatocellular cardnoma, non-small cell lung cancer, melanoma, pancreatic dudal adenocarcinoma, glioma, or 10 breast cancer, or another cancer charaderized in part by having CD38+ cells;

further wherein the multispecific antigen binding molécule or pharmaceutical composition is for use in combination with an anti-PD-1 antibody or antigen binding fragment thereof, wherein the multispecific antigen binding molécule, or pharmaceutical composition is injected intravenousiy, intramuscularly or subcutaneously.