TW202502811A - Immunostimulatory antigen binding molecules that specifically bind to bcma - Google Patents

Abstract

The present invention relates to new humanized antibodies that bind to B-cell maturation antigen (BCMA), and to immunostimulatory antigen binding molecules compromising these BCMA antibodies, in particular to BCMA targeting 4-1BBL trimer-containing antigen binding molecules, methods for their production, as well as their use in the treatment of cancer.

Description

Immunostimulatory antigen binding molecules that specifically bind to BCMA

The present invention relates to novel humanized antibodies that bind to B cell maturation antigen (BCMA), and to immunostimulatory antigen-binding molecules comprising these BCMA antibodies, in particular to antigen-binding molecules containing 4-1BBL trimers that target BCMA, methods for producing the same, and use thereof in treating cancer.

Within the group of hematologic malignancies, multiple myeloma (MM) represents a large, dynamic, competitive disease area and is a disease that presents significant ongoing unmet medical needs and is still not considered curable. Multiple myeloma (MM) is a plasma cell malignancy and the second most common hematologic disorder, primarily affecting the elderly population. Despite significant improvements in the diagnosis, classification, and treatment of MM, patients with high-risk disease have not benefited from advances in treatment and are often primary refractory or experience early relapse. In addition, most patients with low-risk MM eventually develop drug-resistant strains, become difficult to treat, and transition to high-risk disease. Therefore, novel therapies with different mechanisms of action and optimization of treatment efficacy are key to reducing the risk of disease relapse and deepening the durability of response (Caraccio C, Krishna S., Phillips DJ. and Schürch CM, Front Nutrition. 2020, 11, 501).

CAR-T cells are one of the promising new treatment modalities: early data from these agents show significant efficacy and are expected to bring significant improvements in the treatment of MM. However, as has been seen with CAR-T therapy for the treatment of non-Hodgkin's lymphoma (NHL), these treatments are associated with similar disadvantages to the agents already found for the treatment of NHL - in particular, early toxicity from cytokine release syndrome and/or related neurotoxicity and limitations in accessibility.

T cell bispecific antibodies (TCBs) offer another promising immunotherapy approach by binding and stimulating CD3 independently of antigen presentation on class I major histocompatibility complexes (MHC). By simultaneously binding to tumor-specific antigens (TAs), T cells are redirected to TA-positive MM cells to induce their specific lysis. Although some of these TCBs have demonstrated significant antitumor efficacy in patients, their activity still needs to be optimized to ultimately produce robust, lasting cures.

The emerging class of bispecific antibodies that mimic signal 2 includes molecules that target both a tumor-specific antigen (TA) and a co-stimulatory receptor such as OX40, GITR, CD137, ICOS, or CD28. Combining these bispecific antibodies with the emerging class of TCBs could provide an effective “synthetic off-the-shelf CAR T-cell-like” antibody therapy to further deepen and prolong TCB-mediated anti-tumor responses in patients.

B cell maturation antigen (BCMA), a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 (TNFRSF17), is expressed at significantly higher levels in all patient MM cells, but not in other normal tissues except normal plasma cells. BCMA chimeric antigen receptor (CAR) T cells have shown significant clinical activity in patients with relapsed/refractory MM (RRMM) who have received at least three prior therapies, including proteasome inhibitors and immunomodulatory drugs (IMiDs). Other approaches, including anti-BCMA antibody-drug conjugates, have also achieved remarkable clinical responses in patients who have failed at least three prior lines of therapy, including anti-CD38 antibodies, proteasome inhibitors, and immunomodulatory drugs (Cho et al., Front Immunolog. 2018, 9, 1821). However, there remains a need for better treatment options for multiple myeloma.

4-1BB (CD137), a member of the TNF receptor superfamily, was first identified as an inducible molecule expressed by activated T cells (Kwon and Weissman, 1989, Proc Natl Acad Sci USA 86 , 1963-1967). Subsequent studies have shown that many other immune cells also express 4-1BB, including NK cells, B cells, NKT cells, monocytes, neutrophils, mast cells, dendritic cells (DCs), and cells of non-hematopoietic origin, such as endothelial cells and smooth muscle cells (Vinay and Kwon, 2011, Cell Mol Immunol 8 , 281-284). The expression of 4-1BB in different cell types is mainly induced and driven by various stimulation signals, such as T cell receptor (TCR) or B cell receptor triggering, and signal transduction induced by co-stimulatory molecules or receptors of proinflammatory cytokines (Diehl et al., 2002, J Immunol 168 , 3755-3762; Zhang et al., 2010, Clin Cancer Res 13 , 2758-2767).

The 4-1BB ligand (4-1BBL or CD137L) was identified in 1993 (Goodwin et al., 1993, Eur J Immunol 23 , 2631-2641). It has been shown that the expression of 4-1BBL is restricted to professional antigen presenting cells (APCs), such as B cells, DCs, and macrophages. Inducible expression of 4-1BBL is a property of T cells (both T cell subsets) and endothelial cells (Shao and Schwarz, 2011, J Leukoc Biol 89 , 21-29).

Activation of multiple signaling cascades within T cells (both CD4+ and CD8+ subsets) through co-stimulation of the 4-1BB receptor (e.g., via 4-1BBL ligation) results in a potent enhancement of T cell activation. In combination with TCR triggering, agonistic 4-1BB-specific antibodies can enhance T cell proliferation, stimulate lymphokine secretion, and reduce the sensitivity of T lymphocytes to activation-induced cell death (Snell et al., 2011, Immunol Rev 244 , 197-217). This mechanism was further developed as the first proof of concept for cancer immunotherapy. In preclinical models, administration of agonistic antibodies against 4-1BB in tumor-bearing mice resulted in effective antitumor effects (Melero et al., 1997, Nat Med 3 , 682-685). In addition, in hematological tumors, single immunotherapy with anti-CD137 antibodies can rescue 40% to 50% of mice in preclinical models of multiple myeloma (Murillo et al., 2008, Clin. Cancer Res. 2008, 14(21), 6895). Subsequently, increasing evidence showed that 4-1BB was effective as an antitumor agent primarily when administered in combination with other immunomodulatory compounds, chemotherapeutic agents, tumor-specific vaccination, or radiation therapy (Bartkowiak and Curran, 2015, Front Oncol 5 , 117).

Signaling of the TNFR superfamily requires cross-linking of the trimeric ligand for receptor binding, as does the 4-1BB agonist antibody that requires wild-type Fc binding (Li and Ravetch, 2011, Science 333 , 1030-1034). However, systemic administration of 4-1BB-specific agonist antibodies with a functionally active Fc domain resulted in an influx of CD8+ T cells associated with hepatotoxicity (Dubrot et al., 2010, Cancer Immunol Immunother 59 , 1223-1233). Consistently, an Fc-active 4-1BB agonist Ab (BMS-663513) (NCT00612664) caused grade 4 hepatitis, leading to trial termination (Simeone and Ascierto, 2012, J Immunotoxicol 9 , 241-247). Therefore, targeted delivery of 4-1BB agonists to the tumor side may be an option to elicit antitumor efficacy while avoiding systemic toxicity. There is a strong need to provide BCMA-targeted 4-1BB agonists with favorable properties.

The present invention describes novel BCMA-targeted 4-1BB agonists that achieve tumor-dependent T cell activation without systemic toxicity. The BCMA-4-1BBL antigen-binding molecules of the present invention are constructed using the following: an IgG fusion protein, which is composed of a split-trimeric 4-1BB ligand comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 (which corresponds to the natural ligand); and a portion that recognizes the tumor-targeting antigen of BCMA, which is fused to an Fc domain (silent Fc domain) composed of a first unit and a second unit capable of stable binding, and the Fc domain contains one or more amino acid substitutions that reduce the binding affinity and/or effector function of the antigen-binding molecule to the Fc receptor. Fc receptor-mediated cross-linking is thereby abolished and tumor-specific activation is achieved by cross-linking through binding of a second antigen binding domain capable of binding specifically to BCMA. Thus, non-specific FcγR-mediated cross-linking responsible for Fc-mediated toxicity is replaced by BCMA-targeted MM cell-specific cross-linking. The desired mode of action of these molecules is to enhance the effector function of tumor-infiltrating T cells or NK cells after activation by tumor-targeted T cell bispecific antibodies or ADCC antibodies, respectively. Therefore, these molecules are believed to have efficacy in the treatment of multiple myeloma.

In particular, the present invention relates to novel antigen-binding molecules that bind to B cell maturation antigen (BCMA), in particular to novel optimized corresponding humanized BCMA antibodies, and to antigen-binding molecules containing 4-1BBL trimers targeting BCMA comprising such BCMA antibodies and their use in cancer treatment. The present invention further relates to methods for producing such molecules and to methods of use thereof.

BCMA-targeting 4-1BBL trimer-containing antigen binding molecules are active in the presence of BCMA-expressing tumors, contain natural human 4-1BB ligands, and should therefore raise fewer safety concerns than conventional 4-1BB agonistic antibodies or more artificial fusion proteins. The novel antigen binding molecules combine an anti-BCMA antigen binding domain with a moiety that is able to form a co-stimulatory 4-1BBL trimer and is sufficiently stable to be used pharmaceutically. The immunostimulatory antigen binding molecules of the present invention provide a trimer, and therefore biologically active, human 4-1BB ligand, although one of the trimerizing 4-1BBL extracellular domains is located on another polypeptide of the molecule in addition to the other two 4-1BBL extracellular domains.

Therefore, provided herein is an immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) a Fab molecule comprising (i) a heavy chain variable region ( _VH BCMA) comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 1 (GYTFTNYWMH), a CDR-H2 of SEQ ID NO: 2 (IIHPNSGSTNYNEKFQG), and a CDR-H3 of SEQ ID NO: 3 (GIYDYPFAY); and (ii) a light chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 5 (AASSLQS); and (c) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 8 (AASNLQS), and a CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); (b) a first polypeptide and a second polypeptide, which are linked to each other by a disulfide bond, The antigen-binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL connected to each other by a peptide linker, each extracellular domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, and the second polypeptide comprises one extracellular domain of 4-1BBL, the extracellular domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; and (c) an IgG Fc domain.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9. In one aspect, the immunostimulatory antigen binding molecule comprises three extracellular domains of 4-1BBL, each of which comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, in particular the amino acid sequence of SEQ ID NO: 9.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189 and SEQ ID NO: 190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence of SEQ ID NO: 11, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the antigen binding domain comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15, or (b) VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of _SEQ ID NO:16.

In one embodiment, the antibody that specifically binds to BCMA comprises an IgG Fc domain consisting of a first unit and a second unit. In one embodiment, the IgG Fc domain consisting of a first unit and a second unit is an IgG1 Fc domain. In one embodiment, the IgG Fc domain is a human Fc domain.

In one aspect, the IgG Fc domain comprises a modification that promotes association of the first unit and the second unit of the Fc domain. In one aspect, the Fc domain comprises a knob-in-hole modification. In one aspect, the first unit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second unit of the Fc domain comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index numbering).

In a further aspect, the IgG Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. In a specific aspect, the IgG Fc domain is of human IgG1 subclass and comprises amino acid mutations L234A, L235A and P329G (according to Kabat EU index numbering).

In one aspect, an immunostimulatory antigen-binding molecule as described herein comprises (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, fused at the C-terminus to the N-terminus of a CL domain; (aiii) a CL domain fused at the C-terminus to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (aiv) one of the subunits of an Fc domain (e.g., a first subunit); (b) a second polypeptide comprising: (bi) a second extracellular domain of SEQ ID NO: 9 or SEQ ID NO:10, fused at the C-terminus to the N-terminus of the CH1 domain, and (bii) the CH1 domain; (c) a third polypeptide comprising: (ci) a heavy chain of a Fab molecule, fused at the C-terminus to the N-terminus of another subunit of the Fc domain (e.g., the second subunit); and (cii) another subunit of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule.

In one aspect, the immunostimulatory antigen-binding molecule as described above is a molecule wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering).

In a specific aspect, the immunostimulatory antigen binding molecule as described herein comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:25.

In a specific aspect, the immunostimulatory antigen binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 23, and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 24. In a specific aspect, the immunostimulatory antigen binding molecule comprises (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 208, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 207, and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 24. In one aspect, the immunostimulatory antigen-binding molecule consists of: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:208, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:207, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24.

Also provided herein is an immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) a Fab molecule comprising (i) a heavy chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 27 (QITAKSNNYATYYADSVKG), and a CDR-H3 of SEQ ID NO: 28 (DGYH); and (b) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 29 (QITAKSNNYATYYAAPVKG), and a CDR-H3 of SEQ ID NO: 21 (DGYH). NO: 28 (DGYH) CDR-H3; and (ii) a light chain variable region (V _L BCMA) comprising a light chain complementation determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), a CDR-L2 of SEQ ID NO: 31 (NANSLHT) and a CDR-L3 of SEQ ID NO: 32 (EDTSKYPYT); (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL linked to each other by a peptide linker, each extracellular domain comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, and characterized in that the second polypeptide comprises an extracellular domain of 4-1BBL, the extracellular domain comprising SEQ ID NO: 9 or SEQ ID NO: 10. and (c) IgG Fc domain.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9. In one aspect, the immunostimulatory antigen binding molecule comprises three extracellular domains of 4-1BBL, each of which comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, in particular the amino acid sequence of SEQ ID NO: 9.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189 and SEQ ID NO: 190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence of SEQ ID NO: 11, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9.

In one aspect, an immunostimulatory antigen binding molecule as previously defined herein is provided, wherein the antigen binding domain comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 (VH2a) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34 (VL2a), or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 (VH1b) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:36 (VL1a).

In a specific aspect, the antigen-binding domain comprises: _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 (VH2a) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34 (VL2a).

In one aspect, the antibody that specifically binds to BCMA comprises an IgG Fc domain. In one aspect, the Fc domain is an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain.

In one aspect, the IgG Fc domain comprises a modification that promotes the association of the first unit and the second unit of the Fc domain. In one aspect, the IgG Fc domain comprises a knob-in-hole modification. In one aspect, the first unit of the IgG Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second unit of the IgG Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (according to Kabat EU index numbering).

In a further aspect, the IgG Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. In a specific aspect, the IgG Fc domain is of human IgG1 subclass and comprises amino acid mutations L234A, L235A and P329G (according to Kabat EU index numbering).

In one aspect, an immunostimulatory antigen-binding molecule as described herein comprises (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, fused at the C-terminus to the N-terminus of a CL domain; (aiii) a CL domain fused at the C-terminus to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (aiv) one of the subunits of an Fc domain (e.g., a first subunit); (b) a second polypeptide comprising: (bi) a second extracellular domain of SEQ ID NO: 9 or SEQ ID NO:10, fused at the C-terminus to the N-terminus of the CH1 domain, and (bii) the CH1 domain; (c) a third polypeptide comprising: (ci) a heavy chain of a Fab molecule, fused at the C-terminus to the N-terminus of another subunit of the Fc domain (e.g., the second subunit); and (cii) another subunit of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule.

In one aspect, the immunostimulatory antigen-binding molecule as described above is a molecule wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering).

In a specific aspect, the immunostimulatory antigen-binding molecule described herein comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 37, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 38; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 39, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

In a specific aspect, the immunostimulatory antigen-binding molecule described herein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 37, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 38.

In a further aspect, the present invention relates to an antibody that specifically binds to BCMA, wherein the antibody comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:16.

In another aspect, an antibody that specifically binds to BCMA is provided, wherein the antibody comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 33 (VH2a) and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 34 (VL2a), or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 35 (VH1b) and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 36 (VL1a).

According to another aspect of the invention, one or more isolated polynucleotides are provided, encoding an immunostimulatory antigen binding molecule or antibody as previously described herein. The invention further provides a vector, in particular an expression vector, comprising an isolated polynucleotide of the invention, and a host cell comprising an isolated nucleic acid or expression vector of the invention. In some aspects, the host cell is a eukaryotic cell, in particular a mammalian cell. In another aspect, a method of producing an immunostimulatory antigen binding molecule as previously described herein that specifically binds to BCMA or an antibody as previously described herein is provided, the method comprising the steps of: a) culturing a host cell as described above under conditions suitable for expression of the antigen binding molecule, and optionally b) recovering the antigen binding molecule. The present invention also encompasses immunostimulatory antigen-binding molecules that specifically bind to BCMA as produced by the methods of the present invention.

Further provided is a pharmaceutical composition comprising an immunostimulatory antigen binding molecule that specifically binds to BCMA as previously described herein and at least one pharmaceutically acceptable formulation. In one aspect, the pharmaceutical composition comprises an additional therapeutic agent.

Also contemplated are immunostimulatory antigen binding molecules that specifically bind to BCMA or pharmaceutical compositions comprising bispecific BCMA antibodies as described herein for use as medicaments.

In one aspect, an immunostimulatory antigen binding molecule or pharmaceutical composition as previously described herein that specifically binds to BCMA is provided for use in enhancing (a) T cell activation or (b) T cell effector function.

In one aspect, an immunostimulatory antigen binding molecule or pharmaceutical composition as previously described herein that specifically binds to BCMA is provided for use in treating a disease. In one aspect, the disease is cancer, in particular multiple myeloma (MM).

In one embodiment, an immunostimulatory antigen binding molecule or pharmaceutical composition that specifically binds to BCMA as previously described herein is provided for use in treating cancer, particularly multiple myeloma. In another embodiment, an immunostimulatory antigen binding molecule that specifically binds to BCMA as previously described herein is provided for use in treating cancer, wherein the use is for administration in combination with chemotherapy, radiation therapy, and/or other agents used in cancer immunotherapy. In one embodiment, an immunostimulatory antigen binding molecule that specifically binds to BCMA as previously described herein is used in treating cancer, wherein the use is for administration in combination with a T cell activating anti-CD3 bispecific antibody. In a particular aspect, the T cell activating anti-CD3 bispecific antibody is an anti-GPRC5D/anti-CD3 antibody.

In a further aspect, the present invention provides a method of inhibiting the growth of tumor cells in an individual, the method comprising administering to the individual an effective amount of an immunostimulatory antigen binding molecule that specifically binds to BCMA as previously described herein or a pharmaceutical composition of the present invention to inhibit the growth of tumor cells. In another aspect, the present invention provides a method of treating or delaying cancer in an individual, the method comprising administering to the individual an effective amount of an immunostimulatory antigen binding molecule that specifically binds to BCMA as previously described herein or a pharmaceutical composition of the present invention.

Also provided are uses of an immunostimulatory antigen binding molecule that bispecifically binds to BCMA as previously described herein in the manufacture of a medicament for treating a disease in a subject in need thereof, in particular in the manufacture of a medicament for treating cancer, and methods of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an immunostimulatory antigen binding molecule that specifically binds to BCMA of the invention in a pharmaceutically acceptable form. In a specific aspect, the disease is cancer. In any of the above aspects, the subject is a mammal, in particular a human.

Definition

Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly used in the art to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural, and vice versa.

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

As used herein, the term " antigen binding domain that binds to a tumor-associated antigen " or "a portion that is capable of specifically binding to a tumor-associated antigen" refers to a polypeptide molecule that specifically binds to the tumor-associated antigen BCMA. In one aspect, the antigen binding domain is capable of activating a signal through BCMA. In a specific aspect, the antigen binding domain is capable of directing the entity to which it is attached (e.g., 4-1BBL trimer) to cells that express BCMA, for example, to a specific type of tumor cell. Antigen binding domains that are capable of specifically binding to BCMA include antibodies and fragments thereof as further defined herein. Furthermore, an antigen binding domain capable of specifically binding to a tumor-associated antigen may comprise a scaffold antigen binding protein as further defined herein, such as a binding domain based on a designed repeat protein or a designed repeat domain (see, e.g., WO 2002/020565).

With respect to antigen-binding molecules, i.e., antibodies or fragments thereof, the term "antigen-binding domain" refers to a portion of a molecule that comprises a region that specifically binds to part or all of an antigen and complements it. An antigen-binding domain capable of specific antigen binding can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, an antigen-binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In another aspect, an "antigen-binding domain capable of specific binding to a tumor-associated antigen" may also be a Fab fragment or a cross-Fab fragment. As used herein, the terms "first", "second" or "third" with respect to antigen-binding domains, etc., are used to conveniently distinguish when more than one of each type of part is present. Unless expressly stated, the use of these terms is not intended to impose a particular order or direction on the parts.

The term " antibody " herein is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies and multispecific antibodies (eg, bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity.

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

As used herein, the term " monospecific " antibody refers to an antibody having one or more binding sites, each binding site binding to the same epitope of the same antigen. The term " bispecific " means that the antigen binding molecule is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. However, a bispecific antigen binding molecule may also comprise additional antigen binding sites that bind to other antigenic determinants. In certain aspects, the bispecific antigen binding molecule is capable of simultaneously binding to two antigenic determinants, particularly two antigenic determinants expressed on two different cells or on the same cell. Therefore, the term " bispecific " according to the present invention may also include trispecific molecules, for example, a bispecific molecule comprising a 4-1BBL trimer and two antigen binding domains for two different target cell antigens.

The term " valent " as used herein means that there is a certain number of binding sites in an antigen binding molecule that are specific for a different antigenic determinant, and that the binding site is specific for a different antigenic determinant. Thus, the terms "bivalent", "tetravalent" and "hexavalent" respectively indicate that there are two binding sites, four binding sites and six binding sites specific for a certain antigenic determinant in an antigen binding molecule. In a specific aspect of the invention, the bispecific antigen binding molecules according to the invention may be monovalent for a certain antigenic determinant, meaning that they have only one binding site for that antigenic determinant, or they may be bivalent or tetravalent for a certain antigenic determinant, meaning that they have two binding sites or four binding sites, respectively, for that antigenic determinant.

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

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

Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each of which contains a heavy chain variable domain and a light chain variable domain as well as a light chain constant domain and a heavy chain first constant domain (CH1). Therefore, as used herein, the term " Fab fragment " or " Fab molecule " refers to an antibody fragment containing a light chain fragment, which contains a variable light chain (VL) domain and a light chain constant domain (CL), as well as a variable heavy chain domain (VH) and a heavy chain first constant domain (CH1). The Fab' fragment differs from the Fab fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residue in the homeodomain carries a free thiol group. Pepsin treatment produces the F(ab') ₂ fragment, which has two antigen binding sites (two Fab fragments) and a portion of the Fc region. The " conventional Fab fragment " consists of the VL-CL light chain and the VH-CH1 heavy chain.

The term " crossFab fragment " or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy chain and the light chain are exchanged. Two different chain compositions of crossover Fab molecules may exist and are included in the bispecific antibodies of the present invention: on the one hand, the variable regions of the Fab heavy chain and the light chain are exchanged, that is, the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant domain (CH1), and a peptide chain composed of the heavy chain variable domain (VH) and the light chain constant domain (CL). This crossover Fab molecule is also called crossover Fab _(VLVH) . On the other hand, when the constant regions of the heavy chain and light chain of Fab are interchanged, the interchangeable Fab molecule contains a peptide chain composed of the heavy chain variable domain (VH) and the light chain constant domain (CL), and a peptide chain composed of the light chain variable domain (VL) and the heavy chain constant domain (CH1). This type of interchangeable Fab molecule is also called crossover Fab _(CLCH1) .

"Single-chain Fab fragment" or " scFab " is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following in the order from N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragments are stabilized by the native disulfide bonds between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain, according to Kabat numbering) to generate interchain disulfide bonds.

"Interchangeable single-chain Fab fragment" or " x-scFab " is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following in the order from N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen binding site that specifically binds to an antigen, and wherein the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by the insertion of cysteine residues (eg, position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering) to generate interchain disulfide bonds.

" Single-chain variable fragment ( scFv )" is a fusion protein of the variable regions of the heavy chain ( _VH ) and light chain ( _VL ) of an antibody, connected to a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of _VH to the C-terminus of _VL , or vice versa . Despite the removal of the constant regions and the introduction of the linker, this protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96). Furthermore, antibody fragments comprise single polypeptides which have the characteristics of a VH domain (ie, capable of assembling together with a VL domain into a functional antigen binding site) or a VL domain (ie, capable of assembling together with a VH domain into a functional antigen binding site) and thereby provide the antigen binding properties of a full-length antibody.

" Scaffold antigen binding proteins " are known in the art, for example, reticular proteins and designed anchor protein repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one aspect of the invention, the scaffold antigen binding protein is selected from the group consisting of: CTLA-4 (Evibody); lipocalin (Anticalin); protein A-derived molecules such as the Z domain of protein A (affinity antibody); A domain (high affinity multimer/max antibody); serum transferrin ( trans -antibody); designed anchor protein repeat protein (DARPin); variable domain of antibody light or heavy chain (single domain antibody, sdAb); variable domain of antibody heavy chain (nanobody, aVH); V _NAR fragment; fibronectin (AdNectin); C-type lectin domain (Tetranectin); variable domain of novel antigen receptor β-lactamase (V _NAR fragments); human γ-crystallin or ubiquitin (Affilin molecules); Kunitz-type domains of human protease inhibitors, microsomes, such as proteins from the knottin family, peptide aptamers and fibronectins (adnectins). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28 family receptor expressed on primary CD4 ⁺ T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the CDRs of the antibody can be replaced by heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also called Evibodies (e.g. US7166697B1). Evibodies are approximately the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248 (1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. They have a rigid β-sheet secondary structure with many loops at the open end of the cone structure that can be engineered to bind different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633. Avidin is a scaffold derived from protein A of Staphylococcus aureus that can be engineered to bind to an antigen. The domain consists of a three-helix bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818A1. Avimers are multidomain proteins derived from the A-domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide-bonded structure. Diversity is generated by shuffling the natural variation exhibited by the family of A-domains. For additional details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences in permissive surface loops. Examples of engineered transferrin scaffolds include trans-isomers. For additional details, see J. Biol. Chem 274, 24066-24073 (1999). Designer anchoring repeat proteins (DARPins) are derived from anchoring proteins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single anchoring repeat is a 33-residue accumulation motif composed of two α-helices and a β-turn. It can be engineered to bind different target antigens by randomizing the residues in the first α-helix and β-turn of each repeat. Its binding interface can be increased by increasing the number of modules (affinity maturation approach). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1. Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain was derived from the variable domain of the antibody heavy chain from camel (nanobody or _VHH fragment). In addition, the term single domain antibody includes the autonomous human heavy chain variable domain (aVH) or the _VNAR fragment from shark. Fibroin is a scaffold that can be engineered to bind to an antigen. Fibronectin consists of a backbone of the natural amino acid sequence of the 10th domain of 15 repeat units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich structure can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For additional details, see Protein Eng. Des. Sel. 18, 435- 444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, usually thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783–797 (2005). Microbodies are derived from naturally occurring mini-proteins of 25-50 amino acids in length that contain 3-4 cysteine bridges, examples of mini-proteins include KalataBI and conotoxins and knottins. Mini-proteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the mini-protein. For further details on the engineered knotting domain, see WO2008098796.

An " antigen binding domain that binds to the same epitope as a reference molecule" refers to an antigen binding domain that blocks the binding of the reference molecule to its antigen by 50% or more in a competition assay, and conversely, the reference molecule blocks the binding of the antigen binding molecule to its antigen by 50% or more in a competition assay.

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

As used herein, the term " antigenic determinant " is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids). For example, available antigenic determinants may be present on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, not present in serum, and/or present in the extracellular matrix (ECM). Unless otherwise indicated, proteins suitable for use as antigens herein may be any native form of proteins from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). In specific embodiments, the antigen is a human protein. Where reference is made herein to a particular protein, the term encompasses both the "full-length," unprocessed protein and any form of the protein resulting from processing in the cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

" Specific binding " means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding molecule to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent to which an antigen-binding molecule binds to an unrelated protein is less than about 10% of the extent to which the antigen-binding molecule binds to the antigen, for example by SPR. In certain embodiments, the molecule that binds to the antigen has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 ^-8 M or lower, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 to 10 ^-13 M).

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

As used herein, " activating T cell antigen " refers to an antigenic determinant expressed on the surface of T lymphocytes (particularly cytotoxic T lymphocytes) that is capable of inducing T cell activation when interacting with an antibody. Specifically, the interaction of an antibody with an activating T cell antigen can induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In particular embodiments, the activating T cell antigen is CD3, in particular the epsilon subunit of CD3 (see UniProt No. P07766 (version 189), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 167 for human sequences; or UniProt No. Q95LI5 (version 49), NCBI GenBank No. BAB71849.1, SEQ ID NO: 168 for macaque [Macaca mulatta] sequences).

As used herein, " T cell activation " refers to one or more cellular responses of T lymphocytes (particularly cytotoxic T lymphocytes) selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

The term " T cell effector function " refers to the activities of T cells, which play a key role in the adaptive immune system. T cells are responsible for initiating and coordinating the body's immune response to foreign invaders, such as viruses or bacteria, as well as tumor cells. Effector function refers to the various activities that T cells perform to eliminate these invaders, including the release of cytokines, stimulation of other cells, and direct attack and elimination of infected cells.

As used herein, " tumor-associated antigen " or TAA refers to an antigenic determinant presented on the surface of a target cell (e.g., a cell in a tumor, such as a cancer cell, a cell of tumor stroma, a malignant B lymphocyte, a melanoma cell). In certain aspects, the target cell antigen is an antigen on the surface of a tumor cell. In a specific aspect, the TAA is BCMA.

The term " BCMA " refers to B cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRS17) or CD269, and is a type III transmembrane protein without a signal peptide and containing a cysteine-rich extracellular domain. The ligands of BCMA include B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), of which APRIL has a higher affinity for BCMA. BCMA is preferentially expressed by mature B lymphocytes, minimally expressed in hematopoietic stem cells or non-hematopoietic tissues, and is essential for the survival of long-lived bone marrow plasma cells. Membrane-bound BCMA can undergo γ-secretase-mediated shedding from the cell surface, resulting in the circulation of soluble BCMA (sBCMA) and reduced activation of surface BCMA by APRIL and BAFF. BCMA is overexpressed at significantly higher levels in all patient MM cells, but not in other normal tissues except normal plasma cells. BCMA, together with two related TNFR superfamily B cell activating factor receptors (BAFF-R) and transmembrane activator and calcium regulator and cyclophilin ligand interactor (TACI), critically regulates B cell proliferation and survival, as well as maturation and differentiation into plasma cells. These three functionally related receptors support the long-term survival of B cells at different stages of development by binding to their cognate ligands BAFF and/or APRIL. Unless otherwise specified, as used herein, BCMA refers to any BCMA protein from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., macaques), and rodents (e.g., mice and rats). The amino acid sequence of human BCMA is shown in UniProt (www.uniprot.org) Accession No. Q02223 (SEQ ID NO: 93).

Unless otherwise indicated, the term " CD28 " (cluster of differentiation 28, Tp44) refers to any CD28 protein from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., macaques), and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells through CD28 provides potent signals for the production of various leukocyte mediators. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747 (SEQ ID NO:94).

" Agonist antibody " refers to an antibody that contains an agonist function against a given receptor. Generally speaking, when an agonist ligand (factor) binds to a receptor, the tertiary structure of the receptor protein changes and the receptor is activated (when the receptor is a membrane protein, it usually transduces cell growth signals, etc.). If the receptor is a type that forms dimers, the agonist antibody can dimerize the receptor at an appropriate distance and angle, thus acting similarly to the ligand. A suitable anti-receptor antibody can mimic the receptor dimerization effect performed by the ligand, thus becoming an agonist antibody.

A " CD28 agonist antibody " or "CD28 conventional agonist antibody" is an antibody that mimics the role of the natural ligand of CD28 (CD80 or CD86) in enhancing T cell activation in the presence of a T cell receptor signal ("signal 2"). T cells require two signals to be fully activated. Under physiological conditions, "signal 1" comes from the interaction of the T cell receptor (TCR) molecule with the peptide/major histocompatibility complex (MHC) complex on the antigen presenting cell (APC), and "signal 2" is provided by the engagement of a co-stimulatory receptor (e.g., CD28). CD28 agonist antibodies are able to co-stimulate T cells (signal 2). It can also bind to molecules specific for the TCR complex to induce T cell proliferation and cytokine secretion, however, CD28 agonist antibodies cannot fully activate T cells without additional stimulation of the TCR. However, there are subtypes of CD28-specific antigen binding molecules, the so-called CD28 superagonist antibodies. " CD28 superagonist antibodies " are CD28 antibodies that are able to fully activate T cells without additional stimulation of the TCR. CD28 superagonist antibodies can induce T cell proliferation and cytokine secretion without prior activation of the T cells (signal 1).

The term " variable domain " or "variable region" refers to the domain of an antibody heavy chain or light chain that is involved in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three highly variable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

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

Unless otherwise stated, CDRs are determined according to the method described by Kabat et al. in the above-mentioned literature. Those skilled in the art will understand that CDR names may also be determined according to Chothia as described above , McCallum as described above , or any other scientifically accepted naming system. Kabat et al. also defined a variable region sequence numbering system applicable to any antibody. A person of ordinary skill in the art can explicitly specify this "Kabat numbering" system for any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, references to specific amino acid residue positions in antibody variable regions are based on the Kabat numbering system.

As used herein, in the context of antigen binding molecules (e.g., antibodies), the term " affinity matured " refers to an antigen binding molecule derived from a reference antigen binding molecule (e.g., by mutation) that binds to the same antigen, preferably to the same epitope as the reference antibody; and has a higher affinity for the antigen than the reference antigen binding molecule. Affinity maturation typically involves modification of one or more amino acid residues in one or more CDRs of the antigen binding molecule. Typically, the affinity matured antigen binding molecule binds to the same epitope as the initial reference antigen binding molecule.

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

For the purposes of this article, an " acceptor human framework " is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human common framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical to the sequence of the VL human immunoglobulin framework sequence or the human common framework sequence.

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

The " class " of an antibody refers to the type of constant domain or region of its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subtypes (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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

A " human " antibody is an antibody having an amino acid sequence that corresponds to an amino acid sequence produced by a human or human cell or derived from a non-human source using the human antibody repertoire or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. In particular, a "human" or "humanized" antibody comprises a constant region of human origin, in particular of the IgG isotype, more particularly of the IgG1 isotype, which constant region comprises human CH1, CH2, CH3 and/or CL domains.

The term " CL domain " refers to the cognate portion of an antibody light chain polypeptide. Exemplary sequences of human cognate domains are given in SEQ ID Nos: 95 and 96 (human kappa and lambda CL domains, respectively).

The term " CH1 domain " refers to the portion of an antibody heavy chain polypeptide extending approximately from EU position 118 to EU position 215 (EU numbering system according to Kabat). In one aspect, the CH1 domain has an amino acid sequence of ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 97). Typically, a segment having an EPKSC amino acid sequence (SEQ ID NO: 98) then connects the CH1 domain to the hinge region.

The term " hinge region " refers to the portion of the antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in the wild-type antibody heavy chain, for example from about position 216 to about position 230 according to the EU numbering system of Kabat, or from about position 226 to about position 230 according to the EU numbering system of Kabat. Hinge regions of other IgG subtypes can be identified by aligning the hinge region cysteine residues with the IgG1 subtype sequence. The hinge region is usually a dimeric molecule composed of two polypeptides with the same amino acid sequence. The hinge region usually contains up to 25 amino acid residues and is flexible, allowing independent movement of the relevant target binding sites. The hinge region can be subdivided into three domains: the upper, middle and lower hinge domains (see, e.g., Roux, et al., J. Immunol. 161 (1998) 4083).

In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 99), wherein X is S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 100), wherein X is S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 101), wherein X is S or P.

The term " Fc domain " or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In the context of molecules that have been defined by the Fab fragment (including the CH1 domain), the term "Fc domain" may refer only to the IgG CH2 and IgG CH3 domains.

The "CH2 domain" of a human IgG Fc region typically extends from an amino acid residue at about EU position 231 to an amino acid residue at about EU position 340 (according to the EU numbering system of Kabat). In one embodiment, the CH2 domain has an amino acid sequence of APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 102). The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete native Fc region. It is speculated that the carbohydrate may provide an alternative to this domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.

The "CH3 domain" comprises the C-terminal extension residue of the CH2 domain in the Fc region and refers to the portion of the antibody heavy chain polypeptide extending from approximately EU position 341 to EU position 446 (according to the EU numbering system of Kabat). In one aspect, the CH3 domain has an amino acid sequence of GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 103). Herein, the CH3 region may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "knob" ("knob") in one of its chains and a corresponding introduced "cavity" ("hole") in the other of its chains, see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or the cognate region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The " knob -into-hole " technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion (the "knob") at the interface of a first polypeptide and a corresponding cavity (the "hole") in the interface of a second polypeptide so that the protrusion can be placed in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing smaller amino acid side chains on the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Complementary cavities of the same or similar size as the protrusions are formed in the interface of the second polypeptide by replacing larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). The protrusions and cavities can be made by altering the nucleic acid encoding the polypeptide (e.g., by mutagenesis directed to specific sites or by peptide synthesis). In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising the Fc domain with a knob modification additionally comprises the amino acid substitution S354C, and the subunit comprising the Fc domain with a hole modification additionally comprises the amino acid substitution Y349C. The introduction of these two cysteine residues allows the formation of a disulfide bond between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

"A region equivalent to the Fc region of an immunoglobulin" is meant to include naturally occurring allele variants of the Fc region of an immunoglobulin as well as variants with changes that produce substitutions, additions or deletions but do not substantially reduce the ability of the immunoglobulin to mediate effector function (e.g., antibody-dependent cellular cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially losing biological function. Such variants may be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie, J.U. et al., Science 247:1306-10 (1990)).

The term " wild-type Fc domain " refers to an amino acid sequence that is identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc regions include, but are not limited to, native human IgG1 Fc regions (non-A and A allotypes); native human IgG2 Fc regions; native human IgG3 Fc regions; and native human IgG4 Fc regions, as well as naturally occurring variants thereof. Wild-type Fc regions are represented by SEQ ID NO: 104 (IgG1, Caucasian allotype), SEQ ID NO: 105 (IgG1, African American allotype), SEQ ID NO: 106 (IgG2), SEQ ID NO: 107 (IgG3) and SEQ ID NO: 108 (IgG4).

The term " variant ( human ) Fc domain " refers to an amino acid sequence that differs from a "wild-type" (human) Fc domain amino acid sequence by at least one "amino acid mutation". In one aspect, the variant Fc region has at least one amino acid mutation compared to a native Fc region, for example from about 1 to about 10 amino acid mutations in a native Fc region, and in one aspect from about 1 to about 5 amino acid mutations. In one aspect, the (variant) Fc region has at least about 95% homology to a wild-type Fc region. The specific variant Fc domain disclosed herein is a human IgG1 heavy chain constant region having mutations L234A, L235A and P329G, which comprises the amino acid sequence of SEQ ID NO: 109.

The term " effector function " refers to those biological activities attributed to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

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

Receptor cross-linking of the Fc region of IgG antibodies (FcγR) triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cytotoxicity, and release of inflammatory mediators as well as clearance of immune complexes and regulation of antibody production. In humans, three classes of FcγR have been identified, which are: - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modification of at least one of the amino acid residues E233-G236, P238, D265, N297, A327 and P329 (EU index numbering according to Kabat) in the Fc region of IgG reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 to IgG1 and IgG4 reduced binding to FcγRI by 10³-fold and abolished the response of human monocytes to antibody-sensitized erythrocytes (Armour, K.L. et al., Eur. J. Immunol. 29 (1999) 2613–2624). - FcγRII (CD32) binds complexed IgG with moderate to low affinity and is ubiquitously expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in cytotoxicity (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the cytotoxic process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, it appears to function to inhibit further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB inhibits phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B type may help inhibit the activation of these cells through the binding of IgE to its independent receptors. For example, antibodies containing an IgG Fc region with at least one mutation of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (according to the Kabat EU index numbering) have been found to have reduced binding to FcγRIIA. - FcγRIII (CD16) binds IgG with moderate to low affinity and exists in two types. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. For example, antibodies comprising an IgG Fc region having a mutation in at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (according to the Kabat EU index numbering) were found to have reduced binding to FcγRIIIA.

The location of the binding site on human IgG1 for the Fc receptor, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604.

The term " ADCC " or "antibody-dependent cytotoxicity" is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell to which the antibody or its derivative comprises an Fc region, which is typically specifically bound to the Fc region via a protein portion as the N-terminus. The term "reduced ADCC" as used herein refers to a decrease in the number of target cells lysed in a given time by a given concentration of antibody in the culture medium surrounding the target cells via the ADCC mechanism defined above, and/or an increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells in a given time via the ADCC mechanism. The reduction in ADCC is relative to ADCC mediated by the same antibody (but not engineered) produced by the same type of host cells using the same standard production, purification, formulation and storage methods (methods known to those of ordinary skill in the art). For example, ADCC mediated by an antibody comprising an amino acid substitution in the Fc domain that reduces ADCC is reduced relative to ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006/082515 or PCT Publication No. WO 2012/130831). For example, the ability of an antibody to induce the initial step of ADCC is studied by measuring binding of the antibody to cells expressing Fcγ receptors, such as cells expressing recombinant FcγRI and/or FcγRIIA or NK cells (substantially expressing FcγRIIIA). Specifically, binding to FcγR on NK cells is measured.

" Activating Fc receptors " are Fc receptors that, upon binding to the Fc region of an antibody, initiate a signaling event that stimulates the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (SEQ ID NO:110 UniProt accession number P08637, version 141).

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

The term " peptide linker " refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, ( _G4S ) _n , ( _SG4 ) _n or _G4 ( _SG4 ) _n peptide linkers, wherein "n" is typically a number between 1 and 5, typically between 2 and 4, particularly 2, i.e. a peptide selected from the group consisting of GGGGS (SEQ ID NO:111), GGGGSGGGG (SEQ ID NO:112), GGGGSGGGGS (SEQ ID NO:113), SGGGGSGGGG (SEQ ID NO:114) and GGGGSGGGGSGGGG (SEQ ID NO:115), but also including the sequences GSPGSSSSGS (SEQ ID NO:116), (G4S) ₃ (SEQ ID NO:117), (G4S) ₄ (SEQ ID NO:118), GSGSGSGS (SEQ ID NO:119), GSGSGNGS (SEQ ID NO:120). Peptide linkers of particular interest are (G4S) (SEQ ID NO: 111), (G4S)2, GGGGSGGGG (SEQ ID NO: 112) or GGGGSGGGGS (SEQ ID NO: 113), ( _{G4S)3 (SEQ ID NO: 117) and (G4S)4 ( SEQ ID} NO: 118).

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

"Fusion" or "linked" means that the components (e.g., a polypeptide and the extracellular domain of the TNF ligand family member) are linked by a peptide bond (directly or via one or more peptide linkers).

" Percentage (%) of amino acid sequence identity relative to a reference polypeptide sequence (protein) is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the reference polypeptide sequence and the candidate sequence and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN.SAWI or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been deposited with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not varied. In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X/Y, where X is the number of amino acid residues obtained in the alignment of A and B as identical matches by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity between A and B will not be equal to the % amino acid sequence identity between B and A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program, as described just in the previous paragraph.

In certain embodiments, amino acid sequence variants of the BCMA antibodies or immunostimulatory antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the BCMA antibodies or immunostimulatory antigen binding molecules. Amino acid sequence variants of BCMA antibodies or immunostimulatory antigen binding molecules can be prepared by introducing suitable modifications into molecules encoding nucleotide sequences or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antigen binding molecule. Any combination of deletions, insertions and substitutions may be implemented to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding characteristics. Sites of interest for substitutional induction include CDRs and frameworks (FRs). Conservative substitutions are provided in Table B under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into related molecules and the products screened for desired activity, such as maintained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. Table A Original Residue Exemplary substitutions Better replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn；Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; nor-leucine Leu Leu (L) nor-leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; nor-leucine Leu

Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

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

The term " amino acid sequence variant " includes substantial variants, wherein there is an amino acid substitution in one or more highly variable region residues of a parent antigen-binding molecule ( e.g., humanized or human antibodies). Typically, the variants selected for further study will have modifications (e.g., improvements) of certain biological properties compared to the parent antigen-binding molecule (e.g., increased affinity, reduced immunogenicity) and/or will substantially maintain certain biological properties of the parent antigen-binding molecule. Exemplary substitution variants are affinity-matured antibodies, which can be easily produced, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more CDR residues are mutated and the variant antigen-binding molecules are displayed on phage and screened for specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions or deletions may be present within one or more CDRs, as long as such changes do not substantially reduce the ability of the antigen binding molecule to bind to the antigen. For example, conservative changes (e.g., conservative substitutions provided herein) may be made in the CDRs that do not substantially reduce binding affinity. A useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science , 244: 1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. More substitutions can be introduced at amino acid positions, indicating good functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antigen binding molecule is complexed to identify the contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

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

In certain aspects, the immunostimulatory antigen binding molecules provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Glycosylation variants of the molecule can be conveniently obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. When the agonist ICOS binding molecule comprises an Fc domain, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides that are typically attached to Asn297 of the CH2 domain of the Fc region by an N-bond. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in a BCMA antibody or bispecific BCMA antibody can be modified to generate variants with certain improved properties. In one aspect, a BCMA antibody or a variant of a bispecific BCMA antibody is provided that has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function, see, e.g., U.S. Patent Publication Nos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.) Other variants of BCMA antibodies or bispecific BCMA antibodies include variants with bisected oligosaccharides, e.g., wherein the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function, see, e.g., WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Variants in which at least one galactose residue in the oligosaccharide is linked to the Fc region are also provided. Such antibody variants may have improved CDC function and are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, it may be desirable to generate cysteine engineered variants of the immunostimulatory antigen-binding molecules described herein, such as "thioMAbs," in which one or more residues of the molecule are substituted with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the molecule. By replacing these residues with cysteine, reactive thiol groups are thereby placed at sites accessible to the antibody and can be used to conjugate the immunostimulatory antigen-binding molecules to other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain. Cysteine engineered antigen binding molecules can be produced, for example, as described in U.S. Patent No. 7,521,541.

In certain aspects, the immunostimulatory antigen-binding molecules provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable derivatized moieties for antigen-binding molecules include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-triazine, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the bispecific antibody derivative will be used in therapy under defined conditions, and the like. In another aspect, a conjugate of an antibody and a non-protein portion that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, and includes, but is not limited to, a wavelength that does not damage normal cells but heats the non-protein portion to a temperature that kills cells near the antibody-non-protein portion. In another embodiment, an immunoconjugate of the BCMA antibody or bispecific BCMA antibody provided herein can be obtained. An " immunoconjugate " is an antigen binding molecule bound to one or more heterologous molecules, such as a small molecule agent.

The term " polynucleotide " refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid fragments present in a polynucleotide, such as DNA or RNA fragments. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are usually represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses: deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), in particular messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, phosphate backbone linkages, or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules that are suitable as vectors for directly expressing the antibodies of the present invention in vitro and/or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, thereby injecting the mRNA into an individual to produce antibodies (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817 or EP 2 101 823 B1).

By " isolated " nucleic acid molecule or polynucleotide is meant a nucleic acid molecule, DNA or RNA, that is removed from its native environment. For example, for purposes of the present invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or a polynucleotide purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain polynucleotide molecules, but the polynucleotide molecules are present extrachromosomally or at a chromosomal location that is different from the natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive and negative stranded forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, a ribosome binding site, or a transcriptional terminator.

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

The term " expression cassette " refers to a polynucleotide produced recombinantly or synthetically that has a series of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. The recombinant expression cassette can be introduced into a plastid, chromosome, mitochondrial DNA, chromatin DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, in addition to other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette disclosed herein comprises a polynucleotide sequence encoding an immunostimulatory antigen binding molecule or a fragment thereof.

The term " vector " or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene and direct the expression of the gene, which is operably linked to the gene in a target cell. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome that has been introduced into the host cell. The expression vector of the present invention comprises an expression cassette. The expression vector transcribes a large amount of stable mRNA. Once the expression vector enters the target cell, the RNA molecule or protein encoded by the gene is produced by the cell transcription and/or translation machinery. In one embodiment, the expression vector as disclosed herein comprises an expression cassette, which comprises a polynucleotide sequence that encodes an immunostimulatory antigen binding molecule or a fragment thereof.

The terms " host cell ,""host cell strain," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and daughter cells derived therefrom, regardless of the number of passages. The nucleic acid content of daughter cells may not be exactly the same as that of the parent cell, but may contain mutations. Mutant daughter cells having the same function or biological activity as that screened or selected in the initial transformed cell are included herein. Host cells are any type of cell system that can be used to produce the bispecific antigen-binding molecules of the present invention. Host cells include cultured cells, for example mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or fusion tumor cells, yeast cells, insect cells and plant cells (to name a few), but also include transgenic animals, transgenic plants or cells contained in cultured plants or animal tissues.

An " effective amount " of a drug refers to the amount required to produce a physiological change in the cells or tissues to which it is administered.

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

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

The term " pharmaceutical composition " refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effectively exerted, and that does not contain other components that are unacceptably toxic to the subject to which the formulation is to be administered.

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

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

As used herein, " treatment " (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and may be performed for prophylactic or preventive purposes or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or reducing the disease state, relieving or improving prognosis. In some embodiments, the molecules of the invention are used to delay disease development or slow the progression of a disease.

The term " therapy " refers to any regimen, method and/or agent that can be used to prevent, manage, treat and/or ameliorate a disease (or symptoms associated therewith) or cancer. In certain aspects, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies and/or other therapies that can be used to prevent, manage, treat and/or ameliorate a disease or cancer known to those skilled in the art, such as physicians.

As used herein, the term " combination therapy " or " co-administration " includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of an antibody as reported herein may occur prior to, concurrently with, and/or after administration of the additional therapeutic agent(s), preferably one or more antibodies.

The term " cancer " refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Thus, the term cancer as used herein refers to proliferative diseases such as carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin's lymphomas), blastomas, sarcomas, and leukemias. Specifically, the term cancer includes lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, Disease), esophageal cancer, small intestinal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell cancer, renal pelvic cancer, mesothelioma, hepatocellular carcinoma, gallbladder cancer, tumors of the central nervous system (CNS), spinal axonal tumors, brain stem neuroglioma, multiforme glioblastoma, astrocytoma, neurothecoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing's sarcoma. In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer is a hematological cancer, particularly a leukemia, most particularly acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). In a preferred embodiment, the term cancer refers to any cancer in which BCMA is expressed. More preferably, the cancer is multiple myeloma (MM).

Illustrative novelties BCMA antibody

Provided herein are novel antibodies and/or antibody fragments that specifically bind to B cell maturation antigen (BCMA). Provided herein are novel antibodies and/or antibody fragments that specifically bind to the extracellular domain of human BCMA comprising the amino acid sequence of SEQ ID NO: 127. Thus, these antibodies specifically bind to human BCMA.

These antibodies are able to bind to human BCMA as well as to macaque BCMA. Therefore, they also bind to the extracellular domain of macaque BCMA comprising the amino acid sequence of SEQ ID NO:129.

They bind to the extracellular domain of human BCMA (ECD of the amino acid sequence of SEQ ID NO: 127) with a _KD value of less than 5 nM as measured by surface plasmon resonance (SPR) (see Example 1.3).

As shown in Example 3, the novel antibodies are also able to bind to mutant human BCMA variants hu BCMA_R27P (SEQ ID NO: 203), hu BCMA_S30del (SEQ ID NO: 204), hu BCMA_P33S (SEQ ID NO: 205) and hu BCMA_P34del (SEQ ID NO: 206). Therefore, they are not affected by point mutations in the ECD of BCMA, nor do they lose therapeutic activity as observed for other BCMA-targeted molecules such as elenatumab and terituzumab (Lee et al., Nature Medicine 2023, 29, 2295-2306).

Novel antibodies are further characterized in that they are producible in large quantities and at high titers, in that they show high thermal stability (as measured by the aggregation temperature _Tagg ), or in that they are highly human and therefore may be less immunogenic in humans. The percentage of humanness of VH and VL sequences relative to human germline sequences can be determined by the method described in Abhinandan, KR and Martin, Andrew CR 2007, J. Mol. Biol. 2007, 369 , 852-862.

In one aspect, provided herein is an antibody that specifically binds to B cell maturation agent (BCMA), wherein the antibody comprises (i) a heavy chain variable region ( _VH BCMA) comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 1 (GYTFTNYWMH), a CDR-H2 of SEQ ID NO: 2 (IIHPNSGSTNYNEKFQG), and a CDR-H3 of SEQ ID NO: 3 (GIYDYPFAY); and (ii) a light chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 5 (AASSLQS), and a CDR-H3 of SEQ ID NO: 6 (QQSIEDPYT); or (b) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 7 (AASNLES), and a CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); or (c) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 8 (AASNLQS), and a CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT).

In one aspect, provided herein is a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA), wherein the antibody comprises: a _VH BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (VH1a) and SEQ ID NO: 14 (VH1b); and/or a _VL BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15 (VL1f), SEQ ID NO: 16 (VL1a), SEQ ID NO: 17 (VL1b), SEQ ID NO: 18 (VL1c), SEQ ID NO: 19 (VL1d) and SEQ ID NO: 20 (VL1e).

In one aspect, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, wherein the antibody (or antigen-binding domain) comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 16; or (c) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (d) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (e) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 or (h) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: ₁₃₁ ; or (i) _VH BCMA comprising the amino _acid sequence of SEQ ID NO: ₁₄ and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15; or (j _{) VH BCMA} comprising the _amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:16; or (k) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:17; or (l) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:18; or (m) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:19; or (n) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:20; or (o) a VH BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:130 or (p) _{a VH} BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:131.

In one aspect, the BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 16; or (c) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (d) VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (e) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: or (h) _VH BCMA comprising the amino _acid sequence of _SEQ ID NO:13 and _{VL BCMA} comprising the _amino acid sequence of SEQ ID _NO :131.

In one aspect, the BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) comprises (i) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 15; or (j) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 16; or (k) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (l) a VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (m) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: or (p) _VH BCMA comprising the amino _acid sequence of _SEQ ID NO:14 and _{VL BCMA} comprising the _amino acid sequence of SEQ ID _NO :131.

In a specific aspect, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, wherein the antibody (or antigen-binding domain) comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:16.

More specifically, the BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) comprises: _VH BCMA comprising the amino acid sequence of SEQ ID NO:13; and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15.

In another aspect, provided herein is an antibody that specifically binds to B cell maturation agent (BCMA), wherein the antibody comprises (i) a heavy chain variable region ( _VH BCMA) selected from the group consisting of: (a) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 27 (QITAKSNNYATYYADSVKG), and a CDR-H3 of SEQ ID NO: 28 (DGYH); and (b) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 29 (QITAKSNNYATYYAAPVKG), and a CDR-H3 of SEQ ID NO: 29 (DGYH) CDR-H3; and (ii) a light chain variable region (V _L BCMA) comprising a light chain complementation determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), a CDR-L2 of SEQ ID NO: 31 (NANSLHT) and a CDR-L3 of SEQ ID NO: 32 (EDTSKYPYT).

In one aspect, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, wherein the antibody (or antigen-binding domain) comprises: a heavy chain variable region ( _VH BCMA) comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), CDR-H2 of SEQ ID NO: 27 (QITAKSNNYATYYADSVKG), and CDR-H3 of SEQ ID NO: 28 (DGYH); and a light chain variable region ( _VL BCMA) comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), CDR-L2 of SEQ ID NO: 31 (NANSLHT), and CDR-H3 of SEQ ID NO: 32 (EDTSKYPYT) CDR-L3.

In one aspect, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, wherein the antibody (or antigen-binding domain) comprises: a heavy chain variable region ( _VH BCMA) comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), CDR-H2 of SEQ ID NO: 29 (QITAKSNNYATYYAAPVKG), and CDR-H3 of SEQ ID NO: 28 (DGYH); and a light chain variable region ( _VL BCMA) comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), CDR-L2 of SEQ ID NO: 31 (NANSLHT), and CDR-H3 of SEQ ID NO: 32 (EDTSKYPYT) CDR-L3.

In one aspect, provided herein is a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA), wherein the antibody (or antigen-binding domain) comprises: _VH BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33 (VH2a), SEQ ID NO: 35 (VH1b), SEQ ID NO: 53 (VH1a), SEQ ID NO: 54 (VH1a_Y292D), SEQ ID NO: 55 (VH1c_hu CDR2) and SEQ ID NO: 56 (VH1a_W197Y); and/or _VL BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34 (VL2a), SEQ ID NO: 36 (VL1a), SEQ ID NO: 57 (VL1a_L2_GL), SEQ ID NO: 58 (VL2a_L2_GL), SEQ ID NO:59 (VL1a_L1_pGL), SEQ ID NO:60 (VL1a_N651A), SEQ ID NO:61 (VL1a_N651A_N695S), SEQ ID NO:62 (VL1a_H698Q), SEQ ID NO:63 (VL1a_T699S) and SEQ ID NO:64 (VL1a_H698Q_T699S).

In one aspect, provided herein is a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA), wherein the antibody (or antigen-binding domain) comprises: a _VH BCMA comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 33 (VH2a) and SEQ ID NO: 35 (VH1b); and/or a _VL BCMA comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 34 (VL2a) and SEQ ID NO: 36 (VL1a).

In one aspect, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, wherein the antibody (or antigen-binding domain) comprises (a) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:34, or (b) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:36; or (c) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:53 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:57; or (d) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:54 and a VL BCMA comprising the amino acid sequence of SEQ ID NO:36; or (e) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:54 or (i) a V _H BCMA comprising the amino acid sequence of SEQ ID NO:35 and a V _L BCMA comprising the amino acid sequence of SEQ ID NO:60; or (j) a V _H BCMA comprising the amino acid sequence of SEQ ID NO:35 and a V _L BCMA comprising the amino _acid sequence of SEQ ID NO: ₆₁ ; or (j) a _{V H} BCMA comprising the amino acid sequence of SEQ ID NO: ₃₅ and a V _L BCMA comprising the amino acid sequence of SEQ ID NO: ₆₂ . or (k) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:63; or (l) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:34; or (m) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and _{a VL} BCMA comprising the amino acid sequence of SEQ ID NO:36; or (n) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:59; or (o) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and a VL _BCMA comprising the amino acid sequence of SEQ ID NO:63; BCMA.

In a specific aspect, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, wherein the antibody (or antigen-binding domain) comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:36.

More specifically, the BCMA antibody comprises: _VH BCMA comprising the amino acid sequence of SEQ ID NO:33; and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34.

In one embodiment, the antibody that specifically binds to BCMA is a full-length antibody, specifically of the human IgG1 subclass. In a specific embodiment, it comprises amino acid mutations L234A, L235A, and P329G (according to the Kabat EU index number).

Contains novel BCMA Antibody immunostimulatory antigen binding molecules

This patent application provides novel BCMA-targeted 4-1BBL antigen-binding molecules, which include three 4-1BB ligands or fragments thereof fused to the antigen-binding molecules, wherein two 4-1BB ligands or fragments thereof are fused to each other via a linker, and one 4-1BB ligand is present in different chains of the molecule (split-type 4-1BBL trimer). These BCMA-targeted 4-1BBL antigen-binding molecules have advantageous properties, such as excellent manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, and an expanded dosage range that can be administered to patients and thus can observe possible enhanced efficacy. Novel BCMA-targeted 4-1BBL antigen-binding molecules comprise an Fc domain composed of a first unit and a second unit capable of stably binding, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity and/or effector function (Fc silencing) of the antigen-binding molecule to the Fc receptor, and thus avoid non-specific cross-linking via the Fc receptor. Instead, they comprise a specific antigen-binding domain capable of specific binding to BCMA, which causes cross-linking at the tumor site. Surprisingly, the inventors have discovered that, based on its binding properties, the BCMA antigen-binding domain as described herein has advantageous properties that make it more useful in this format than other BCMA antigen-binding domains. This can be seen, for example, in the Jurkat NFκB/4-1BB reporter cell assay (Example 4.1), where 4-1BBL antigen binding molecules comprising BCMA antibody PR were less able to induce T cell activation than 4-1BBL antigen binding molecules comprising the novel BCMA antibodies described herein. It has been found that 4-1BBL antigen binding molecules comprising these BCMA antigen binding domains have improved functionality and ability to increase T cell activation, particularly in the presence of T cell activating anti-CD3 bispecific antibodies. Thus, enhanced tumor-specific T cell activation is achieved.

In one aspect, provided herein is an immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising a) an antigen-binding domain comprising (i) a heavy chain variable region ( _VH BCMA) comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 1 (GYTFTNYWMH), CDR-H2 of SEQ ID NO: 2 (IIHPNSGSTNYNEKFQG), and CDR-H3 of SEQ ID NO: 3 (GIYDYPFAY); and (ii) a light chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), CDR-L2 of SEQ ID NO: 5 (AASSLQS); and (c) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 8 (AASNLQS), and a CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); (b) a first polypeptide and a second polypeptide, which are linked to each other by a disulfide bond, The antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL or a fragment thereof connected to each other by a peptide linker, and the second polypeptide comprises one extracellular domain of 4-1BBL or a fragment thereof; and (c) an Fc domain, which is composed of a first unit and a second unit.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9. In one aspect, the immunostimulatory antigen binding molecule comprises three extracellular domains of 4-1BBL, each of which comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, in particular the amino acid sequence of SEQ ID NO: 9.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189 and SEQ ID NO: 190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence of SEQ ID NO: 11, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9.

In one embodiment, the peptide linker that connects the extracellular domains of 4-1BBL or fragments thereof to each other comprises the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 113. In a specific embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO: 112.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the antigen binding molecule comprises: a _VH BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (VH1a) and SEQ ID NO: 14 (VH1b); and/or a _VL BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15 (VL1f), SEQ ID NO: 16 (VL1a), SEQ ID NO: 17 (VL1b), SEQ ID NO: 18 (VL1c), SEQ ID NO: 19 (VL1d) and SEQ ID NO: 20 (VL1e).

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the antigen binding molecule comprises (a) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 15, or (b) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 16; or (c) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (d) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and a VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (e) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 19. or (i) _{a VH} BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:15; or (j) _{a VH BCMA} comprising the amino acid sequence of SEQ ID NO: ₁₄ and _{a VL BCMA} comprising the amino acid _sequence of SEQ ID NO:16. or (k) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (l) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _{VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (m) a VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a VL BCMA comprising the amino acid sequence of SEQ ID NO: 19; or (n) a VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a VL BCMA comprising the} amino acid sequence of SEQ ID NO: 20; or (o) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 130; or (p) a VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 131; The invention also provides a _VH BCMA comprising the amino acid sequence of SEQ ID NO:14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:131.

In one aspect, the immunostimulatory antigen-binding molecule as defined above comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 16; or (c) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (d) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (e) VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 19. or (f) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 20; or ( _g ) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 130; or (h) _VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO: 131.

In one aspect, the immunostimulatory antigen-binding molecule as defined above comprises (i) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 15; or (j) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 16; or (k) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 17; or (l) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a VL BCMA comprising the amino acid sequence of SEQ ID NO: 18; or (m) a _VH BCMA comprising the amino acid sequence of SEQ ID NO: 14 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO: 19. _L BCMA; or (n) V _H BCMA comprising the amino acid sequence of SEQ ID NO: 14 and V _L BCMA comprising the amino acid sequence of SEQ ID NO: 20; or (o) V _H BCMA comprising the amino acid sequence of SEQ ID NO: 14 and V _L BCMA comprising the amino acid sequence of SEQ ID NO: 130; or (p) V _H BCMA comprising the amino acid sequence of SEQ ID NO: 14 and V _L BCMA comprising the amino acid sequence of SEQ ID NO: 131.

In a specific aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the antigen binding molecule comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:16.

More specifically, the immunostimulatory antigen-binding molecule comprises: _VH BCMA comprising the amino acid sequence of SEQ ID NO:13; and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15.

In one particular aspect, the antigen binding domain is a Fab molecule.

In one embodiment, the immunostimulatory antigen binding molecule as defined above comprises an Fc domain consisting of a first unit and a second unit. In one embodiment, the Fc domain consisting of a first unit and a second unit is an IgG, in particular an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

In one aspect, the Fc domain comprises a modification that promotes the association of the first unit and the second unit of the Fc domain. In one aspect, the Fc domain comprises a knob-in-hole modification. In one aspect, the first unit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second unit of the Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

In one embodiment, the immunostimulatory antigen binding molecule as described herein comprises (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CL domain; (aiii) a CL domain, fused at the C-terminus to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (aiv) one of the subunits of an Fc domain (e.g., a first subunit); (b) a second polypeptide comprising: (bi) a third extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CH1 domain; and (bii) a CH1 domain; (c) a third polypeptide comprising: (ci) the Fab The heavy chain of the molecule is fused at the C-terminus to the N-terminus of another of the subunits of the Fc domain (e.g., the second subunit); and (cii) another of the subunits of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule.

In one aspect, the immunostimulatory antigen-binding molecule as described above is a molecule wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering).

In one aspect, an immunostimulatory antigen binding molecule as described herein comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:21, a peptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:22, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:23, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:24. In a further aspect, an immunostimulatory antigen binding molecule as described herein comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:21, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:22, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:23, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:25.

In a specific aspect, the immunostimulatory antigen binding molecule as described herein comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:25.

In a specific aspect, the immunostimulatory antigen-binding molecule comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 23, and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 24. In one aspect, the immunostimulatory antigen-binding molecule comprises: (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 208, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising an amino acid sequence of SEQ ID NO: 207, and a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 24. In a specific aspect, the immunostimulatory antigen-binding molecule consists of: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:208, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:207, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24.

Also provided herein is an immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) an antigen-binding domain comprising (i) a heavy chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 27 (QITAKSNNYATYYADSVKG), and a CDR-H3 of SEQ ID NO: 28 (DGYH); and (b) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 29 (QITAKSNNYATYYAAPVKG), and a CDR-H3 of SEQ ID NO: 21 (DGYH). NO: 28 (DGYH) CDR-H3; and (ii) a light chain variable region (V _L BCMA) comprising a light chain complementation determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), CDR-L2 of SEQ ID NO: 31 (NANSLHT) and CDR-L3 of SEQ ID NO: 32 (EDTSKYPYT); (b) a first polypeptide and a second polypeptide, which are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains or fragments thereof of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one extracellular domain or fragment thereof of 4-1BBL; and (c) an Fc domain, which is composed of a first unit and a second unit.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9. In one aspect, the immunostimulatory antigen binding molecule comprises three extracellular domains of 4-1BBL, each of which comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, in particular the amino acid sequence of SEQ ID NO: 9.

In one aspect, an immunostimulatory antigen binding molecule as defined above is provided, wherein the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189 and SEQ ID NO: 190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In a specific aspect, the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence of SEQ ID NO: 11, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9.

In one embodiment, the peptide linker that connects the extracellular domains of 4-1BBL or fragments thereof to each other comprises the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 113. In a specific embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO: 112.

In one aspect, an immunostimulatory antigen binding molecule as previously defined herein is provided, wherein the antigen binding molecule comprises: a _VH BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33 (VH2a), SEQ ID NO: 35 (VH1b), SEQ ID NO: 53 (VH1a), SEQ ID NO: 54 (VH1a_Y292D), SEQ ID NO: 55 (VH1c_hu CDR2) and SEQ ID NO: 56 (VH1a_W197Y); and/or a _VL BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34 (VL2a), SEQ ID NO: 36 (VL1a), SEQ ID NO: 57 (VL1a_L2_GL), SEQ ID NO: 58 (VL2a_L2_GL), SEQ ID NO: 59 (VL1a_L1_pGL), SEQ ID NO:60 (VL1a_N651A), SEQ ID NO:61 (VL1a_N651A_N695S), SEQ ID NO:62 (VL1a_H698Q), SEQ ID NO:63 (VL1a_T699S) and SEQ ID NO:64 (VL1a_H698Q_T699S).

In one aspect, an immunostimulatory antigen binding molecule as described above is provided, wherein the antigen binding molecule comprises: a _VH BCMA comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 33 (VH2a) and SEQ ID NO: 35 (VH1b); and/or a _VL BCMA comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 34 (VL2a) and SEQ ID NO: 36 (VL1a).

In one aspect, an immunostimulatory antigen-binding molecule is provided, wherein the antigen-binding molecule comprises (a) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:34, or (b) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:36; or (c) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:53 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:57; or (d) a VH BCMA comprising the amino acid sequence of SEQ ID NO:54 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:36; or (e) a _VH BCMA comprising the amino acid sequence of SEQ ID NO:54 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:57. or (i) _{a VH} BCMA comprising the amino acid sequence of SEQ ID NO:35 and a _VL BCMA comprising the amino acid sequence of SEQ ID NO:60; or (j) _{a VH BCMA} comprising the amino acid sequence of SEQ ID NO: ₃₅ and _{a VL BCMA} comprising the amino acid _sequence of SEQ ID NO:61. or (n) _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and _VL BCMA comprising the amino _acid sequence of SEQ _ID NO: ₅₉ ; or (o) _VH BCMA comprising the amino acid sequence of SEQ ID _NO :33 and _{VL BCMA} comprising the amino acid sequence of _SEQ ID NO:63.

In a specific aspect, an immunostimulatory antigen binding molecule as described above is provided, wherein the antigen binding molecule comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:36.

More specifically, the immunostimulatory antigen-binding molecule comprises: _VH BCMA comprising the amino acid sequence of SEQ ID NO:33; and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34.

In one particular aspect, the antigen binding domain is a Fab molecule.

In one embodiment, the antigen-binding molecule that specifically binds to BCMA comprises an Fc domain consisting of a first unit and a second unit. In one embodiment, the Fc domain consisting of a first unit and a second unit is IgG, specifically an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

In one aspect, the Fc domain comprises a modification that promotes the association of the first unit and the second unit of the Fc domain. In one aspect, the Fc domain comprises a knob-in-hole modification. In one aspect, the first unit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second unit of the Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

In one embodiment, the immunostimulatory antigen binding molecule as described herein comprises (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CL domain; (aiii) a CL domain, fused at the C-terminus to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (aiv) one of the subunits of an Fc domain (e.g., a first subunit); (b) a second polypeptide comprising: (bi) a third extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CH1 domain; and (bii) a CH1 domain; (c) a third polypeptide comprising: (ci) the Fab The heavy chain of the molecule is fused at the C-terminus to the N-terminus of another of the subunits of the Fc domain (e.g., the second subunit); and (cii) another of the subunits of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule.

In one aspect, the immunostimulatory antigen-binding molecule as described above is a molecule wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering).

In one aspect, an immunostimulatory antigen binding molecule as described herein comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:21, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:22, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:37, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:38. In a further aspect, an immunostimulatory antigen binding molecule as described herein comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:21, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:22, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:39, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:40.

In a specific aspect, the immunostimulatory antigen-binding molecule described herein comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 37, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 38; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 39, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

In a specific aspect, the immunostimulatory antigen-binding molecule described herein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 37, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 38.

In any of the above-mentioned aspects, the immunostimulatory antigen-binding molecule comprises an Fc domain. In one aspect, the Fc domain is an IgG, in particular an IgG1 Fc domain. In a particular aspect, the Fc domain is a human Fc domain, in particular a human IgG1 Fc domain. In any of the above-mentioned aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. In a particular aspect, it comprises amino acid mutations L234A, L235A and P329G (according to Kabat EU index numbering).

Reduce Fc Receptor binding and / Or effect function Fc Domain Modification

The Fc domain of the immunostimulatory antigen-binding molecule consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are able to stably associate with each other. The Fc domain confers favorable pharmacokinetic properties to the antigen-binding molecules of the present invention, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in target tissues. On the other hand, however, it may result in the undesirable targeting of the antigen-binding molecule to cells expressing Fc receptors rather than to cells that better carry the antigen.

Thus, the Fc domain of the immunostimulatory antigen binding molecule that binds to BCMA exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native IgG1 Fc domain. In one aspect, the Fc domain does not substantially bind to an Fc receptor and/or does not induce effector function. In a particular aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc domain does not induce effector function. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen capture by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling that induces apoptosis, reduced dendritic cell maturation, or reduced T cell activation.

In certain aspects, one or more amino acid modifications can be introduced into the Fc region of the antigen binding molecules provided herein, thereby generating Fc region variants. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In one particular aspect, an immunostimulatory antigen binding molecule is provided that specifically binds to BCMA, wherein the Fc region comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly to an Fcγ receptor. In one aspect, an immunostimulatory antigen binding molecule is provided that specifically binds to BCMA, wherein the Fc region comprises one or more amino acid substitutions, and wherein ADCC induced by the immunostimulatory antigen binding molecule is reduced to 0% to 20% of ADCC induced by an antibody comprising a wild-type human IgG1 Fc region.

In one aspect, the Fc domain of the antigen-binding molecule described herein comprises one or more amino acid mutations that reduce the binding affinity and/or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. Specifically, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329 (EU numbering). In particular, the Fc domain comprises amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chain. More specifically, an immunostimulatory antigen-binding molecule that specifically binds to BCMA is provided, comprising an Fc domain having amino acid substitutions L234A, L235A and P329G ("P329G LALA", EU number) in the IgG heavy chain. The amino acid substitutions L234A and L235A are referred to as the so-called LALA mutation. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of human IgG1 Fc domains and is described in International Patent Application Publication No. WO 2012/130831 A1, which also describes methods for making such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function).

Fc domains with reduced Fc receptor binding and/or effector function also include domains having substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

In another aspect, the Fc domain is an IgG4 Fc domain. IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), specifically, the amino acid substitution S228P. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E and S228P and P329G (EU numbering). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are also described in WO 2012/130831.

Variant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods known in the art. Genetic methods may include site-specific mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. The correctness of the nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined by ELISA, or by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and the Fc receptor can be obtained, for example, by recombinant expression. Alternatively, the binding affinity of an Fc domain or a cell-activating antibody comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

The effector function of an Fc domain or an antigen binding molecule as described herein comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of molecules of interest are described in the following references: U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo in an animal model such as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some aspects, the binding of the Fc domain to complement components is reduced, specifically to C1q. Thus, in some aspects, wherein the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether the bispecific antibodies of the invention are able to bind to C1q and therefore have CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

In a specific aspect, the Fc domain that exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising amino acid substitutions L234A, L235A and, optionally, P329G, or a human IgG4 Fc domain comprising amino acid substitutions S228P, L235E and, optionally, P329G (numbered according to the Kabat EU index). More specifically, it is a human IgG1 Fc domain comprising amino acid substitutions L234A, L235A and P329G (numbered according to the Kabat EU index).

Promote heterodimerization Fc Domain Modification

The immunostimulatory antigen-binding molecules that specifically bind to BCMA comprise different antigen-binding sites fused to one or the other of the two subunits of the Fc domain, which can therefore be contained in two different polypeptide chains. The recombinant co-expression of these polypeptides and the subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antigen-binding molecules described herein in recombinant production, it would be advantageous to introduce modifications in the Fc domain of the antibodies described herein that promote the association of the desired polypeptides.

Therefore, in a specific embodiment, an immunostimulatory antigen-binding molecule that specifically binds to BCMA is provided, which comprises an Fc domain composed of a first unit and a second unit capable of stably binding, wherein the Fc domain comprises a modification that promotes the binding of the first unit and the second unit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is performed in the CH3 domain of the Fc domain.

In a specific aspect, the modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" in the other of the two subunits of the Fc domain. Therefore, an immunostimulatory antigen-binding molecule that specifically binds to BCMA is provided, wherein the antibody comprises an Fc domain composed of a first unit and a second unit capable of stably binding, and the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity and/or effector function of the antigen-binding molecule to the Fc receptor, wherein according to the knob-into-hole method, the first unit of the Fc domain comprises a knob and the second unit of the Fc domain comprises a hole. In a specific aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index numbering).

The "knob-and-hole" technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be placed in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing smaller amino acid side chains on the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a complementary cavity of the same or similar size as the protrusion is formed in the interface of the second polypeptide.

Thus, in one aspect, in the CH3 domain of the first unit of the Fc domain of the immunostimulatory antigen-binding molecule that specifically binds to BCMA, amino acid residues are substituted with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first unit, and the protrusion can be positioned in the cavity in the CH3 domain of the second unit, and in the CH3 domain of the second unit of the Fc domain, amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second unit, and the protrusion in the CH3 domain of the first unit can be positioned in the cavity. The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide (e.g., by mutations directed to specific sites or by peptide synthesis). In a specific embodiment, in the CH3 domain of the first unit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second unit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, in addition in the second unit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A).

In another embodiment, the serine residue at position 354 is replaced by a cysteine residue (S354C) in the first subunit of the Fc domain, and the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) in the second subunit of the Fc domain. The introduction of these two cysteine residues leads to the formation of a disulfide bond between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index numbering).

In an alternative aspect, modifications that promote the association of the first and second Fc domain subunits include modifications that mediate electrostatic switching, such as described in PCT Publication No. WO 2009/089004. Typically, this approach involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, thereby making homodimer formation electrostatically unfavorable, but heterodimerization electrostatically favorable.

The C-terminus of the heavy chain of the antibody as reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus, in which one or both C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with P. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one embodiment of all embodiments reported herein, an antigen-binding molecule comprising a heavy chain including a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all the embodiments reported herein, the antigen binding molecule comprises a heavy chain comprising a C-terminal CH3 domain as specified herein, which comprises a C-terminal glycine residue (G446, numbering according to the Kabat EU index).

Fab Modifications in the domain

In one embodiment, an immunostimulatory antigen-binding molecule that specifically binds to BCMA and is characterized by monovalently binding to BCMA is provided, comprising: (a) a first antigen-binding domain that is capable of specifically binding to BCMA; (b) a first polypeptide and a second polypeptide that are linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL or fragments thereof that are linked to each other by a peptide linker, and the second polypeptide comprises one extracellular domain of 4-1BBL or fragments thereof; and (c) an Fc domain composed of a first unit and a second unit that are capable of stably binding, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity and/or effector function of the antigen-binding molecule to an Fc receptor, wherein 4-1BBL The two extracellular domains of 4-1BBL and one extracellular domain of 4-1BBL are fused to the homeostatic domains CL and CH1 via a peptide linker, as appropriate, and they are exchanged with each other so that CH1 is part of a shorter polypeptide ("light chain") and CL is part of a longer polypeptide ("heavy chain"). The homeostatic domains CH1 and CL are exchanged according to the Crossmab technology.

Multispecific antibodies with domain swapping/interchanging in one binding arm (cross-MabVH-VL or cross-MabCH-CL) are described in detail in WO2009/080252 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. They significantly reduce side products caused by mispairing of a light chain targeting a first antigen with an incorrect heavy chain targeting a second antigen (compared to approaches without such domain swapping).

In another aspect, and to further enhance correct pairing, the immunostimulatory antigen binding molecules that specifically bind to BCMA contain various charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the cross-linked or non-cross-linked CH1 and CL domains. In a specific aspect, the present invention relates to an immunostimulatory antigen-binding molecule that specifically binds to BCMA, wherein in the CL domain of the Fab molecule that binds to BCMA, the amino acid at position 123 (EU numbering) is substituted with arginine (R) and the amino acid at position 124 (EU numbering) is substituted with lysine (K), and wherein in one of the CH1 domains, the amino acid at position 147 (EU numbering) and the amino acid at position 213 (EU numbering) are substituted with glutamine (E). In a specific aspect, in the CL domain of the Fab molecule capable of specifically binding to BCMA, the amino acid at position 123 (EU numbering) is substituted with arginine (R) and the amino acid at position 124 (EU numbering) is substituted with lysine (K), and in the CH1 domain of the Fab molecule capable of specifically binding to BCMA, the amino acids at position 147 (EU numbering) and position 213 (EU numbering) are substituted with glutamine (E).

Polynucleotide

The present invention further provides isolated polynucleotides encoding BCMA antibodies or immunostimulatory antigen binding molecules that specifically bind to BCMA as described herein. One or more isolated polynucleotides encoding BCMA antibodies or immunostimulatory antigen binding molecules that specifically bind to BCMA can be expressed as a single polynucleotide encoding the entire antigen binding molecule or as multiple (e.g., two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides can be associated, for example, by disulfide bonds or other means, to form a functional antigen binding molecule. For example, the light chain portion of an immunoglobulin can be encoded by a separate polynucleotide from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an immunoglobulin. In certain aspects, the isolated polynucleotide encodes a complete immunostimulatory antigen binding molecule that specifically binds to BCMA as described herein. In other aspects, the isolated polynucleotide encodes a polypeptide contained in an immunostimulatory antigen binding molecule that specifically binds to BCMA as described herein. In certain aspects, the polynucleotide or nucleic acid is DNA. In other aspects, the polynucleotide of the invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the invention can be single-stranded or double-stranded RNA.

Recombination method

BCMA antibodies or immunostimulatory antigen-binding molecules that specifically bind to BCMA as described herein can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding antigen-binding molecules that specifically bind to BCMA or polypeptide fragments thereof (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such polynucleotides can be easily isolated and sequenced using known methods. In one aspect, a vector comprising one or more of the polynucleotides described herein is provided, preferably a vector is expressed. Expression vectors containing the antibody (fragment) and appropriate transcription/translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, for example, the techniques described in: Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector can be a part of a plasmid, a virus, or a nucleic acid fragment. The expression vector includes an expression cassette in which a polynucleotide encoding an antibody or polypeptide fragment thereof (i.e., a coding region) is cloned in operable association with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it can be considered part of the coding region (if present), but any flanking sequences (e.g., promoters, ribosomal binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, etc.) are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are translated or co-translated into final proteins after proteolysis. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode heterologous coding regions, which may or may not be fused to a polynucleotide encoding an antibody or polypeptide fragment thereof of the invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs (e.g., secretory signaling peptides) or heterologous functional domains. Operably associated means that the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product and the nature of the linker between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide if the promoter is able to affect transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs bulk transcription of DNA only in predetermined cells. In addition to the promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription.

Suitable promoters and other transcription control regions are disclosed herein. Various transcription control regions are known to those skilled in the art. Such regions include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate early promoter, together with intron A), simian virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include regions derived from vertebrate genes (e.g., actin, heat shock protein, bovine growth hormone, and rabbit â-globulin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., promoter-inducing tetracyclins). Similarly, various translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from viral systems (specifically, internal ribosome entry sites or IRES, also known as CITE sequences). Expression cassettes may also include other features, such as origins of replication and/or chromosomal integration elements, such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

The polynucleotides and nucleic acid coding regions as described herein may be combined with other coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if secretion of an antibody or polypeptide fragment thereof is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the antibody or polypeptide fragment thereof as described herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein as the growing protein chain is exported through the rough endoplasmic reticulum. Those of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. In certain aspects, a natural signal peptide is used, such as an immunoglobulin heavy or light chain signal peptide or a functional derivative of the sequence that retains the ability to direct secretion to which it is operably associated. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by the leader sequence of human histoplasminogen activator (TPA) or mouse β-glucuronidase. DNA encoding a short protein sequence that can be used to facilitate later purification (e.g., a histidine tag) or to aid in labeling an antibody as described herein may be included within or at the end of a polynucleotide encoding an antibody or polypeptide fragment thereof as described herein.

In a further aspect, a host cell comprising one or more polynucleotides as described herein is provided. In certain aspects, a host cell comprising one or more vectors as described herein is provided. Polynucleotides and vectors may combine any of the features described herein with respect to polynucleotides and vectors, respectively, alone or in combination. In one aspect, the host cell comprises a vector (e.g., transformed or transfected with the vector) comprising a polynucleotide encoding (a portion of) an antibody as described herein. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce a fusion protein or fragment thereof of the present invention. Host cells suitable for replication and support of expression of antigen binding molecules are well known in the art. Such cells may be transfected or transduced with a particular expression vector, where appropriate, and large numbers of cells containing the vector may be grown to inoculate large-scale fermenters to obtain sufficient amounts of antibody for clinical use. Suitable host cells include prokaryotic microorganisms (e.g., E. coli) or various eukaryotic cells (e.g., Chinese hamster ovary cells (CHO), insect cells, etc.). For example, the polypeptide may be produced in bacteria, particularly where glycosylation is not required. After expression, the polypeptide may be separated from the soluble portion of the bacterial cell paste and may be further purified. In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable hosts for the cloning or expression of polypeptide encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of polypeptides with partially or fully human glycosylation patterns. See: Gerngross, Nat Biotech 22, 1409-1414 (2004); and Li et al., Nat Biotech 24, 210-215 (2006).

Suitable host cells for expression of (glycosylated) polypeptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many bacilliform virus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing the PLANTIBODIES ^™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be used. Other examples of mammalian host cell lines that can be used are: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (e.g., 293 or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)); baby hamster kidney cells (BHK); mouse testicular Sertoli cells (e.g., TM4 cells as described in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumor cells (MMT 060562); TRI cells (as described by Mather et al., Annals NY Acad Sci 383, 44-68 (1982)); MRC 5 cells; and FS4 cells. Other mammalian host cell lines that can be used include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines, such as YO, NS0, P3X63, and Sp2/0. For a review of some mammalian host cell strains suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, and also include transgenic animals, transgenic plants, or cells in cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). Standard techniques are known in the art, and foreign genes can be expressed in these systems. Cells expressing polypeptides comprising heavy or light chains of immunoglobulins can be engineered to also express the other of the immunoglobulin chains, so that the expressed product is an immunoglobulin having both heavy and light chains.

In one aspect, a method for producing an immunostimulatory antigen-binding molecule or a polypeptide fragment thereof that specifically binds to BCMA is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding an antibody or a polypeptide fragment thereof as provided herein under conditions suitable for expression of the antibody or polypeptide fragment thereof, and recovering the antibody or polypeptide fragment thereof as described herein from the host cell (or host cell culture medium).

In certain aspects, the antigen-binding domain (e.g., Fab fragment) capable of specifically binding to BCMA that forms part of an immunostimulatory antigen-binding molecule that specifically binds to BCMA comprises at least an immunoglobulin variable region capable of binding to an antigen. The variable region may form and be derived from a part of a naturally or non-naturally occurring antibody and its fragments. Methods for producing polyclonal antibodies and monoclonal antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly (e.g., as described in U.S. Patent No. 4,186,567), or can be obtained, for example, by screening a combinatorial library comprising variable heavy chains and variable light chains (see, e.g., U.S. Patent No. 5,969,108 to McCafferty).

Immunoglobulins of any animal species can be used in the methods described herein. Non-limiting immunoglobulins that can be used can be of murine, mammalian, or human origin. If the antibody is intended for human use, a chimeric form of an immunoglobulin in which the constant region of the immunoglobulin is from a human can be used. Humanized or fully human forms of immunoglobulins can also be prepared according to methods well known in the art (see, e.g., U.S. Patent No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods, including but not limited to: (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) framework and invariant regions with or without retention of key framework residues (e.g., those important for maintaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interactions) onto human framework and invariant regions, or (c) grafting entire non-human variable domains but "cloaking" them within the human-like segments by replacing surface residues. Humanized antibodies and methods for making them are generally described in, e.g., Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further disclosed in, e.g., Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing FR The specific immunoglobulins according to the present invention are human immunoglobulins. Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are generally described in: van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001); and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and can be derived from human monoclonal antibodies made by the fusion tumor method (see, for example, Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering immunogens to modified transgenic animals, thereby producing intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see, e.g., Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be produced by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

In certain aspects, the antigen binding domains contained in the antigen binding molecules described herein are engineered to have enhanced binding affinity according to methods disclosed, for example, in PCT Publication WO 2012/020006 (see examples related to affinity maturation) or U.S. Patent Application Publication No. 2004/0132066. The ability of the antibodies of the present invention to bind to specific epitopes can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding analysis (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antigen-binding molecules that compete with a reference antibody for binding to a particular antigen. In certain aspects, such competing antigen-binding molecules bind to the same epitope (e.g., a linear or conformational epitope) as that bound by the reference antigen-binding molecule. Detailed exemplary methods for localization of epitopes bound by antigen-binding molecules are provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In an exemplary competition assay, the immobilized antigen is incubated in a solution comprising a first labeled antigen-binding molecule that binds to the antigen and a second unlabeled antigen-binding molecule that is tested for its ability to compete with the first antigen-binding molecule for binding to the antigen. The second antigen-binding molecule may be present in the fusion tumor supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antigen-binding molecule but not the second unlabeled antigen-binding molecule. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample compared to the control sample, this indicates that the second antigen-binding molecule is competing with the first antigen-binding molecule for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Antigen binding molecules that specifically bind to BCMA as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, particle size filtration chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens that bind to the antigen binding molecule can be used. For example, for affinity chromatography purification of the antigen binding molecules of the present invention, a matrix having protein A or protein G can be used. Sequence protein A or G affinity analysis and size exclusion analysis can be used to separate antigen-binding molecules substantially as described in the Examples. The purity of the antibody or fragment thereof can be determined by any of a number of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, etc. For example, the bispecific antibodies shown to be expressed as described in the Examples are intact and properly assembled, as demonstrated by reducing and non-reducing SDS-PAGE.

Measurement

The BCMA antibodies or immunostimulatory antigen-binding molecules that specifically bind to BCMA provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Affinity determination

The affinity of the antigen binding molecules provided herein for the relative target can be determined by surface plasmon resonance (SPR) according to the methods described in the Examples using standard test equipment (e.g., protein instrumentation (Bio-rad)) and, for example, a receptor or target protein that can be obtained by recombinant expression. The affinity of the antigen binding molecules for the target cell antigen can also be determined by surface plasmon resonance (SPR) using standard test equipment (e.g., protein instrumentation (Bio-rad)) and, for example, a receptor or target protein that can be obtained by recombinant expression. According to one aspect, _KD is measured by surface plasmon resonance at 25°C using a Proteon® machine (Bio-Rad).

2. Binding assays and other assays

The binding of antibodies or immunostimulatory antigen binding molecules specifically binding to BCMA provided herein to cells expressing the corresponding receptor can be assessed by, for example, flow cytometry (FACS) using cell lines expressing the specific receptor or target antigen. In one aspect, a CHO-K1 cell line expressing the extracellular domain of BCMA and its non-truncated mutants is used in the binding assay.

In a further aspect, Jurkat-NFkB-luc2_4-1BB cells were used to demonstrate the binding of the antigen binding molecules to 4-1BBL.

3. Activity assay

In one aspect, an assay for identifying the biological activity of a BCMA-4-1BBL antigen binding molecule is provided. The biological activity may include, for example, T cell proliferation and cytokine secretion measured by the methods described in Example 4. Antigen binding molecules having such biological activity in vivo and/or in vitro are also provided.

Pharmaceutical compositions, formulations and routes of administration

In a further aspect, the present invention provides a pharmaceutical composition comprising any of the immunostimulatory antigen-binding molecules specifically binding to BCMA provided herein, which is used, for example, in any of the following treatment methods. In one aspect, the pharmaceutical composition comprises an immunostimulatory antigen-binding molecule specifically binding to BCMA provided herein and at least one pharmaceutically acceptable excipient. In another aspect, the pharmaceutical composition comprises an immunostimulatory antigen-binding molecule specifically binding to BCMA provided herein and at least one additional therapeutic agent (e.g., as described below).

Pharmaceutical compositions as disclosed herein comprise a therapeutically effective amount of one or more antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable formulation. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic or other untoward reactions when administered to animals (e.g., humans) (where appropriate). Based on the present disclosure, one skilled in the art will recognize the preparation of pharmaceutical compositions containing at least one immunostimulatory antigen binding molecule that specifically binds to BCMA and, optionally, additional active ingredients, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. In particular, the composition is a lyophilized formulation or an aqueous solution. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers, and combinations thereof, as known to those skilled in the art.

Parenteral compositions include those designed for administration by injection, such as subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the antigen-binding molecule containing the 4-1BBL trimer can be formulated in an aqueous solution, preferably in a physiologically compatible buffer (such as Hanks' solution, Ringer's solution or physiological saline buffer). The solution may contain a formulating agent, such as a suspending agent, a stabilizer and/or a dispersant. Alternatively, the immunostimulatory antigen-binding molecule specifically bound to BCMA may be in powder form to be reconstituted with a suitable carrier (e.g., sterile pyrogen-free water) before use. Sterile injectable solutions are prepared by incorporating the desired amount of the fusion protein of the present invention into an appropriate solvent with a variety of other ingredients listed below as needed. Sterility can be easily achieved, for example, by filtering through a sterile filter membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and/or other ingredients. For sterile powders for preparing sterile injections, suspensions or emulsions, the preferred preparation method is vacuum drying or freeze drying technology, which can obtain a powder of the active ingredient and any other desired ingredients from a previously filtered sterile liquid medium. If necessary, the liquid medium should be appropriately buffered, and the liquid diluent should be isotonic before injecting sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage and must be resistant to the contaminating action of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be minimized to safe concentrations, for example, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable excipients include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexahydroxyammonium chloride; benzoylmethane chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextrose, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to prepare high concentration solutions. In addition, the suspension of the active compound may be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or carriers include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes.

The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Publishing Company, 1990). Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which are in the form of shaped articles, such as films or microcapsules. In certain embodiments, prolonged absorption of the injectable composition can be produced by using a substance that delays absorption in the composition, such as aluminum monostearate, gelatin, or a combination thereof. Exemplary pharmaceutically acceptable excipients herein further include interstitial drug dispersions, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one embodiment, sHASEGP is conjugated to one or more additional glycosaminoglycanases such as chondroitinase. Exemplary freeze-dried antibody formulations are described in U.S. Patent No. 6,267,958. Water-soluble antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer. In addition to the compositions described above, immunostimulatory antigen binding molecules that specifically bind to BCMA can also be formulated as storage preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, an immunostimulatory antigen binding molecule that specifically binds to BCMA can be formulated using a suitable polymeric or hydrophobic substance (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising immunostimulatory antigen binding molecules that specifically bind to BCMA can be manufactured using conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing methods. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the protein into a pharmaceutically acceptable preparation. The appropriate formulation depends on the chosen route of administration. Immunostimulatory antigen binding molecules that specifically bind to BCMA can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acids or bases. Such salts include acid addition salts, such as those formed with free amino groups of protein components, or with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acids. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous solvents and other protic solvents than the corresponding free base forms. The composition herein may also contain more than one active ingredient required for treating a specific indication, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Formulations for intravital administration are generally sterile. Sterility can be easily achieved, for example, by filtering through a sterile filter membrane.

Treatment methods and compositions

Any immunostimulatory antigen binding molecule that specifically binds to BCMA provided herein can be used alone or in combination in a therapeutic method.

In one embodiment, an immunostimulatory antigen binding molecule that specifically binds to BCMA is provided, which is used as a drug. In a further embodiment, an antigen binding molecule that specifically binds to BCMA and 4-1BB is provided, which is used to treat cancer. In a specific embodiment, an immunostimulatory antigen binding molecule that specifically binds to BCMA is provided, which is used to treat hematological malignancies. The term "hematological malignancies" includes diseases selected from the group consisting of: multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma, as well as acute myeloid leukemia and acute lymphocytic leukemia. In a specific embodiment, an antigen binding molecule that specifically binds to BCMA and 4-1BB is provided for use in treating multiple myeloma (MM).

In certain aspects, an immunostimulatory antigen binding molecule that specifically binds to BCMA is provided for use in a method of treatment. In certain aspects, an immunostimulatory antigen binding molecule that specifically binds to BCMA is provided herein for use in a method of treating an individual with cancer, the method comprising administering to the individual an effective amount of the immunostimulatory antigen binding molecule that specifically binds to BCMA. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.

In one aspect, an immunostimulatory antigen binding molecule that specifically binds to BCMA as described herein is used to inhibit the growth of cancer cells that express BCMA.

In certain aspects, an immunostimulatory antigen binding molecule that specifically binds to BCMA is provided for use in a method of treatment. In certain aspects, provided herein is an immunostimulatory antigen binding molecule that specifically binds to BCMA for use in a method of treating an individual with cancer, the method comprising administering to the individual an effective amount of an immunostimulatory antigen binding molecule that specifically binds to BCMA. In another aspect, an immunostimulatory antigen binding molecule that specifically binds to BCMA is provided for use in a method of treating an individual with a cancer expressing BCMA, in particular a hematological malignancy selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, acute myeloid leukemia, and acute lymphoblastic leukemia, the method comprising administering to the individual an effective amount of an immunostimulatory antigen binding molecule that specifically binds to BCMA. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.

In a further aspect, provided herein is a use of an immunostimulatory antigen binding molecule that specifically binds to BCMA as described herein in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in the treatment of cancer, in particular BCMA expressing cancer. In a further aspect, the medicament is for use in a method of treating cancer, the method comprising administering an effective amount of the medicament to an individual having cancer. In one such aspect, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, e.g., as described below. In another aspect, the medicament is for use in the treatment of BCMA expressing cancer. In a further aspect, the medicament is for use in a method of treating cancer, in particular BCMA expressing cancer, the method comprising administering an effective amount of the medicament to an individual having cancer. In a further aspect, provided herein is a method for treating cancer, in particular BCMA-expressing cancer. In one aspect, the method comprises administering to an individual having cancer an effective amount of an immunostimulatory antigen binding molecule that specifically binds to BCMA. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The "individual" according to any of the above aspects may be a human.

In a further aspect, the present invention provides a pharmaceutical formulation comprising any immunostimulatory antigen binding molecule specifically binding to BCMA as reported herein, which is used, for example, in any of the above treatment methods. In one aspect, the pharmaceutical formulation comprises an immunostimulatory antigen binding molecule specifically binding to BCMA as reported herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical formulation comprises an immunostimulatory antigen binding molecule specifically binding to BCMA as reported herein and at least one additional therapeutic agent.

The immunostimulatory antigen binding molecules specifically binding to BCMA as reported herein can be used alone or in combination with other agents for therapy. For example, the immunostimulatory antigen binding molecules specifically binding to BCMA as reported herein can be co-administered with at least one additional therapeutic agent. Therefore, immunostimulatory antigen binding molecules specifically binding to BCMA as described herein are provided for use in cancer immunotherapy. In certain aspects, an immunostimulatory antigen binding molecule specifically binding to BCMA is provided for use in cancer immunotherapy. The "individual" according to any of the above aspects is preferably a human.

Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the antibodies as reported herein may be administered before, simultaneously and/or subsequently to the administration of the additional therapeutic agent or agents. In one aspect, the administration of the immunostimulatory antigen binding molecule that specifically binds to BCMA and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

Antigen binding molecules as reported herein (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if local treatment is desired, intralesional. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

The immunostimulatory antigen binding molecules that specifically bind to BCMA as described herein will be formulated, dosed and administered in a manner consistent with good medical practice. In this case, factors to be considered include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The immunostimulatory antigen binding molecules that specifically bind to BCMA do not necessarily need to be, but can optionally be formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other therapeutic agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These agents are generally used in the same dosages and by any route as described herein, or about 1% to 99% of the dosages described herein, or in any dosage and by any route determined empirically/clinically to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an immunostimulatory antigen binding molecule that specifically binds to BCMA as described herein (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity or course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. An immunostimulatory antigen binding molecule that specifically binds to BCMA is suitable for administration to the patient once or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.5 mg/kg to 10 mg/kg) of an immunostimulatory antigen binding molecule that specifically binds to BCMA may be an initial candidate dose for administration to a patient, for example, by one or more divided administrations, or by continuous infusion. Depending on the above factors, a typical daily dose may range from about 1 μg/kg to 100 mg/kg or more. For repeated dosing over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the antigen binding molecule would range from 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or, for example, about 6 doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be used. The progress of this treatment is readily monitored by customary techniques and assays.

Other medications and treatments

As previously described, immunostimulatory antigen binding molecules that specifically bind to BCMA can be administered in combination with one or more other agents in therapy. For example, an antigen binding molecule as described herein can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent that can be administered for the treatment of a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may contain any active ingredients suitable for the specific indication being treated, preferably, those having complementary active ingredients that do not adversely affect each other. In some aspects, the additional therapeutic agent is another anticancer agent, such as a microtubule disruptor, an anti-metabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis promoter, or an anti-angiogenic agent. In some aspects, the additional therapeutic agent is an immunomodulator, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent or a cytostatic agent, a cell apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis-inducing agents.

Thus, provided are immunostimulatory antigen binding molecules that specifically bind to BCMA as described herein or pharmaceutical compositions comprising the same for use in treating cancer, wherein the bispecific antibody is administered in combination with chemotherapy, radiation and/or other agents used in cancer immunotherapy.

Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of fusion protein used, the type of disease or treatment, and other factors as described above. The bispecific antigen-binding molecules or antibodies of the present invention are generally used in the same dosage and by the administration route as described herein, or about 1% to 99% of the dosage described herein, or in any dosage and by any route determined to be appropriate empirically/clinically. Such combination therapies described above include combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case administration of the bispecific antigen-binding molecules or antibodies of the invention may be carried out before, simultaneously with, and/or after administration of the additional therapeutic agents and/or adjuvants.

In a further aspect, an immunostimulatory antigen binding molecule as described herein that specifically binds to BCMA is provided for use in treating cancer, in particular cancer expressing BCMA, wherein the bispecific antigen binding molecule is administered in combination with another immunomodulator. The term "immunomodulator" refers to any substance including monoclonal antibodies that affect the immune system. The molecules of the present invention may be considered immunomodulators. Immunomodulators may be used as anti-tumor agents for the treatment of cancer. In one embodiment, immunomodulators include, but are not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivolumab or pembrolizumab), PD-L1 antibodies (e.g., atezolizumab, avelumab or durvalumab), OX-40 antibodies, 4-1BB antibodies, and GITR antibodies. Such combination therapies described above include combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case administration of the bispecific antigen-binding molecule may be prior to, concurrently with, and/or after administration of the additional therapeutic agents and/or adjuvants.

and T Cell Bispecific Antibody Panel

In one aspect, an immunostimulatory antigen binding molecule that specifically binds to BCMA can be administered in combination with a T cell activating anti-CD3 bispecific antibody. The T cell activating anti-CD3 bispecific antibody is specific for a tumor-associated antigen, such as GPRC5D, CD38 or BCMA. In one aspect, the T cell activating anti-CD3 bispecific antibody is an anti-GPRC5D/anti-CD3 bispecific antibody.

In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises at least one antigen-binding domain capable of specifically binding to GPRC5D, the at least one antigen-binding domain comprising: a heavy chain variable region ( _VH GPRC5D) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 140, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 141, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 142; and a light chain variable region ( _VL GPRC5D) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 144, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 145. In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises at least one antigen-binding domain capable of specifically binding to GPRC5D, the at least one antigen-binding domain comprising: a heavy chain variable region ( _VH GPRC5D) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150; and a light chain variable region ( _VL GPRC5D) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154 and SEQ ID NO: 155.

In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises at least one antigen-binding domain capable of specifically binding to GPRC5D, the at least one antigen-binding domain comprising: a heavy chain variable region (VH GPRC5D) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 146 and a light chain variable region ( _VL GPRC5D) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ _{ID NO} : 147. Specifically, the antigen-binding domain capable of specifically binding to GPRC5D comprises a heavy chain variable region ( _VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 146, and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 147.

In another aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises at least one antigen-binding domain capable of specifically binding to GPRC5D, the at least one antigen-binding domain comprising: a heavy chain variable region ( _VH GPRC5D) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 156, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 157, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region ( _VL GPRC5D) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 159, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 160, and (vi) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 161. In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises at least one antigen-binding domain capable of specifically binding to GPRC5D, the at least one antigen-binding domain comprising: a heavy chain variable region ( _VH GPRC5D) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, and SEQ ID NO: 167; and a light chain variable region ( _VL GPRC5D) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, and SEQ ID NO: 172. In another aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to GPRC5D, the at least one antigen-binding domain comprising (a) a heavy chain variable region (VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 146 and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 147; or (b) a heavy chain variable region ( _VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 153; or (c) a heavy chain variable region ( _VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 146 and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 153. or (d) a heavy chain variable region ( _VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 170; or (e) a heavy chain variable region ( _VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 164 _and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 169.

In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises an antigen-binding domain capable of specifically binding to CD3, the antigen-binding domain comprising: a heavy chain variable region ( _VH CD3) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; and a light chain variable region ( _VL CD3) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178. In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises an antigen-binding domain capable of specifically binding to CD3, wherein the antigen-binding domain comprises: a heavy chain variable region ( _VH CD3) comprising the amino acid sequence of SEQ ID NO: 179; and a light chain variable region ( _VL CD3) comprising the amino acid sequence of SEQ ID NO: 180.

In another aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises an antigen-binding domain capable of specifically binding to CD3, the antigen-binding domain comprising: a heavy chain variable region ( _VH CD3) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 181, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 182, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 183; and a light chain variable region ( _VL CD3) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 184, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 185, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 186. In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises an antigen-binding domain capable of specifically binding to CD3, wherein the antigen-binding domain comprises: a heavy chain variable region ( _VH CD3) comprising the amino acid sequence of SEQ ID NO: 187; and a light chain variable region ( _VL CD3) comprising the amino acid sequence of SEQ ID NO: 188.

In one aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises: an antigen-binding domain capable of specifically binding to CD3, comprising a heavy chain variable region ( _VH CD3) comprising the amino acid sequence of SEQ ID NO: 179 and a light chain variable region ( _VL CD3) comprising the amino acid sequence of SEQ ID NO: 180; and two antigen-binding domains capable of specifically binding to GPRC5D, wherein the antigen-binding domains capable of specifically binding to GPRC5D each comprise a heavy chain variable region ( _VH GPRC5D) comprising the amino acid sequence of SEQ ID NO: 146 and a light chain variable region ( _VL GPRC5D) comprising the amino acid sequence of SEQ ID NO: 147. In a specific aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises: a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 132, two polypeptides at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 133, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 134, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 135. In a further specific aspect, the bispecific antibody comprises a polypeptide sequence of SEQ ID NO: 132, two polypeptide sequences of SEQ ID NO: 133, a polypeptide sequence of SEQ ID NO: 134, and a polypeptide sequence of SEQ ID NO: 135 (GPRC5D CD3 TCB). In a specific aspect, the anti-GPRC5D/anti-CD3 bispecific antibody is forimtamig.

In another aspect, the anti-GPRC5D/anti-CD3 bispecific antibody comprises a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 136, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 137, a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 138, and a polypeptide at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 139. In one aspect, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 136, the polypeptide sequence of SEQ ID NO: 137, the polypeptide sequence of SEQ ID NO: 138, and the polypeptide sequence of SEQ ID NO: 139 (GPRC5D CD3 1+1 bispecific antibody).

In a further aspect, the T cell activating anti-CD3 bispecific antibody is an anti-BCMA/anti-CD3 bispecific antibody. In one aspect, the anti-BCMA/anti-CD3 bispecific antibody is in a 2+1 format. In one aspect, the anti-BCMA/anti-CD3 bispecific antibody comprises: an amino acid sequence of SEQ ID NO: 191, an amino acid sequence of SEQ ID NO: 192, an amino acid sequence of SEQ ID NO: 193, and an amino acid sequence of SEQ ID NO: 194. In another aspect, the anti-BCMA/anti-CD3 bispecific antibody is in a 1+1 format. In one aspect, the anti-BCMA/anti-CD3 bispecific antibody comprises: an amino acid sequence of SEQ ID NO: 195, an amino acid sequence of SEQ ID NO: 196, an amino acid sequence of SEQ ID NO: 197, and an amino acid sequence of SEQ ID NO: 198. In one aspect, the anti-BCMA/anti-CD3 bispecific antibody comprises: an amino acid sequence of SEQ ID NO: 199, an amino acid sequence of SEQ ID NO: 200, an amino acid sequence of SEQ ID NO: 201, and an amino acid sequence of SEQ ID NO: 202.

In another aspect, the T cell activating anti-CD3 bispecific antibody is an anti-FcRH5/anti-CD3 bispecific antibody. Anti-FcRH5/anti-CD3 bispecific antibodies are disclosed, for example, in WO 2016/205520 A1.

Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the therapeutic agent can be administered before, simultaneously and/or subsequently to the administration of the other therapeutic agent or agents. In one embodiment, administration of the therapeutic agent and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

Finished Products

In another aspect of the invention, an article of manufacture containing materials useful for treating, preventing and/or diagnosing the above-mentioned conditions is provided. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Such containers can be formed from a variety of materials, such as glass or plastic. The container contains a composition alone or in combination with another composition that is effective for treating, preventing and/or diagnosing the symptoms, and may have a sterile access hole (for example, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an immunostimulatory antigen binding molecule that specifically binds to BCMA as disclosed herein. The label or drug package insert indicates that the composition is used to treat the selected condition. Furthermore, the article of manufacture may comprise (a) a first container containing a composition, wherein the composition comprises an immunostimulatory antigen binding molecule that specifically binds to BCMA; and (b) a second container containing a composition, wherein the composition comprises other additional toxic agents or other therapeutic agents. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a specific disease. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it may further comprise other materials, including other buffers, diluents, filters, needles, and syringes. Table B ( Sequence ) : SEQ ID NO: Name sequence 1 BCMA (E04_VH1a)- CDR-H1 GYTFTNYWMH 2 BCMA (E04_VH1a)- CDR-H2 IIHPNSGSTNYNEKFQG 3 BCMA (E04_VH1a)- CDR-H3 GIYDYPFAY 4 BCMA (E04_VL1f)- CDR-L1 RASESVSIHGTHLMH 5 BCMA (E04_VL1f)- CDR-L2 AASSLQS 6 BCMA (E04_VL1f)- CDR-L3 QQSIEDPYT 7 BCMA (E04_VL1a)- CDR-L2 AASNLES 8 BCMA (E04_VL1c)- CDR-L2 AASNLQS 9 Human (hu) 4-1BBL (71-248) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 10 Human (hu) 4-1BBL (71-254) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE 11 Dimeric hu 4-1BBL (71-248) linked by G4SG4 linker REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGG SGGGGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 12 Dimeric hu 4-1BBL (71-254) linked by G4SG4 linker REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSEGGGG SGGGGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE 13 BCMA (E04_VH1a) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQGTLVTVSS 14 BCMA (E04_VH1b) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQGTLVTVSS 15 BCMA (E04_VL1f) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 16 BCMA (E04_VL1a) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 17 BCMA (E04_VL1b) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 18 BCMA (E04_VL1c) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 19 BCMA (E04_VL1d) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 20 BCMA (E04_VL1e) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK twenty one Dimeric 4-1BB ligand (71-248) - CL Fc knob REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPA GLGGGGSGGGGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLG LFRVTPEIPAGLGGGGSGGGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP twenty two Monomer 4-1BB ligand (71-248)-CH1 REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC twenty three BCMA (E04_VH1a) VH CH1 (EE)- Fc PGLALA QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP twenty four BCMA (E04_VL1f) VL- CL (P1AG7400) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25 BCMA (E04_VL1a) VL- CL (P1AG7409) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 26 BCMA (54_VH2a)- CDR-H1 GFTFSNAWMD 27 BCMA (54_VH2a)- CDR-H2 QITAKSNNYATYYADSVKG 28 BCMA (54_VH2a)- CDR-H3 DG 29 BCMA (54_VH1b)- CDR-H2 QITAKSNNYATYYAAPVKG 30 BCMA (54_VL2a)- CDR-L1 RASEDIRNGLA 31 BCMA (54_VL2a)- CDR-L2 NANSLHT 32 BCMA (54_VL2a)- CDR-L3 EDTSKYPYT 33 BCMA (54_VH2a) - VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVSQITAKSNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDDGYHWGQGTLVTVSS 34 BCMA (54_VL2a) - VL DIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 35 BCMA (54_VH1b) - VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWIAQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 36 BCMA (54_VL1a) - VL AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 37 BCMA (54_VH2a) VH CH1 (EE)- Fc PGLALA (P1AG7397) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVSQITAKSNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 38 BCMA (54_VL2a) VL- CL (RK) (P1AG7397) DIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 39 BCMA (54_VH1b) VH CH1 (EE)- Fc PGLALA (P1AG7422) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWIAQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 40 BCMA (54_VL1a) VL- CL (RK) (P1AG7422) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 41 Parental BCMA (E04) VH QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGIIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGIYDYPFAYWGQGTLVTVSS 42 Parental BCMA (E04) VL DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSETDFTLNIHPVEEEDAATYFCQQSIEDPYTFGGGTKLEIK 43 Human IGHJ germline IGHJ4*01 CDR3 back frame YFDYWGQGTLVTVSS 44 Human IGKJ germline IGKJ4*01 CDR3 back frame LTFGGGTKVEIK 45 BCMA (E04_VL1g) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVEIK 46 BCMA (E04_VL1h) VL DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVEIK 47 BCMA-54 VH EVQLVESGGGLVKPGESLRLSCAASGFTFSNAWMDWVRQAPGKRLEWIAQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKKEDTAVYYCTDDGYHWGQGTLVTVSS 48 BCMA-54 VL AIQMTQSPSSSLSASVGDRVTIACRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDEAIYYCEDTSKYPYTFGQGTKLEIK 49 Human IGHJ germline IGHJ1*01 CDR3 back frame AEYFQHWGQGTLVTVSS 50 Human IGKJ germline IGKJ2*01 CDR3 back frame YTFGQGTKLEIK 51 BCMA CDR-H2 RIKSKTDGGTTDYAAPVKG 52 BCMA CDR-L2 AASSLQS 53 BCMA (54_VH1a) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 54 BCMA (54_VH1a_Y292D) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGQITAKSNNYATDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 55 BCMA (54_VH1c_huCDR2) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 56 BCMA (54_VH1a_W197Y) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAYMDWVRQAPGKGLEWVGQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 57 BCMA (54_VL1a_L2_GL) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 58 BCMA (54_VL2a_L2_GL) DIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 59 BCMA (54_VL1a_L1_pGL) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNDLGWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 60 BCMA (54_VL1a_N651A) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 61 BCMA (54_VL1a_N651A_N695S) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 62 BCMA (54_VL1a_H698Q) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 63 BCMA (54_VL1a_T699S) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 64 BCMA (54_VL1a_H698Q_T699S) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 65 BCMA (54_VH1a) VH-CH1-Fc 杵PGLALA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 66 BCMA (54_VL1a_L1_pGL) VL-Cκ AIQMTQSPSSSLSASVGDRVTITCRASEDIRNDLGWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 67 Fc fragment DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSP 68 BCMA (54_VH1a_Y292D) VH-CH1-Fc 杵PGLALA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGQITAKSNNYATDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 69 BCMA (54_VL1a) VL-Cκ AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 70 BCMA (54_VL1a_T699S) VL-Cκ AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 71 BCMA (54_VL2a) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 72 BCMA (54_VH1b) VH-CH1-Fc 杵PGLALA EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWIAQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 73 BCMA (54_VL1a_N651A) VL-Cκ AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 74 BCMA (54_VL1a_N651A_N695S) VL-Cκ AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 75 BCMA (54_VH2a) VH-CH1-Fc Knob PGLALA EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVSQITAKSNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 76 BCMA (54) heavy chain (Fc-knob PGLALA) EVQLVESGGGLVKPGESLRLSCAASGFTFSNAWMDWVRQAPGKRLEWIAQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKKEDTAVYYCTDDGYHWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 77 BCMA (54) Light Chain AIQMTQSPSSSLSASVGDRVTIACRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDEAIYYCEDTSKYPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 78 BCMA (E04_VH1a) VH-CH1-Fc 杵PGLALA QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 79 BCMA (E04_VL1a) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 80 BCMA (E04_VL1b) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 81 BCMA (E04_VL1d) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 82 BCMA (E04_VL1e) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 83 BCMA (E04_VL1f) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 84 BCMA (E04_VL1g) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 85 BCMA (E04_VL1h) VL-Cκ DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 86 BCMA (E04_VH1b) VH-CH1-Fc Knob PGLALA QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 87 BCMA (E04) heavy chain (Fc knob PGLALA) QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGIIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSSLTSEDSAVYYCARGIYDYPFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 88 BCMA (E04) Light Chain DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSETDFTLNIHPVEEEDAATYFCQQSIEDPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 89 5D5 LM4 heavy chain (P1AH1062) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGLSWVRQAPGKGLEWVSLIDSSGSSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEHGLFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGRRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSA SLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS 90 5D5 LM4 Light Chain (P1AH1062) EIVLTQSPGTLSLSPGERATLSCKASQDIDDAINWYQQKPGQAPRLLIYDASLRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSLRTPITFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 91 BCMA (PR) VH CH1 –Fc PGLALA (P1AF7814) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAITASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMSLWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 92 BCMA (PR) VL-CL (P1AF7814) EIVLTQSPGTLSLSPGERATLSCRASQSVSAYYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYERWPLTFGQGTKVE IKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 93 Human BCMA Uniprot Accession Number Q02223 MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR 94 Human CD28 Uniprot Accession No. P10747 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIY FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 95 Human kappa CL domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 96 Human λ CL domain QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 97 IgG CH1 domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 98 CH1 domain linker EPKSC 99 Hinge DKTHTCPXCP, where X is S or P 100 Hinge Medium HTCPXCP, where X is S or P 101 Short hinge CPXCP, where X is S or P 102 IgG CH2 domain APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQESTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 103 IgG CH3 domain GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 104 IgG1, Caucasian allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 105 IgG1, African American allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 106 IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 IgG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 108 IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 109 hu IgG1 heavy chain constant region with mutations L234A, L235A and P329G ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 110 Human FcγRIIIa, Uniprot Accession No. P08637 MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK 111 Peptide linker (G4S) GGGGS 112 Peptide linker G4SG4 GGGGSGGGG 113 Peptide linker (G4S)2 GGGGSGGGGS 114 Peptide Linker (SG4)2 SGGGGSGGGG 115 Peptide Linker G4(SG4)2 GGGGSGGGGSGGGG 116 Peptide linker GSPGSSSSGS 117 (G4S)3 peptide linker GGGGSGGGGSGGGGS 118 (G4S)4 Peptide Linker GGGGSGGGGSGGGGSGGGGS 119 Peptide linker GSGSGSGS 120 Peptide linker GSGSGNGS 121 Peptide linker GGSGSGSG 122 Peptide linker GGSGSG 123 Peptide linker GGS 124 Peptide linker GGSGNGSG 125 Peptide linker GGNGSGSG 126 Peptide linker GGNGSG 127 Human BCMA extracellular domain MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA 128 Mascot BCMA (Cercopithecus edulis) NCBI XP_005591343.1 mlqmarqcsqneyfdsllhdckpcqlrcsstppltcqrycnasmtnsvkgmnailwtclglsliislavfvltfllrkmsseplkdefkntgsgllgmanidlekgrtgdeivlprgleytveectcedciknkpkvdsdhcfplpameegatilvttktndycnslsaalsvteieksisar 129 Mascot BCMA extracellular domain mlqmarqcsqneyfdsllhdckpcqlrcsstppltcqrycnasmtnsvkgmna 130 BCMA (E04_VL1g) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVEIK 131 BCMA (E04_VL1h) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVEIK 132 GPRC5D (5E11) hu IgG1 VH-CH1 "EE" Fc PGLALA (P1AE6625) EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGT MVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 133 GPRC5D (5E11)VL-Cκ「RK」 (P1AE6625) EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTRESPLTFGQGTRLE IKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 134 GPRC5D (5E11) VH-CH1(EE)-CD3 (Cl22) VL-CH1-Fc (Pestrel, P329G LALA) (P1AE6625) EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGT KLTVLSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 135 CD3 (Cl22) VH-CL (P1AE6625) EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWG QGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 136 GPRC5D hu IgG1 VH-CH1 "EE" Fc PGLALA (P1AE3357) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 137 GPRC5D VL-Cκ「RK」 (P1AE3357) DIQMTQSPSSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK RTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 138 CD3 VL-CH1-Fc (PESTLE, P329G LALA) (P1AE3357) QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 139 CD3 VH-CL (P1AE3357) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWG QGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 140 GPRC5D (5E11) - CDR-H1 KYAMA 141 GPRC5D (5E11) - CDR-H2 SISTGGVNTYYADSVKG 142 GPRC5D (5E11) - CDR-H3 HTGDYFDY 143 GPRC5D (5E11) - CDR-L1 RASQSVSISGINLMN 144 GPRC5D (5E11) - CDR-L2 HASILAS 145 GPRC5D (5E11) - CDR-L3 QQTRESPLT 146 GPRC5D (5E11) VH1c EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 147 GPRC5D (5E11) VL2b EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTRESPLTFGQGTRLEIK 148 GPRC5D (5E11) VH1a EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYRDSVKARFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 149 GPRC5D (5E11) VH1b ELQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYRDSVKARFTISRDNAKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 150 GPRC5D (5E11) VH1d ELQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 151 GPRC5D (5E11) VL1a DIVMTQSPDSLAVSLGERATINCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTRESPLTFGQGTRLEIK 152 GPRC5D (5E11) VL1c DIVMTQSPDSLAVSLGERATINCKSSQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTRESPLTFGQGTRLEIK 153 GPRC5D (5E11) VL2a EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPRLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTRESPLTFGQGTRLEIK 154 GPRC5D (5E11) VL3a DIQMTQSPSSSLSASVGDRVTITCRASQSVSISGINLMNWYQQKPGKQPKLLIYHASILASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRESPLTFGQGTRLEIK 155 GPRC5D (5E11) VL3b DIQMTQSPSSSLSASVGDRVTITCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRESPLTFGQGTRLEIK 156 GPRC5D (5F11) - CDR-H1 NYGMA 157 GPRC5D (5F11) - CDR-H2 SISTGGGNTYYRDSVKG 158 GPRC5D (5F11) - CDR-H3 HDR 159 GPRC5D (5F11) - CDR-L1 RSSKSLLHSNGITYVY 160 GPRC5D (5F11) - CDR-L2 RMSNRAS 161 GPRC5D (5F11) - CDR-L3 GQLLENPYT 162 GPRC5D (5F11) VH1a QVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRHDRGGLYWGQGTMVTVSS 163 GPRC5D (5F11) VH1b EVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRHDRGGLYWGQGTMVTVSS 164 GPRC5D (5F11) VH1c QVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRHDRGGLYWGQGTMVTVSS 165 GPRC5D (5F11) VH1d EVQLVESGGGVVQPGRSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRHDRGGLYWGQGTMVTVSS 166 GPRC5D (5F11) VH2b EVQLVESGGGLVQPGGSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYRDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRHDRGGLYWGQGTMVTVSS 167 GPRC5D (5F11) VH2d EVQLVESGGGLVQPGGSLRLSCAASGFSFSNYGMAWVRQAPGKGLEWVASISTGGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRHDRGGLYWGQGTMVTVSS 168 GPRC5D (5F11) VL1a DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYVYWYLQKPGQSPQVLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHCGQLLENPYTFGQGTKLEIK 169 GPRC5D (5F11) VL1b DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYVYWYLQKPGKSPQVLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHCGQLLENPYTFGQGTKLEIK 170 GPRC5D (5F11) VL2a DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYVYWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHCGQLLENPYTFGQGTKLEIK 171 GPRC5D (5F11) VL2b DIVMTQSPDSLAVSLGERATINCKSSSLLHSNGITYVYWYQQKPGQPPKLLIYRMSNLASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYHCGQLLENPYTFGQGTKLEIK 172 GPRC5D (5F11) VL2c EIVLTQSPGTLSLSPGERATLSCRASKSLLHSNGITYVYWYQQKPGQAPRLLIYRMSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYHCGQLLENPYTFGQGTKLEIK 173 CD3 (Cl22) CDR-H1 SYAMN 174 CD3 (Cl22) CDR-H2 RIRSKYNNYATYYADSVKG 175 CD3 (Cl22) CDR-H3 HTTFPSSYVSYYGY 176 CD3 (Cl22) CDR-L1 GSSTGAVTTSNYAN 177 CD3 (Cl22) CDR-L2 GTNKRAP 178 CD3 (Cl22) CDR-L3 ALWYSNLWV 179 CD3 (Cl22) VH EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS 180 CD3 (Cl22) VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 181 CD3 (V9) CDR-H1 GYSFTGYTMN 182 CD3 (V9) CDR-H2 LINPYKGVSTYNQKFKD 183 CD3 (V9) CDR-H3 SGYYGDSDWYFDV 184 CD3 (V9) CDR-L1 RASQDIRNYLN 185 CD3 (V9) CDR-L2 YTSRLES 186 CD3 (V9) CDR-L3 QQGNTLPWT 187 CD3 (V9) VH EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS 188 CD3 (V9) VL DIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK 189 Dimeric hu 4-1BBL (71-248) linked by a (G4S) ₂ linker REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGS GGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 190 Dimeric hu 4-1BBL (71-254) linked by a (G4S) ₂ linker REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGS GGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE 191 Anoukitumomab hu IgG1_Fc PGLALA (P1AF0105) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 192 Anoukitumumab VL-Cκ TNFRSF17 (P1AF0105) EIVLTQSPGTLSLSPGERATLSCRASQSVSDEYLSWYQQKPGQAPRLLIHSASTRATGIPDRFSGSGSGTDFTLAISRLEPEDFAVYYCQQYGYPPDFTFGQGTKVEI KRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 193 Anoukitumomab hu IgG1_Fc pestle PGLALA (P1AF0105) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDNAMGWVRQAPGKGLEWVSAISGPGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVLGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTK LTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 194 Anoukitumumab VL-Cκ CD3 (P1AF0105) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 195 Elenatumab hu IgG1_Fc PGLALA (P1AH5054) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 196 Elenatumab VL-Cκ TNFRSF17 (P1AH5054) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEI KRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 197 Elenatumab hu IgG1_Fc knob PGLALA (P1AH5054) DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLE IKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 198 Elenatumab VL-Cκ CD3 (P1AH5054) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCardRPSYYVLDYWGQG TTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 199 Terituzumab anti-TNFRSF17 heavy chain QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 200 Terituzumab anti-TNFRSF17 light chain SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 201 Terituzumab anti-CD3 heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFA YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 202 Terituzumab anti-CD3 light chain QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 203 huBCMA_R27P MLQMAGQCSQNEYFDSLLHACIPCQLPCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKISSEPLEDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISARTRTRPLEQKLISEEDLAANDILDYKDDDDKV 204 huBCMA_S30del MLQMAGQCSQNEYFDSLLHACIPCQLRCS_NTPPLTCQRYCNASVTNSVKGTNAILWTCLLGLSLIISLAVFVLMFLLRKISSEPLEDEFKNTGSGLLGMANIDLEKS RTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISARTRTRPLEQKLISEEDLAANDILDYKDDDDKV 205 huBCMA_P33S MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTSPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKISSEPLEDEFKNTGSGLLGMANIDLEKSRTGDEIIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISARTRTRPLEQKLISEEDLAANDILDYKDDDDKV 206 huBCMA_P34del MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTP_LTCQRYCNASVTNSVKGTNAILWTCLLGLSLIISLAVFVLMFLLRKISSEPLEDEFKNTGSGLLGMANIDLEKS RTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISARTRTRPLEQKLISEEDLAANDILDYKDDDDKV 207 BCMA (E04_VH1a) VH CH1 (EE)- Fc PGLALA (P1AG7400) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 208 Dimeric 4-1BB ligand (71-248) - CL Fc knob (P1AG7400) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPA GLGGGGSGGGGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLG LFRVTPEIPAGLGGGGSGGGGRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

For general information on human immunoglobulin light and heavy chain nucleotide sequences, see: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chains are numbered and referenced according to the numbering system of Kabat as defined above (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

The following numbered paragraphs (paras) describe aspects of the invention: 1. An immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) an antigen-binding domain comprising (i) a heavy chain variable region (VH BCMA) comprising a heavy chain complement determining region CDR-H1 of SEQ ID NO: 1 (GYTFTNYWMH), a CDR-H2 of SEQ ID NO: 2 (IIHPNSGSTNYNEKFQG), and a CDR-H3 of SEQ ID NO: 3 (GIYDYPFAY); and (ii) a light chain variable region (VL BCMA) selected from the group consisting of: (a) a VL comprising SEQ ID NO: 4 (RASESVSIHGTHLMH) : a light chain complementary determining region CDR-L1 of SEQ ID NO: 5 (AASSLQS), CDR-L2 of SEQ ID NO: 6 (QQSIEDPYT); and (b) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), CDR-L2 of SEQ ID NO: 7 (AASNLES), and CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); and (c) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), CDR-L2 of SEQ ID NO: 8 (AASNLQS), and CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); (b) The first polypeptide and the second polypeptide are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL or fragments thereof linked to each other by a peptide linker, and the second polypeptide comprises one extracellular domain of 4-1BBL or fragment thereof, and (c) an Fc domain, which is composed of a first unit and a second unit. 2. The immunostimulatory antigen binding molecule of paragraph 1, wherein the extracellular domain of 4-1BBL or fragment thereof comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. 3. The immunostimulatory antigen binding molecule of paragraph 1 or 2, wherein the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189 and SEQ ID NO: 190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 4. The immunostimulatory antigen binding molecule of any of paragraphs 1 to 3, wherein the antigen binding domain comprises (a) VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and VL BCMA comprising the amino acid sequence of SEQ ID NO: 15, or (b) VH BCMA comprising the amino acid sequence of SEQ ID NO: 13 and VL BCMA comprising the amino acid sequence of SEQ ID NO: 16. 5. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 4, wherein the antigen binding domain is a Fab molecule. 6. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 5, wherein the Fc domain composed of the first unit and the second unit is IgG, in particular an IgG1 Fc domain. 7. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 6, wherein the Fc domain is a human Fc domain. 8. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 7, wherein the Fc comprises a modification that promotes the association of the first unit and the second unit of the Fc domain. 9. The immunostimulatory antigen binding molecule of paragraph 8, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index). 10. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 9, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. 11. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 10, wherein the Fc domain is of the human IgG1 subtype and comprises the amino acid mutations L234A, L235A and P329G (numbering according to the Kabat EU index). 12. The immunostimulatory antigen-binding molecule of any one of paragraphs 1 to 11, comprising (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CL domain; (aiii) a CL domain, fused at the C-terminus to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (aiv) one of the subunits of an Fc domain (e.g., a first subunit); (b) a second polypeptide comprising: (bi) a third extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CH1 domain; and (bii) a CH1 domain; (c) a third polypeptide comprising: (ci) a Fab The invention relates to a Fab molecule comprising a heavy chain, which is fused at the C-terminus to the N-terminus of another one of the subunits of the Fc domain (e.g., the second subunit); and (cii) another one of the subunits of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule. 13. The immunostimulatory antigen-binding molecule of paragraph 12, wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). 14. An immunostimulatory antigen-binding molecule according to any one of claims 1 to 13, comprising (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:25. 15. An immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) an antigen-binding domain comprising (i) a heavy chain variable region (VL BCMA) selected from the group consisting of: (a) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 27 (QITAKSNNYATYYADSVKG), and a CDR-H3 of SEQ ID NO: 28 (DGYH); and (b) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 29 (QITAKSNNYATYYAAPVKG), and a CDR-H3 of SEQ ID NO: 2 ID NO: 28 (DGYH) CDR-H3; and (ii) a light chain variable region (VL BCMA) comprising a light chain complementation determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), CDR-L2 of SEQ ID NO: 31 (NANSLHT) and CDR-L3 of SEQ ID NO: 32 (EDTSKYPYT); (b) a first polypeptide and a second polypeptide, which are linked to each other by a disulfide bond, wherein the antigen binding molecule is characterized in that the first polypeptide comprises two extracellular domains or fragments thereof of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one extracellular domain or fragment thereof of 4-1BBL, and (c) an Fc domain, which is composed of a first unit and a second unit. 16. The immunostimulatory antigen binding molecule of paragraph 15, wherein the extracellular domain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 17. The immunostimulatory antigen binding molecule of paragraph 15 or 16, wherein the immunostimulatory antigen binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189 and SEQ ID NO: 190, and characterized in that the second polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 18. The immunostimulatory antigen binding molecule of any one of paragraphs 15 to 17, wherein the antigen binding domain comprises (a) VH BCMA comprising the amino acid sequence of SEQ ID NO: 33 (VH2a) and VL BCMA comprising the amino acid sequence of SEQ ID NO: 34 (VL2a), or (b) VH BCMA comprising the amino acid sequence of SEQ ID NO: 35 (VH1b) and VL BCMA comprising the amino acid sequence of SEQ ID NO: 36 (VL1a). 19. The immunostimulatory antigen binding molecule of any one of paragraphs 15 to 19, wherein the antigen binding domain is a Fab molecule. 20. The immunostimulatory antigen binding molecule of any one of paragraphs 15 to 19, wherein the Fc domain composed of the first unit and the second unit is IgG, in particular an IgG1 Fc domain. 21. The immunostimulatory antigen binding molecule of any of paragraphs 15 to 20, wherein the Fc domain is a human Fc domain. 22. The immunostimulatory antigen binding molecule of any of paragraphs 15 to 21, wherein the Fc comprises a modification that promotes association of the first unit and the second unit of the Fc domain. 23. The immunostimulatory antigen binding molecule of paragraph 22, wherein the first unit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second unit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index). 24. The immunostimulatory antigen binding molecule of any one of paragraphs 15 to 23, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. 25. The immunostimulatory antigen binding molecule of any one of paragraphs 15 to 24, wherein the Fc domain is of human IgG1 subtype and comprises the amino acid mutations L234A, L235A and P329G (according to the Kabat EU index numbering). 26. The immunostimulatory antigen-binding molecule of any one of paragraphs 15 to 25, comprising (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CL domain; (aiii) a CL domain, fused at the C-terminus to the N-terminus of one of the subunits of an Fc domain (e.g., a first subunit); and (aiv) one of the subunits of an Fc domain (e.g., a first subunit); (b) a second polypeptide comprising: (bi) a third extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CH1 domain; and (bii) a CH1 domain; (c) a third polypeptide comprising: (ci) The heavy chain of the Fab molecule is fused at the C-terminus to the N-terminus of another one of the subunits of the Fc domain (e.g., the second subunit); and (cii) another one of the subunits of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule. 27. The immunostimulatory antigen-binding molecule of paragraph 26, wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). 28. The immunostimulatory antigen binding molecule of any one of paragraphs 15 to 27, comprising (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 37, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 38; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 39, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 40. 29. One or more isolated polynucleotides encoding the immunostimulatory antigen binding molecule of any one of claims 1 to 28. 30. One or more vectors, in particular expression vectors, comprising the polynucleotide of claim 29. 31. A host cell comprising a polynucleotide as described in paragraph 29 or a vector as described in paragraph 30. 32. A method for producing an immunostimulatory antigen binding molecule that specifically binds to BCMA, comprising the steps of: a) culturing the host cell as described in paragraph 31 under conditions suitable for expressing the immunostimulatory antigen binding molecule, and optionally b) recovering the immunostimulatory antigen binding molecule. 33. An immunostimulatory antigen binding molecule that specifically binds to BCMA, produced by the method as described in paragraph 32. 34. A pharmaceutical composition comprising the immunostimulatory antigen binding molecule as described in any one of paragraphs 1 to 28 or 33 and at least one pharmaceutically acceptable excipient. 35. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 28 or 33 or the pharmaceutical composition of paragraph 34 for use as a medicament. 36. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 28 or 33 or the pharmaceutical composition of paragraph 34 for use in enhancing (a) T cell activation or (b) T cell effector function. 37. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 28 or 33 or the pharmaceutical composition of paragraph 34 for use in treating a disease. 38. The immunostimulatory antigen binding molecule or the pharmaceutical composition for use of paragraph 37, wherein the disease is cancer. 39. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 28 or 33 or the pharmaceutical composition of paragraph 34 for use in treating cancer, wherein the use is for administration in combination with chemotherapy, radiation therapy and/or other agents used in cancer immunotherapy. 40. The immunostimulatory antigen binding molecule of any one of paragraphs 1 to 28 or 33 or the pharmaceutical composition of paragraph 34 for use in treating cancer, wherein the use is for administration in combination with a T cell activating anti-CD3 bispecific antibody. 41. The immunostimulatory antigen binding molecule or pharmaceutical composition for use of paragraph 40, wherein the T cell activating anti-CD3 bispecific antibody is an anti-GPRC5D/anti-CD3 antibody. 42. Use of an immunostimulatory antigen binding molecule as described in any one of paragraphs 1 to 28 or 33 or a pharmaceutical composition as described in paragraph 34 in the manufacture of a medicament for treating a disease, in particular for treating cancer. 43. A method for treating a disease, in particular cancer, in an individual, comprising administering to the individual an effective amount of an immunostimulatory antigen binding molecule as described in paragraphs 1 to 28 or 33 or a pharmaceutical composition as described in paragraph 34. 44. The method as described in paragraph 43, further comprising administering in combination with chemotherapy, radiotherapy and/or other agents used in cancer immunotherapy, in particular in combination with a T cell activating anti-CD3 bispecific antibody. *** Example

The following are examples of methods and compositions of the present invention. It should be understood that various other aspects may be implemented in view of the general description given above.

Recombinant DNA Techniques: DNA was manipulated using standard methods as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. General information on the nucleotide sequences of human immunoglobulin light and heavy chains is provided in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242.

DNA sequencing: Determine the DNA sequence through double-strand sequencing.

Gene Synthesis: When necessary, the desired gene fragments were generated by PCR using appropriate templates or by automated gene synthesis at Geneart AG (Regensburg, Germany) or Genscript (New Jersey, USA) from synthesized oligonucleotides and PCR products. The gene fragments flanked by individual restriction endonuclease cleavage sites were cloned into standard cloning/sequencing vectors. Plasmid DNA was purified from transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene fragments were designed with appropriate restriction sites to allow subcloning into the respective expression vectors. All constructs were designed with a 5' end DNA sequence coding for a leader peptide that targets the protein for secretion in eukaryotic cells.

Production of IgG and Bispecific Antibodies: DNA sequences encoding BCMA and the heavy and light chain variable regions of the antibodies were cloned into mammalian expression vectors using conventional cloning techniques. The antibodies described herein were produced in FedBatch format using shaker flasks. Recombinant production was performed by transient transfection of Expi293™ cells in defined serum-free medium. Transfections were performed using the ExpiFectamine™ 293 Transfection Kit (Gibco). Cell culture supernatants were harvested 7 to 12 days after transfection.

Quantification of protein titer: The protein titer of the supernatant samples was determined by affinity chromatography using a POROS A 20 µm column, 2.1 x 30 mm (Life Technologies, Carlsbad, CA, USA) on a high performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, MA, USA). The supernatant was loaded onto the column equilibrated with 0.2 M Na ₂ HPO ₄ (pH 7.4) and then eluted with 0.1 M citric acid, 0.2 M NaCl (pH 2.5). The titer was quantified by measuring the absorbance at 280 nm and the protein concentration was subsequently calculated by comparing the eluted peak area (under the curve) of the analyte with that of a reference standard curve.

Purification of IgG and bispecific antibodies: Proteins were purified from cell culture supernatants following standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5, or PBS; elution buffer: 20 mM, 25 mM, or 50 mM sodium citrate, pH 3.0). Elution was performed at pH 3.0, and the pH of the samples was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size-selective chromatography in 20 mM histidine, 140 mM NaCl, pH 6.0.

Analysis of IgG and bispecific antibodies: The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. The purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of reducing agents using LabChipGXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using analytical size-screening columns (TSKgel G3000 SW XL or UP-SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH ₂ PO ₄ , 250 mM KCl pH 6.2, 0.02% NaN ₃ ). Final quality was very good for all molecules with monomer content ranging from approximately 70% to almost 100% and CE-SDS purity >90%. In summary, all IgG and bispecific antibodies were produced with high quality.

Mass Spectrometry : This section describes the characterization of multispecific antibodies with VH/VL exchange (VH/VL crossover Mabs), with emphasis on their correct assembly. The expected primary structure was analyzed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated intact crossover Mabs and deglycosylated/fibrinolytic digested or otherwise deglycosylated/restricted LysC digested crossover Mabs. VH/VL crossover Mabs were deglycosylated with N-glycosidase F for 17 hours at 37°C in phosphate or Tris buffer at a protein concentration of 1 mg/ml. Fibrolysin or restricted LysC (Roche) digestion with 100 µg of deglycosylated VH/VL crossover Mab was performed in Tris buffer pH 8 for 120 h at room temperature and 40 min at 37°C, respectively. Samples were desalted by HPLC on a Sephadex G25 column (GE Healthcare) prior to mass spectrometry analysis. Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Determination of binding and binding affinity of multispecific antibodies to the respective antigens using surface plasmon resonance (SPR) (BIACORE): The binding of the generated antibodies to the respective antigens was investigated by surface plasmon resonance using a BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, goat anti-human IgG, JIR 109-005-098 antibody was immobilized on a CM5 chip via amine coupling for presentation of antibodies against the respective antigens. Binding was measured in HBS buffer (HBS-P) (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4) at 25°C (or at 37°C). Antigen (R&D Systems or purified in-house) was added to the solution at various concentrations. Association was measured by antigen injection for 80 seconds to 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3 - 10 minutes and the KD value was estimated using a 1:1 Langmuir binding model. Negative control data (e.g., buffer curve) were subtracted from the sample curve for correction of system endogenous baseline shift and noise signal reduction. Analysis of sensing spectra and calculation of affinity data were performed using the respective Biacore Evaluation software. Example 1 Generation and production of optimal anti- BCMA antibodies 1.1 Generation of humanized variants of anti -BCMA antibody E04 1.1.1 Methods

Anti-BCMA antibody E04 is disclosed in WO 2012/163805 and has a VH domain of SEQ ID NO:41 and a VL domain of SEQ ID NO:42. Its optimized variants were generated as described below. To identify suitable human acceptor frameworks during humanization, a combination of two approaches was used. On the one hand, a classical approach was performed by querying the mouse input sequence (trimmed for variable parts) in a BLASTp database of human V and J region sequences. The selection criteria for selecting a human acceptor framework were sequence homology, identical or similar CDR lengths, estimated frequency of human germline, and conservation of certain amino acids at the VH-VL domain interface. After the germline identification step, the CDRs of the mouse input sequence were grafted into the human acceptor framework region. Each amino acid difference between these initial CDR grafts and the parental antibody was evaluated to assess the possible impact on the structural integrity of the corresponding variable regions, and "back mutations" to the parental sequence were introduced when deemed appropriate. The structural evaluation was based on homology models of the Fv region of the parental antibody and the humanized variants, which were built using an in-house antibody structural homology modeling program implemented using BIOVIA Discovery Studio Environment version 17R2. In some of the humanized variants, "forward mutations" were included, i.e., amino acid exchanges that changed the original amino acid occurring at a given CDR position of the parental binder to an amino acid found at the equivalent position in the human receptor germline. The goal is to increase the overall human character of the humanized variants (beyond the framework regions) to further reduce the risk of immunogenicity.

On the other hand, an in-house open-source computer-run tool was used to predict the VH-VL domain orientation of paired VH and VL humanized variants (WO 2016/062734). The results were compared with the predicted VH-VL domain orientation of the parental binder to select framework combinations with a geometry close to the original antibody. A reasonable approach is to detect possible amino acid exchanges in the VH-VL interface region, which may lead to disruptive changes in the pairing of the two domains and thus have an adverse effect on the binding properties. 1.1.2 Selection and adaptation of the receptor framework

The selection of receptor framework is shown in Table 1 below: Table 1 : Receptor framework Mouse (mus musculus) V zone reproductive series Selection of human receptor V region germline VH1ab IGHV1-64*01 IGHV1-46*01 VL1abcdef IGKV3-3*01 IGKV1-39*01

The CDR3 rear framework region is from human IGHJ germline IGHJ4*01 (YFDY WGQGTLVTVSS , SEQ ID NO: 41) and human IGKJ germline IGKJ4*01 (LT FGGGTKVEIK , SEQ ID NO: 42). The portion related to the receptor framework is underlined below.

Based on structural considerations, reverse mutations from the human receptor framework to amino acids in the parent strain were introduced at certain positions of the E04 humanized variants. In addition, certain positions were identified as promising candidates for forward mutations, where amino acids in the CDRs of the parent binder were replaced with amino acids found in the human receptor germline. The changes are detailed in Table 2 below. Table 2 : List of variants Variant Name mutation Identity to human V region germline (BLASTp) VH1a fK64Q、fS65G 91.8 VH1b fK64Q, fS65G, bR71V, bT73K 89.8 VL1a - 84.8 V L1 oeLh 83.8 V L QUR 85.9 V L fE55Q、bG68E 84.8 V L fN53S、fE55Q 86.9 V L fN53S, fE55Q, bG68E 85.9 Note: Back mutations are prefixed with b and reverse mutations are prefixed with f , e.g. bS49A refers to a reverse mutation from serine to alanine at position 49 (human germline amino acid to parental antibody amino acid). All residue indices are given in Kabat numbering. 1.1.3 T cell epitope prediction

To assess the presence of potential T-cell epitopes in the humanized sequences, the NetMHCIIpan 4.0 predictor was used (Reynisson B et al.: NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data, Nucl. Acids Res., 48(W1): W449–W454 (2020)). Predictions were made for the following human MHC class II alleles: DRB1*01:01, DRB1*03:01, DRB1*04:01, DRB1*07:01, DRB1*08:01, DRB1*09:01, DRB1*11:01, DRB1*13:01, and DRB1*15:01.

The thresholds for strong and weak binding 15mer peptides were set at percentile rankings 1 and 5, respectively. Binding 15mer peptides with percentile rankings higher than 5 were not considered. Similarly, all binding 15mer peptides with 9mer core peptides that appeared in 10 or more human V region germline sequences were not considered. Germline sequences were obtained from the IMGT database (Giudicelli, V. et al.: IMGT/LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences. Nucl. Acids Res., 34(S1):D781-D784 (2006)). Since many predicted 15mer binders share the same 9mer core peptide, Table 3 below also details the number of unique 9mer cores present in the corresponding sequences and predicted to bind in the ≤ 5 percentile range. Table 3 : T cell epitopes sequence #Strongly binds 15mer peptide #Weakly binding 15mer peptide #Total bound 15mer peptides #Unique 9mer peptide core E04_VH_Parenting 6 53 59 9 VH1a 0 12 12 5 VH1b 0 17 17 7 E04_VL_Parenting 10 37 47 8 VL1a 10 29 39 7 V L1 10 29 39 7 V L 11 28 39 7 V L 11 28 39 7 V L 8 twenty one 29 5 V L 8 twenty one 29 5 1.1.4 VH and VL domains of humanized BCMA antibodies obtained

The VH domain of the obtained humanized BCMA antibody can be found in Table 4 below, and the VL domain of the obtained humanized BCMA antibody is listed in Table 5 below. Table 4 : Amino acid sequences of the VH domain of the humanized BCMA antibody describe sequence Seq ID No BCMA (E04_VH1a) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQGTLVTVSS 13 BCMA (E04_VH1b) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGIIHPNSGSTNYNEKFQGRVTMTVDKSTSTVYMELSSLRSEDTAVYYCARGIYDYPFAYWGQGTLVTVSS 14 Table 5 : Amino acid sequences of the VL domain of humanized BCMA antibodies describe sequence Seq ID No BCMA (E04_VL1a) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 16 BCMA (E04_VL1b) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 17 BCMA (E04_VL1c) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 18 BCMA (E04_VL1d) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 19 BCMA (E04_VL1e) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 20 BCMA (E04_VL1f) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSETDFTLTISSLQPEDFATYYCQQSIEDPYTFGGGTKVEIK 15 BCMA (E04_VL1g) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVEIK 130 BCMA (E04_VL1h) DIQMTQSPSSSLSASVGDRVTITCRASESVSIHGTHLMHWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSETDFTLTISSLQPEDFATYFCQQSIEDPYTFGGGTKVEIK 131

The humanized amino acid sequences of the heavy chain and light chain variable domains of the E04 humanized variants were fused to a one-armed human IgG1 backbone/human CH1-hinge-CH2-CH3 with an effective silencing Fc domain (P329G; L234A, L235A) to abolish binding to the Fcγ receptor and formation of the light chain according to the method described in WO 2012/130831 A1. For the correct assembly of the one-armed IgG1, a human Fc containing an effective silencing Fc domain was used. The amino acid sequence was reverse translated into DNA, and the resulting cDNA was synthesized (GeneArt or Twist Biosciences) and then cloned into a heavy chain expression vector as a fusion protein with a human IgG1 backbone expression vector and as a fusion protein with human C-κ. The light chain (LC) and heavy chain (HC) plasmids were then co-transfected into HEK293 cells and purified from the supernatant 7 days later using standard antibody purification methods. 1.2 Generation of humanized variants of anti -BCMA antibody 54 1.2.1 Methods

Anti-BCMA antibody 54 is disclosed in WO 2013/072415 and has a VH domain of SEQ ID NO: 47 and a VL domain of SEQ ID NO: 48. BCMA-54 is a humanized antibody with 84.8% identity to the most similar human HV germline (IGHV3-15*01) and 83.2% identity to the most similar human KV germline (IGKV1-6*01). Although the variable regions of BCMA-54 are based on framework regions of human origin, there are several positions in these regions that do not correspond to human germline amino acids. Examples include positions VH-16 (Ala), VH-44 (Arg), VH-84 (Lys), VL-22 (Ala), VL-83 (Glu), and VL-95 (Ile). In addition, there is the possibility of humanizing portions of the CDRs to reduce the immunogenic potential of the conjugate and reduce the number of potential T-cell epitopes.

To achieve this optimization potential, suitable human receptor frameworks were identified by querying the original BCMA-54 sequence against a BLASTp database of human V- and J-region sequences. The selection criteria for the human receptor frameworks were sequence homology, identical or similar CDR lengths, estimated frequency of human germlines, and conservation of certain amino acids at the VH-VL domain interface. Following a germline identification step, the CDRs of the BCMA-54 input sequence were grafted into the human receptor framework regions. The differences between these initial CDR grafts and the parental antibody were evaluated for each amino acid to assess the possible impact on the structural integrity of the corresponding variable regions, and "back mutations" to the parental sequence were introduced when deemed appropriate. The structural evaluation was based on homology models of the Fv regions of BCMA-54 and optimized variants, which were built using an in-house antibody structural homology modeling program implemented using BIOVIA Discovery Studio Environment version 17R2. In some of the humanized variants, "forward mutations" were included, i.e., amino acid exchanges that changed the original amino acid occurring at a given CDR position of the parental binder to an amino acid found at the equivalent position in the human receptor germline.

An in-house open-source computer-run tool was used to predict the VH-VL domain orientation of paired VH and VL humanized variants (WO 2016/062734). The results were compared with the predicted VH-VL domain orientation of the parental binder to select framework combinations with a geometry close to the original antibody. A reasonable approach would be to detect possible amino acid exchanges in the VH-VL interface region, which could lead to disruptive changes in the pairing of the two domains and thus adversely affect the binding properties. 1.2.2 Choice of receptor framework and its adaptation

The selection of the following receptor frameworks is shown in Table 6 : Table 6 : Receptor frameworks V region reproductive series in BCMA-54 Germline selection of human receptor V regions VH1ab IGHV3-15*01 IGHV3-15*01 VH2a IGHV3-23*01 VL1a IGKV1-6*01 IGKV1-6*01 V L IGKV1-39*01

The CDR3 rear framework region is from human IGHJ germline IGHJ1*01 (AEYFQHWGQGTLVTVSS, SEQ ID NO:49) and human IGKJ germline IGKJ2*01 (YTFGQGTKLEIK, SEQ ID NO:50). The portion associated with the receptor framework is underlined.

Based on structural considerations, back mutations from the human receptor framework to amino acids in the original BCMA-54 sequence were introduced at certain positions in the optimized variants. In addition, certain positions were identified as promising candidates for forward mutations, where amino acids in the CDRs of the parent binder were replaced with amino acids found in the human receptor germline. On top of the base variants (VH1a, VH1b, VH2a, VL1a, and VL2a), additional sequence variants were defined that typically introduced additional forward mutations at various positions or stretches of the corresponding base sequence ("germline"). One variant (VH1a_W197Y) was designed to improve the predicted hydrophobic surface patch, thereby potentially improving the biophysical properties of the VH region. The changes are detailed in Table 7 below. Table 7 : List of variants Variant Name mutation Identity to human V region germline (BLASTp) VH1a ikB 89.9 VH1b bV48I, bG49A, bT94D 87.9 VH2a fA61D, fP62S, bA93T, bK94D 87.8 VH1a_Y292D bT94D、fY58D 90.9 VH1c_huCDR2 bT94D and CDR-H2 were cloned into RIKSKTDGGTTDYAAPVKG (SEQ ID NO:51) (IGHV3-15*01) 99.0 VH1a_W197Y bT94D、fW33Y 88.9 VL1a 86.3 V L 86.2 VL1a_L2_GL CDR-L2 was germline-coded as AASSLQS (SEQ ID NO:52) (IGKV1-6*01) 90.5 VL2a_L2_GL CDR-L2 was germline-coded as AASSLQS (SEQ ID NO:52) (IGKV1-39*01) 90.4 VL1a_L1_pGL fG32D、fA34G 88.4 VL1a_N651A fN40 87.4 VL1a_N651A_N695S fN40A, fN52S 88.4 VL1a_H698Q QUR 87.4 VL1a_T699S fT56S 87.4 VL1a_H698Q_T699S fH55Q, fT56S 88.4 Note: Back mutations are prefixed with b and reverse mutations are prefixed with f , e.g. bS49A refers to a reverse mutation from serine to alanine at position 49 (human germline amino acid to parental antibody amino acid). All residue indices are given in Kabat numbering. 1.2.3 T cell epitope prediction

To assess the presence of potential T-cell epitopes in the humanized sequences, the NetMHCIIpan 4.0 predictor was used (Reynisson B et al.: NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data, Nucl. Acids Res., 48(W1): W449–W454 (2020)). Predictions were made for the following human MHC class II alleles: DRB1*01:01, DRB1*03:01, DRB1*04:01, DRB1*07:01, DRB1*08:01, DRB1*09:01, DRB1*11:01, DRB1*13:01, and DRB1*15:01.

The thresholds for strong and weak binding 15mer peptides were set at percentile rankings 1 and 5, respectively. Binding 15mer peptides with percentile rankings higher than 5 were not considered. Similarly, all binding 15mer peptides with 9mer core peptides that appeared in 10 or more human V region germline sequences were not considered. Germline sequences were obtained from the IMGT database (Giudicelli, V. et al.: IMGT/LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences. Nucl. Acids Res., 34(S1):D781-D784 (2006)). Since many predicted 15mer binders share the same 9mer core peptide, Table 8 below also details the number of unique 9mer cores present in the corresponding sequences and predicted to bind in the ≤ 5 percentile range. Table 8 : T cell epitopes sequence #Strongly binds 15mer peptide #Weakly binding 15mer peptide #Total bound 15mer peptides #Unique 9mer peptide core 54_VH_Parenting 15 60 75 8 VH1a 15 52 67 6 VH1b 15 53 68 6 VH2a 7 54 61 6 VH1a_Y292D 14 54 68 5 VH1c_huCDR2 10 twenty four 34 4 VH1a_W197Y 14 53 67 6 54_VL_Parenting 12 17 29 5 VL1a 12 15 27 5 V L 12 15 27 5 VL1a_L2_GL 9 12 twenty one 4 VL2a_L2_GL 9 12 twenty one 4 VL1a_L1_pGL 12 twenty three 35 7 VL1a_N651A 11 twenty three 34 6 VL1a_N651A_N695S 9 19 28 6 VL1a_H698Q 12 16 28 6 VL1a_T699S 13 16 29 5 VL1a_H698Q_T699S 14 15 29 6 1.2.4 VH and VL domains of humanized BCMA antibodies obtained

The VH domain of the obtained humanized BCMA antibody can be found in Table 9 below, and the VL domain of the obtained humanized BCMA antibody is listed in Table 10 below. Table 9 : Amino acid sequences of the VH domain of the humanized BCMA antibody describe sequence Seq ID No BCMA (54_VH1a) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 53 BCMA (54_VH1b) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWIAQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 35 BCMA (54_VH2a) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVSQITAKSNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDDGYHWGQGTLVTVSS 33 BCMA (54_VH1a_Y292D) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGQITAKSNNYATDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 54 BCMA (54_VH1c_huCDR2) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMDWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 55 BCMA (54_VH1a_W197Y) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAYMDWVRQAPGKGLEWVGQITAKSNNYATYYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTDDGYHWGQGTLVTVSS 56 Table 10 : Amino acid sequences of the VL domain of humanized BCMA antibodies describe sequence Seq ID No BCMA (54_VL1a) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 36 BCMA (54_VL2a) DIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 34 BCMA (54_VL1a_L2_GL) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 57 BCMA (54_VL2a_L2_GL) DIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 58 BCMA (54_VL1a_L1_pGL) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNDLGWYQQKPGKAPKLLIYNANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 59 BCMA (54_VL1a_N651A) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAANSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 60 BCMA (54_VL1a_N651A_N695S) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYAASSLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 61 BCMA (54_VL1a_H698Q) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 62 BCMA (54_VL1a_T699S) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 63 BCMA (54_VL1a_H698Q_T699S) AIQMTQSPSSSLSASVGDRVTITCRASEDIRNGLAWYQQKPGKAPKLLIYNANSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEDTSKYPYTFGQGTKLEIK 64

The humanized amino acid sequences of the heavy and light chain variable domains of the E04 humanized variants were fused to a one-armed human IgG1 backbone/human CH1-hinge-CH2-CH3 with an effective silencing Fc domain (P329G; L234A, L235A) to abolish binding to the Fcγ receptor according to the method described in WO 2012/130831 A1, and contained knob mutations and formed the light chain according to the knob-in-hole technique. For the correct assembly of the one-armed IgG1, a human Fc containing an effective silencing Fc domain was used. The amino acid sequence was reverse translated into DNA, and the resulting cDNA was synthesized (GeneArt or Twist Biosciences) and then cloned into a heavy chain expression vector as a fusion protein with a human IgG1 backbone expression vector and as a fusion protein with human C-κ. The light chain (LC) and heavy chain (HC) plasmids were then co-transfected into HEK293 cells and purified from the supernatant 7 days later using standard antibody purification methods. 1.3 Characterization of humanized anti- BCMA variants

To characterize anti-BCMA antibody variants, all clones were expressed as monovalent one-armed IgG-like constructs ( Figure 1A ). This format was chosen to characterize binding to BCMA in a 1:1 model.

76 variants were generated to select the two best humanized variants of anti-BCMA antibody E04 and the two best humanized variants of anti-BCMA antibody 54. From these 76 variants, 29 variants with the highest affinity to huBCMA (15 BCMA 54 variants and 14 BCMA E04 variants) were pre-selected and further characterized by measuring affinity to cyBCMA.

The corresponding VH/VL pairs, construct IDs (TaPIR IDs) and corresponding SEQ ID NOs of the 29 variants and parental antibodies are listed in Table 11 below. Table 11 : Summary of the monovalent anti -BCMA variants expressed Variant VH/VL (one-armed IgG construct) Tapir ID SEQ ID NO: SEQ ID NO: SEQ ID NO: BCMA (54_VH1a) / BCMA (54_VL1a_L1_pGL) P1AG5067 65 66 67 BCMA (54_VH1a_Y292D) / BCMA (54_VL1a) P1AG5057 68 69 67 BCMA (54_VH1a_Y292D) / BCMA (54_VL1a_L1_pGL) P1AG5014 68 66 67 BCMA (54_VH1a_Y292D) / BCMA (54_VL1a_T699S) P1AG5065 68 70 67 BCMA (54_VH1a_Y292D) / BCMA (54_VL2a) P1AG5080 68 71 67 BCMA (54_VH1b) / BCMA (54_VL1a) P1AG5072 72 64 67 BCMA (54_VH1b) / BCMA (54_VL1a_L1_pGL) P1AG5027 72 66 67 BCMA (54_VH1b) / BCMA (54_VL1a_N651A) P1AG5056 72 73 67 BCMA (54_VH1b) / BCMA (54_VL1a_N651A_N695S) P1AG5064 72 74 67 BCMA (54_VH1b) / BCMA (54_VL1a_T699S) P1AG5046 72 70 67 BCMA (54_VH1b) / BCMA (54_VL2a) P1AG5030 72 71 67 BCMA (54_VH2a) / BCMA (54_VL1a) P1AG5028 75 64 67 BCMA (54_VH2a) / BCMA (54_VL1a_L1_pGL) P1AG5013 75 61 67 BCMA (54_VH2a) / BCMA (54_VL1a_T699S) P1AG5071 75 65 67 BCMA (54_VH2a) / BCMA (54_VL2a) P1AG5031 75 71 67 BCMA (54) Parent P1AG5061 76 77 67 BCMA (E04_VH1a) /BCMA (E04_VL1a) P1AG5063 78 79 67 BCMA (E04_VH1a) / BCMA (E04_VL1b) P1AG5004 78 80 67 BCMA (E04_VH1a) / BCMA (E04_VL1d) P1AG5043 78 81 67 BCMA (E04_VH1a) / BCMA (E04_VL1e) P1AG5007 78 82 67 BCMA (E04_VH1a) / BCMA (E04_VL1f) P1AG5036 78 83 67 BCMA (E04_VH1a) / BCMA (E04_VL1g) P1AG5059 78 84 67 BCMA (E04_VH1a) / BCMA (E04_VL1h) P1AG5053 78 85 67 BCMA (E04_VH1b) / BCMA (E04_VL1a) P1AG5026 86 79 67 BCMA (E04_VH1b) / BCMA (E04_VL1b) P1AG5044 86 80 67 BCMA (E04_VH1b) / BCMA (E04_VL1d) P1AG5078 86 81 67 BCMA (E04_VH1b) / BCMA (E04_VL1e) P1AG5034 86 82 67 BCMA (E04_VH1b) / BCMA ((E04_VL1f) P1AG5060 86 83 67 BCMA (E04_VH1b) / BCMA (E04_VL1g) P1AG5021 86 84 67 BCMA (E04_VH1b) / BCMA (E04_VL1h) P1AG5076 86 85 67 BCMA (E04) Parent P1AG5062 87 88 67

The protein concentration of the purified constructs was determined by measuring the optical density (OD) at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science 1995 (4) 2411-1423. The purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChipGXII (Perkin Elmer). Aggregate content was determined by HPLC at 25°C using an analytical size-screening column (TSKgel G3000 SW _XL or UP-SW3000) equilibrated in running buffer (25 mM K ₂ HPO ₄ , 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7; or 200 mM KH ₂ PO 4 , 250 mM KCl pH 6.2, respectively). A summary of the yield and purification parameters for all monovalent anti-BCMA variants is given in Table 12 .

The binding kinetics of monovalent humanized BCMA antibody variants to human BCMA and macaque BCMA were studied by surface plasmon resonance (SPR) using a BIACORE T200 instrument (GE Healthcare). All experiments were performed at 25°C using HBS-P buffer (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) as running buffer and dilution buffer. Anti-Fc IgG capture antibody (specific for the Fc region of the PGLALA variant) was immobilized on a Series S Sensor Chip CM5 (Cytiva) using standard amine coupling chemistry to give a surface density of approximately 15,000 resonance units (RU). Anti-BCMA antibodies were captured on the surface at a flow rate of 5 μl/min for 30 s, resulting in capture reactions of 50 to 200 RU. A series of diluted antigens (human BCMA Fc homodimer (R&D Systems) or macaque BCMA Fc homodimer (R&D Systems), respectively) were injected onto the surface at concentrations ranging from 3 to 300 nM at 30 μl/min for 120 s (association phase). The dissociation phase was monitored for 300 to 600 s by washing with running buffer. The surface was regenerated by injecting 5 mM NaOH (freshly prepared) 2 x 30 s. Global refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from a reference flow cell without captured antibody. The resulting curves were fit to a 1:1 Langmuir binding model using BIAevaluation software.

The _KD values of the monovalent constructs including the 29 best BCMA variants are shown in Table 12 below. Table 12 : Production, purification and binding properties of the monovalent anti -BCMA variants shown Tapir ID Product amount [µg] Analyte SEC monomer [%] CE SDS main peak [%] K _D [M] huBCMA K _D [M] cyBCMA P1AG5067 2231 97.2 93.7 3.4E-09 1.7E-09 P1AG5057 1184 96.7 90.6 1.7E-09 4.0E-09 P1AG5014 1246 97 92.2 1.7E-09 1.1E-10 P1AG5065 1110 95.8 88.9 2.6E-09 6.4E-09 P1AG5080 2195 97 89.9 1.3E-09 4.3E-09 P1AG5072 3394 97.2 93.8 1.3E-09 3.2E-09 P1AG5027 3492 98.3 95.4 1.7E-09 1.2E-09 P1AG5056 3200 97.4 94 4.2E-09 1.3E-08 P1AG5064 1760 100 89.7 3.5E-09 1.3E-08 P1AG5046 2685 97.9 92.6 1.9E-09 3.9E-09 P1AG5030 3344 97.6 91 1.3E-09 2.2E-09 P1AG5028 3405 98.7 96.1 2.7E-09 3.8E-09 P1AG5013 2900 97.6 94.5 2.4E-09 1.9E-09 P1AG5071 2508 96.5 91.4 3.4E-09 6.5E-09 P1AG5031 3155 97.5 91.1 2.4E-09 4.7E-09 P1AG5061 3481 95.5 82.6 1.4E-09 1.9E-09 P1AG5063 3041 99.6 96.3 1.6E-09 2.8E-08 P1AG5004 2454 98.6 95.2 2.5E-09 1.6E-08 P1AG5043 2485 100 96.7 2.6E-09 1.4E-08 P1AG5007 393 98.6 100 3.0E-09 1.2E-08 P1AG5036 2735 99.6 97.4 2.9E-09 9.5E-09 P1AG5059 2520 99.1 95.4 2.3E-09 3.0E-08 P1AG5053 1222 99.6 99.3 2.4E-09 1.3E-08 P1AG5026 569 98.9 93.1 2.7E-09 3.3E-08 P1AG5044 1398 97.7 91.8 1.8E-09 2.0E-08 P1AG5078 1236 99.8 96 2.4E-09 2.2E-08 P1AG5034 286 100 96.5 3.2E-09 3.2E-08 P1AG5060 702 96.4 86.5 3.2E-09 1.4E-08 P1AG5021 344 97.3 92.2 2.8E-09 3.5E-08 P1AG5076 944 99.7 99.5 1.9E-09 2.3E-08 P1AG5062 1995 99.8 97.2 3.1E-09 3.3E-08

Based on the results of an in silico assessment of the occurrence of potential T-cell epitopes in the humanized sequences using the NetMHCIIpan 4.0 predictor (see Example 1.2.3), antibodies P1AG5080, P1AG5072, P1AG5028, P1AG5031, P1AG5063 and P1AG5036 (4 BCMA 54 variants and 2 BCMA E04 variants) were selected as molecules with the lowest potential T-cell epitopes. In combination with their binding behaviors, P1AG5072 and P1AG5031 were selected as BCMA 54 variants, and P1AG5063 and P1AG5036 were selected as BCMA E04 variant antibodies to be included in the bispecific antibodies. Example 2 Generation and production of antigen binding molecules containing 4-1BBL trimers targeting BCMA 2.1 Selection and cloning of antigen binding molecules containing 4-1BBL trimers targeting BCMA

For the generation of expression plasmids, the sequences of the respective variable domains were used and subcloned in a framework with the respective constant regions that were previously inserted into the respective receptor mammalian expression vectors. In the Fc domain, Pro329Gly, Leu234Ala and Leu235Ala mutations (PG-LALA) have been introduced into the constant region of the human IgG1 heavy chain to abolish binding to Fcγ receptors, according to the method described in International Patent Application Publication No. WO 2012/130831. To generate the constructs, the Fc fragment contained either a "knob" (S354C/T366W mutation, numbered according to the Kabat EU index) or a "hole" mutation (Y349C/T366S/L368A/Y407V mutation, numbered according to the Kabat EU index) to avoid re-chain mispairing. To improve correct pairing, charges were introduced into the CH1 and Cκ domains of the BCMA antigen-binding portion as described in International Patent Application Publication No. WO 2015/150447.

A polypeptide comprising two extracellular domains of the 4-1BB ligand separated by a (G4S)2 linker and fused to a human IgG1 - CL domain was cloned as follows: human 4-1BB ligand, (G4S)2 linker, human 4-1BB ligand, (G4S)2 linker, human CL, Fc hole IgG1. A polypeptide comprising one extracellular domain of the 4-1BB ligand and fused to a human IgG1-CH domain was cloned as follows: human 4-1BB ligand, (G4S)2 linker, human CH.

A schematic diagram of an antigen binding molecule containing a 4-1BBL trimer targeting BCMA is shown in Figure 1B . Table 13 summarizes the specific antigen binding molecules prepared, their identifiers, and the sequences of the heavy chain (Fc1 knob/fusion protein 2 and Fc2 hole) and light chain (LC and fusion protein 1). Table 13 : Summary of the anti- BCMA-4-1BBL antigen binding molecules presented describe TAPIR ID SEQ ID NO: (Fusion protein 1) SEQ ID NO: (Fc1 knob/fusion protein 2) SEQ ID NO: (Fc2 ) SEQ ID NO: (LC) BCMA (E04_VH1a/VL1f) -4-1BBL P1AG7400 twenty two twenty one twenty three twenty four BCMA (E04-VH1a/VL1a)-4-1BBL P1AG7409 twenty two twenty one twenty three 25 BCMA (54_VH2a/VL2a) -4-1BBL P1AG7397 twenty two twenty one 37 38 BCMA (54_VH1b/VL1a)-4-1BBL P1AG7422 twenty two twenty one 39 40 BCMA (PR) -4-1BBL P1AF7814 twenty two twenty one 91 92 2.2 Production and purification of BCMA - targeting 4-1BBL trimer-containing antigen-binding molecules

DNA sequences encoding the heavy chain variable region and light chain variable region of the BCMA antigen binding domain and 4-1BBL fusion protein were cloned into mammalian expression vectors using conventional cloning techniques. The bispecific antibodies described herein were produced in FedBatch format using shaker flasks. Recombinant production was performed by transient transfection of Expi293™ cells in defined serum-free medium. Transfections were performed using the ExpiFectamine™ 293 Transfection Kit (Gibco). Cell culture supernatants were harvested 7 to 12 days after transfection.

Quantification of protein titer: The protein titer of the supernatant samples was determined by affinity chromatography using a POROS A 20 µm column, 2.1 x 30 mm (Life Technologies, Carlsbad, CA, USA) on a high performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, MA, USA). The supernatant was loaded onto the column equilibrated with 0.2 M Na ₂ HPO ₄ (pH 7.4) and then eluted with 0.1 M citric acid, 0.2 M NaCl (pH 2.5). The titer was quantified by measuring the absorbance at 280 nm and the protein concentration was subsequently calculated by comparing the eluted peak area (under the curve) of the analyte with that of the reference standard curve.

Purification of bispecific antibodies: Proteins were purified from cell culture supernatants following standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5, or PBS; elution buffer: 20 mM, 25 mM, or 50 mM sodium citrate, pH 3.0). Elution was performed at pH 3.0, and the pH of the samples was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size-selective chromatography in 20 mM histidine, 140 mM NaCl, pH 6.0.

Analysis of antigen binding molecules containing 4-1BBL trimer targeting BCMA: The concentration of purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. The purity and molecular weight of the protein were analyzed by CE-SDS in the presence and absence of reducing agents using LabChipGXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using an analytical size screening column (TSKgel G3000 SW XL or UP-SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH ₂ PO ₄ , 250 mM KCl pH 6.2, 0.02% NaN ₃ ).

A summary of the purification parameters of the selected molecules is given in Table 14. Table 14 : Summary of the production and purification of antigen binding molecules containing 4-1BBL trimers targeting BCMA TAPIR ID describe Yield [mg/l] Analytical SEC (HMW/monomer/LMW) [%] Purity measured by CE-SDS [%] P1AG7400 BCMA (E04_VH1a/VL1f) -4-1BBL 99 100 P1AG7409 BCMA (E04-VH1a/VL1a)-4-1BBL 99 100 P1AG7397 BCMA (54_VH2a/VL2a) -4-1BBL 99 100 P1AG7422 BCMA (54_VH1b/VL1a)-4-1BBL 99 98.5 2.3 Biophysical and biochemical characterization of BCMA - targeting 4-1BBL trimer-containing antigen-binding molecules

To predict the developability of BCMA-4-1BBL antigen-binding molecules, their biophysical and biochemical properties were assessed by computational methods and assays.

Predicting the protein's net charge as a function of pH based on the sequence using standard software (e.g., EMBOSS tools) and therefore its isoelectric point (calculated pI) helps estimate whether the bispecific antibody is suitable for binding and not binding to cation and anion exchange chromatography media and therefore suitable for general purification methods during production. All BCMA-4-1BBL constructs produced had pI values in the range of approximately 8, which is considered suitable for developability (see Table 15 below).

The thermal stability of the prepared BCMA-4-1BBL construct was monitored by dynamic light scattering (DLS) and by monitoring the temperature-dependent endogenous protein fluorescence using an Optim 2 instrument (Avacta Analytical, UK) by applying a temperature ramp. 10 µg of filtered protein sample at a protein concentration of 1 mg/ml was applied to the Optim 2 instrument in duplicate. The temperature was increased from 25°C to 85°C at a rate of 0.1°C/min and the fluorescence intensity ratio at 350 nm/330 nm and the scattering intensity at 266 nm were collected. The results are shown in Table 15. The aggregation temperatures (T _agg ) of the tested BCMA-4-1BBL constructs P1AG7397 and P1AG7422 were favorable compared to constructs P1AG7409 and P1AG7400, but the aggregation temperatures of all constructs were considered high enough for further development.

The apparent hydrophobicity of the bispecific antibodies was assessed by hydrophobic interaction chromatography (HIC) as the relative retention time compared to a hydrophobicity standard (90% of the immunoglobulins in an IVIG preparation have a relative retention time <0.35). In detail, 20 μg of sample was injected onto a HIC-Ether-5PW (Tosoh) column equilibrated with 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0. Elution was performed using a linear gradient from 0% to 100% buffer B (25 mM sodium phosphate, pH 7.0) in 60 min. The retention time was then compared to a protein standard of known hydrophobicity (e.g., Avastin). All constructs were more hydrophobic than typical antibodies.

For FcRn affinity chromatography, FcRn was expressed, purified and biotinylated as described (Schlothauer et al., MAbs 2013, 5(4), 576-86). For coupling, the prepared receptor was added to avidin-argonose (GE Healthcare). The resulting FcRn-argonose matrix was filled in the column shell. The column was equilibrated with 20 mM 2-(N-morpholino)-ethanesulfonic acid (MES) and 140 mM NaCl (pH 5.5) (Eluent A) at a flow rate of 0.5 ml/min. 30 μg of antibody sample was diluted with Eluent A at a volume ratio of 1:1 and applied to the FcRn column. The column was washed with 5 column volumes of Eluent A, followed by elution with a linear gradient from 20% to 100% 20 mM Tris/HCl and 140 mM NaCl, pH 8.8 (Eluent B) over 35 column volumes. Analysis was performed in a column oven at 25°C. The elution profile was monitored by continuous measurement of absorbance at 280 nm. Retention times were compared with those of protein standards of known affinity.

Heparin affinity was determined by injecting 30 to 50 μg of sample onto a TSKgel Heparin-5PW (Tosoh) column equilibrated with 50 mM Tris (pH 7.4). Elution was performed using a linear gradient from 0% to 100% buffer B (50 mM Tris, 1M NaCl, pH 7.4 mM) in 37 minutes. Retention times were compared to protein standards of known affinity. Retention times for specific BCMA-4-1BBL constructs and expected values for measured retention times are shown in Table 15. Table 15 : In vitro developability assessment of BCMA-4-1BBL antigen binding molecules Parameters Expected values for an “ideal” antibody P1AG7409 BCMA E04 1a 1a 4-1BBL P1AG7400 BCMA E04 1a 1f 4-1BBL P1AG7397 BCMA 54 2a 2a 4-1BBL P1AG7422 BCMA 54 1b 1a 4-1BBL Calculated pI, charge distribution >8: OK 7…8: Orange mark <7: Red mark 7.99 7.99 7.8 8.16 Thermal stability (T _agg ) _Tagg ＞ 64℃ 55.50 56.30 64.70 65.90 Apparent hydrophobicity (HIC) ¹ Rel. ret. ＜0.34 (＜Avastin) 0.54 0.54 0.50 0.50 ^FcRn2 heparin 0.07 0.19 0.02 0.28 -0.21 0.57 -0.23 0.54 Affinity analysis 1 - Relative to universal hydrophobicity standards. 90% of immunoglobulins in IVIG preparations have a relative retention time < 0.35. 2 - Relative to universal FcRn affinity standards. Normal IgG has a relative retention time between 0 and 2.75.

The binding potency was characterized by surface plasmon resonance (SPR) after stress using specific BCMA-4-1BBL constructs. The results are shown in Table 16. The reduction in binding potency caused by incubation of the molecules for 14 days at 37°C, pH 7.4 and 40°C, pH 6 was quantified by surface plasmon resonance using a Biacore T200 instrument (GE Healthcare). Samples stored at -80°C, pH 6 were used as reference. The reference sample and samples stressed at 40°C in 20 mM histidine buffer, 140 mM NaCl, pH 6.0, and samples stressed at 37°C in PBS buffer, pH 7.4, were all at a concentration of 1.0 mg/ml. After the stress period (14 days), samples in PBS buffer were dialyzed back into 20 mM histidine buffer, 140 mM NaCl, pH 6.0 for further analysis.

All SPR experiments were performed using a BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden) at 25°C with HBS-P+ buffer (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) as running and dilution buffer. Antigens (R&D Systems or purified in-house) were added to the solution at various concentrations. Biotinylated human BCMA and 4-1BB and biotinylated anti-hu IgG (Capture Select, Thermo Scientific, #7103262100) were immobilized on S-Series Sensor Chips SA (GE Healthcare, #29104992) to a surface density of at least 1000 resonance units (RU). The BCMA-4-1BBL construct was injected at a concentration of 2 μg/ml over 30 s at a flow rate of 5 μl/min and dissociation was monitored over 120 s. The surface was regenerated by injecting 10 mM glycine buffer, pH 1.5 over 60 s. Correction for bulk refractive index differences was performed by subtracting a blank injection and subtracting the response obtained from a blank flow cell. For evaluation, the binding response was taken 5 s after the end of the injection.

To normalize the binding signal, BCMA and 4-1BB binding was divided by the anti-hu IgG response (signal (RU) obtained after capturing the BCMA-4-1BBL construct on the immobilized anti-hu IgG antibody). Relative binding activity was calculated by comparing each temperature-stressed sample to the corresponding unstressed sample. As shown in Table 16, all BCMA-4-1BBL constructs showed stable binding to BCMA and 4-1BB after stress. Table 16 : In vitro developability assessment of bispecific antibodies targeting BCMA and 4-1BB Parameters Expected value of "ideal" leader P1AG7409 BCMA E04 1a 1a 4-1BBL P1AG7400 BCMA E04 1a 1f 4-1BBL P1AG7397 BCMA 54 2a 2a 4-1BBL P1AG7422 BCMA 54 1b 1a 4-1BBL CE-SDS [%] Monomer (non-red) References Compared with reference <2%. 97.93 98.11 98.31 94.56 PBS 37℃ 90.42 89.76 85.02 86.83 His 40℃ 88.82 86.19 91.73 90.78 CE-SDS [%] LC+HC (red) References Compared with reference <2%. 99.87 99.86 99.81 99.82 PBS 37℃ 97.08 98.48 96.63 98.87 His 40℃ 98.75 98.86 98.1 99.55 SEC monomer [%] References Compared with reference <2%. 98.20 97.80 97.20 97.50 PBS 37℃ 93.70 93.00 92.00 91.50 His 40℃ 98.3 98.3 98.1 97.7 Target binding ± stress. By SPR RAC [%] References >90% 100 100 100 100 100 100 100 100 PBS 37℃ 100 97 101 99 101 98 101 98 His 40℃ 97 97 99 98 98 94 99 96 BCMA 4-1BB BCMA 4-1BB BCMA 4-1BB BCMA 4-1BB

Compared to classical antibodies or bispecific antibodies, the BCMA-41BBL antigen binding molecules showed lower stability in in vitro assessments. However, these data are within the range of other fusion proteins currently being tested in clinical trials and are therefore considered acceptable. 2.4 Production and purification of bispecific antigen binding molecules targeting BCMA and CD3

For comparison, the well-characterized BCMA-targeted CD3 T-cell engagers Aneumax and Aneumax were prepared. Aneumax is a 2+1 format BCMA x CD3 bispecific antibody based on an IgG1 Fc with L234A/L235A/P329G (EU numbering) mutations. The sequences are identified in the International List of Nonproprietary Nomenclature (recommended INN: List 85; WHO Drug Information, Vol. 35, No. 1, 2021). Anutakizumab comprises the amino acid sequence of SEQ ID NO: 191, the amino acid sequence of SEQ ID NO: 192, the amino acid sequence of SEQ ID NO: 193, and the amino acid sequence of SEQ ID NO: 194 (2+1 version).

Alenmab is a BCMA x CD3 bispecific antibody of 1+1 format. The sequence is identified in the International Nonproprietary Nomenclature List (Suggested INN: List 87, WHO Drug Information, Vol. 36, No. 1, 2022). Alenmab comprises an amino acid sequence of SEQ ID NO: 195, an amino acid sequence of SEQ ID NO: 196, an amino acid sequence of SEQ ID NO: 197, and an amino acid sequence of SEQ ID NO: 198.

To produce anukitumab (P1AF0105), DNA sequences encoding the variable heavy and light chain regions of the corresponding binding domains were cloned into mammalian expression vectors using conventional cloning techniques. Antibodies were produced by transient transfection of Expi293F cells. Cells were seeded at a density of 2.5 x 10 ⁶ /mL in Expi293 medium (Gibco, #1435101). Expression plasmids and ExpiFectamine (Gibco, ExpiFectamine Transfection Kit, #13385544) were mixed separately in OptiMEM (Gibco, #11520386). After 5 minutes, the two solutions were combined, mixed by pipetting and incubated at room temperature for 15 to 20 minutes. Cells were added to the plasmid/ExpiFectamine solution and incubated for 24 hours in a shaking incubator at 37°C and 5% CO _2. One day after transfection, supplements (Enhancer 1+2, ExpiFectamine Transfection Kit) were added. After 4 to 5 days, cell supernatants were harvested by centrifugation and subsequent filtration (0.2 μm filter) and proteins were purified from the harvested supernatants by standard methods as described below.

Elenatumab (P1AH5054) was produced and purified by Proteros according to their standard methods and protocols.

Quantification of Fc-containing constructs in supernatants was performed by Protein A-HPLC on an Agilent HPLC System with UV detection. Supernatants were injected onto POROS 20 A (Applied Biosystems), washed with 10 mM Tris, 50 mM glycine, 100 mM NaCl, pH 8.0, and eluted in the same buffer at pH 2.0. Titers were quantified by measuring the absorbance at 280 nm, and protein concentrations were subsequently calculated by comparing the eluted peak area (under the curve) of the analyte with that of a reference standard curve.

Proteins were purified from filtered cell culture supernatants following standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was performed at pH 3.0, followed by immediate pH neutralization of the sample. Proteins were concentrated by centrifugation using a Millipore Amicon® ULTRA-15 (Merck, #UFC903096), and aggregated proteins were separated from monomeric proteins by size-selective chromatography in 20 mM histidine, 140 mM NaCl, pH 6.0. The concentration of purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. The purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChip GXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH ₂ PO ₄ , 250 mM KCl pH 6.2, 0.02% NaN ₃ ) at 25°C. A summary of the purification parameters is given in Table 16A. Table 16A: Summary of Production and Purification of Bispecific BCMA x CD3 Antigen Binding Molecules TAPIR ID describe Yield [mg/l] Analytical SEC (monomer) [%] Purity measured by CE-SDS [%] P1AF0105 Anoukitumab 16.8 94.8 88.7 P1AH5054 Elenatumab 7.6 ＞99 ＞98

Terituzumab is a BCMA x CD3 bispecific antibody with IgG4-F234A/L235A/S228P (EU number) Fc. The sequence is identified in the International Nonproprietary Nomenclature List (Recommended INN: List 82, WHO Drug Information, Vol. 33, No. 3, 2019). It was obtained from a supplier (FarmaMondo, lot number AT1334P1). Terituzumab comprises an amino acid sequence of SEQ ID NO: 199, an amino acid sequence of SEQ ID NO: 200, an amino acid sequence of SEQ ID NO: 201, and an amino acid sequence of SEQ ID NO: 202. Example 3 Binding of antigen-binding molecules containing 4-1BBL trimers targeting BCMA

To measure binding to BCMA or 4-1BB, we performed FACS-based binding assays on CHO transfectants that were transduced to stably express either human BCMA or on Jurkat reporter cells expressing human 4-1BB, respectively.

Generation of a CHO-K1 cell line expressing a non-truncating mutant of the BCMA extracellular domain

Full-length cDNA encoding human BCMA (UniProt: Q02223) and its corresponding mutants (R27P, S30del, P33S, and P34del) were subcloned into a lentiviral transfer vector controlled by the CMV promoter. Lentiviral particles were prepared by transiently co-transfecting HEK 293-derived virus production cells (Gibco, #A35347) with the transfer plasmid and lentiviral packaging mix (pRSV-Rev, pCgpV, and pCMV-VSV-G) using the LV-MAX Transfection Kit (Gibco, #A35346) according to the manufacturer's protocol. Viral supernatants were harvested 48 h after transfection, filtered through a 0.45 μm low protein binding filter, and stored at -80°C until use.

One day before transduction, 5 x 10 ⁴ CHO-K1 (ATCC CRL-9618) cells were seeded into each well of a 24-well plate. The next day, the medium was replaced with 300 µL of purified lentiviral supernatant and 100 µL of fresh medium DMEM/F-12 (Gibco, #11320033) supplemented with 10% fetal bovine serum (Gibco, #16140063) and 1% GlutaMAX supplement (Gibco; #31331-028). To facilitate viral transduction, the infectious medium was further supplemented with 2.5 µL of TransDux™ reagent and 100 µL of MAX Enhancer (SBI; #LV860A-1). The cells were then incubated at 37°C for 24 hours. After the incubation period, the virus medium was discarded and the cells were subsequently maintained in fresh medium.

Three days after transduction, the culture medium was supplemented with 6 µg/mL puromycin (Invivogen; #ant-pr-1). After initial selection, cells displaying human BCMA surface expression were isolated by BD FACSAria III cell sorter (BD Biosciences) and subsequently cultured to generate stable clones. After 4 weeks of stability testing, surface expression and stability were verified by flow cytometric analysis using mouse PE-conjugated anti-human BCMA (BioLegend, #357503).

Stable CHO transfectants (parental cell line CHO-k1 ATCC #CCL-61) were cultured in DMEM/F-12 (Gibco, #10565018) supplemented with: 10% fetal bovine serum (Gibco, #16140063 or Sigma-Aldrich F4135) and 1% GlutaMAX supplement (Gibco; #31331-028), including 6 μg/ml puromycin (Invivogen; #ant-pr-1). Adherent CHO cells were detached using Cell Dissociation Buffer (Gibco, #13151014) or trypsin (Gibco, TrypLE™ Express Enzyme #2605-010 from ThermoFisher Scientific), counted, and checked for viability. All subsequent steps were performed at 4°C.

BCMA Combination Solution

To assess binding to BCMA, CHO-huBCMA cells were resuspended at 1 Mio cells per ml in FACS buffer (PBS, 2% fetal bovine serum; 1% 0.5 M EDTA pH 8; 0.25% NaN ₃ nitride). 0.1 Mio cells were plated per well of a round-bottom 96-well plate and washed once with 150 μl per well of cold FACS buffer. Cells were stained with 50 μl per well to total volume and increasing concentrations (0.48 pM to 2000 nM) of the indicated BCMA-4-1BBL antigen binding molecules for 30 minutes at 4°C. Then, cells were centrifuged and washed twice with 150 μl FACS buffer.

Pre-diluted secondary antibody (PE-AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific; Jackson Immunoresearch, 109-116-170, diluted 1:50 in FACS buffer) was added to each well and the plate was incubated at 4°C for 30 minutes. Cells were washed twice and then fixed by adding 50μl 1% PFA (in PBS) and incubating at room temperature for 20 min. Cells were washed twice with 150 μl FACS buffer and analyzed on a BD Fortessa flow cytometer equipped with software FACS Diva. Binding curves and _EC50 values were obtained using GraphPadPrism6. Data measured for various BCMA-4-1BBL antigen binding molecules and controls (eg, "secondary antibody" refers only to samples containing only fluorophore-labeled secondary antibodies) are shown in Figures 2A and 3A .

To assess binding to BCMA variants with indicated point mutations in the hu BCMA extracellular domain, CHO transfectants were resuspended in PBS (Gibco by ThermoFisher Scientific, #20012050) and counted. Live-dead staining of target cells was performed by incubating cells at 1.5 Mio cells per ml with Zombie Aqua Viability dye (BioLegend #42310277143) at a dilution of 1:1000 in PBS for 10 minutes in the dark, followed by two wash steps with PBS and centrifugation at 400 x g for 4 minutes at 4°C. CHO-K1 cells were adjusted to 1.5 Mio cells per ml in PBS and 40 μl was plated per well of a 384-well U-bottom plate (ThermoFisher Scientific #264573). The plate was centrifuged again at 400 x g for 4 minutes at 4°C and 20 μl of supernatant was removed.

Cells were stained in a total volume of 20 to 40 μl per well by adding 20 μl of 2x concentrated BCMA-4-1BBL antigen binding molecules at indicated dilutions (final concentration range 0.008 to 125 nM) for 30 min at 4°C in the dark. Cells were then centrifuged and washed twice with 40 μl PBS buffer.

Pre-diluted secondary antibody (R-phycoerythrin AffiniPure Fab fragment goat anti-human IgG, Fcγ fragment specific; Jackson Immunoresearch, 109-117-008, diluted 1:100 in PBS) was added to the plate containing 20 ml (final dilution 1:200) in a total of 20 ml per well. The plate was incubated at 4°C for 30 minutes. The cells were washed twice with 40 μl PBS per well and then fixed overnight at 4°C by adding 40 μl of 1% PFA (in PBS). The cells were washed twice with 40 μl FACS buffer and analyzed on a BD FACSymphony™ A5 cytometer. Binding curves and _EC50 values were obtained using GraphPadPrism 6. Data measured for various BCMA-4-1BBL antigen binding molecules and controls (e.g., “secondary antibody only” refers to samples containing only fluorophore-labeled secondary antibodies) are shown in Figures 3C to 3H .

4-1BB Combination Solution

The agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces 4-1BB downstream signaling and promotes CD8 T cell survival and activity via activation of nuclear factor κB (NFκB) (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS, J Immunol 2002; 169, 4882-4888). To monitor this NFκB activation mediated by the 4-1BBL antigen binding molecule, the Jurkat-NFκB-luc2_4-1BB reporter cell line was developed by Promega in collaboration with Roche Diagnostics GmbH and stably transfected with two plasmids encoding the human 4-1BB and NFκB-driven luciferase reporter genes. The cell lines were grown in RPMI1640 medium supplemented with 10% FCS, 25 mM HEPES (Thermofisher), 2 mM GlutaMax, 0.1 mM non-essential amino acids (Thermofisher), and 1 mM sodium pyruvate. In addition, the medium was supplemented with 400 µg/ml hygromycin B and 600 µg/mL lysozyme (Sigma-Aldrich). Jurkat cells were cultured twice a week to maintain a density between 0.2 and 1.0 x10 ⁶ cells/ml.

All subsequent steps were performed at 4°C. Jurkat-NFkB-luc2_4-1BB cells were resuspended in PBS at 1.0 x 10 ⁶ cells/ml. Cells were seeded at 0.1 x10 ⁶ cells per well in a 96-well round-bottom plate and stained with a fixable viability dye (LIVE/DEAD™ Fixable Near-Infrared Dead Cell Stain Kit (APC/Cy7), L34976 MolecularProbes, diluted 1:1000 in PBS) at 4°C for 20 min. After washing the cells once with PBS, they were incubated with 50 μl per well of 1000 nM to 0.0001 pM (1:8 serial dilutions) of the indicated BCMA-4-1BBL constructs, a negative reference (non-targeting 4-1BBL) or isotype control, or no construct for 30 min at 4°C. Cells were washed twice with cold FACS buffer (PBS, 2% fetal bovine serum; 1% 0.5 m EDTA, pH 8; 0.25% NaN ₃ azidopropene (20%)) and then stained with PE-conjugated secondary antibody (PE-AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific; Jackson Immunoresearch, 109-116-170) that was diluted 1/100 in cold FACS buffer for 30 min at 4°C. After the last two wash steps, cells were fixed with 1% PFA for 20 min at room temperature. Flow cytometry acquisition was performed using a custom-designed BD Biosciences Fortessa and analysis was performed using a BD Diva. _EC50 values were calculated using GraphPad Prism software. Data measured for various BCMA-4-1BBL antigen binding molecules and controls (eg, "secondary antibody" refers to samples containing only fluorophore-labeled secondary antibodies) are shown in Figures 2B and 3B .

All antigen-binding molecules containing 4-1BBL trimers targeting BCMA are able to bind to human BCMA ( Figures 2A and 3A ) and to human 4-1BB ( Figures 2B and 3B ) in a concentration-dependent manner. The BCMA-4-1BBL construct showed significantly stronger binding to human BCMA than the construct P1AH1062 (BCMA 5D5 LM4) prepared according to WO 2021/118246 A1. P1AH1062 is a bivalent BCMA antibody that contains two scFv fragments that bind to 4-1BB at their C-termini. Therefore, P1AH1062 contains two similar heavy chains of SEQ ID NO:89 and two light chains of SEQ ID NO:90. For all constructs, binding to human 4-1BB was comparable ( Figures 2B and 3B ). _EC50 values are listed in Table 17 below. Table 17 : _EC50 values (nM) for binding of the indicated antigen binding molecules to human 4-1BB or BCMA expressed on cells BCMA Antibody The part that binds to 4-1BB Name EC ₅₀ for binding to 4.1BB (nM) EC ₅₀ for BCMA binding (nM) BCMA (5D5 LM4) scFv (4-1BB) P1AH1062 1.89 103 BCMA (E04_1a1f) 4-1BBL P1AG7400 0.51 41.5 BCMA (54_2a2a) 4-1BBL P1AG7397 0.27 23.7 BCMA (E04_1a1a) 4-1BBL P1AG7409 0.45 21.6 BCMA (54_1a1a) 4-1BBL P1AG7422 0.29 10.8 - 4-1BBL Negative reference (non-targeted 4-1BBL) 0.23 -

In the second assay, the bispecific antigen-binding molecules tested included the trimeric 4-1BBL and the BCMA binders BCMA (54_2a2a) or BCMA (E04_1a1f). As one of the relapse mechanisms of MM may be non-truncated, missense mutations or in-frame deletions in the extracellular domain of BCMA, these molecules were compared head-to-head with the well-described BCMA-targeted CD3 T cell binders, terituzumab, aneutumomab and elenatumab (Lee et al., Nature Medicine 2023, 29, 2295-2306).

The binding properties of the various BCMA-targeted molecules are summarized in Table 17A . Both the BCMA-targeted 4-1BBL bispecific molecule and Anouketumab were able to bind to wild-type and all mutant forms of human BCMA in a concentration-dependent manner. In contrast, elenatumab had significantly reduced binding to the point mutation R27P, which only translated into a weak binding signal at the highest concentration. Terituzumab was unable to bind to the point mutations R27P and S30del. Efficient binding to BCMA is critical for the therapeutic activity of the BCMA-targeted bispecific molecules, indicating an advantage for molecules that are not affected by point mutations in the ECD of BCMA. The difference in the maximum values for wild-type and mutant BCMA transfectants is due to the different expression levels of human BCMA wild-type and variants on the transfectants and is consistent for all molecules tested. Table 17A: High-level summary of the binding properties of the indicated BCMA -targeting molecules to human wild-type (wt) BCMA or the indicated point mutations in the extracellular domain of human BCMA BCMA Antibody Name wt BCMA CHO-K1-BCMA R27P_ strain_12 CHO-K1-BCMA P34del_ clone_C10 CHO-K1-BCMA P33S_ strain_D5 CHO-K1-BCMA S30del_ strain_H1 BCMA (54_2a2a) P1AG7397 yes yes yes yes yes BCMA (E04_1a1f) P1AG7400 yes yes yes yes yes Elenatumab P1AH5054 yes Reduced and only at high concentrations yes yes yes Terituzumab FarmaMondoLot No. AT1334P1 yes no yes yes no Anoukitumab P1AF0105 yes yes yes yes yes

"Yes" means binding to BCMA variants. Example 4 In vitro functional characterization of antigen binding molecules containing 4-1BBL trimers targeting BCMA

Several cell-based in vitro and ex vivo assays were performed to evaluate the ability of BCMA-4-1BBL antigen binding molecules to promote bispecific mediator activation of T cells. (Jurkat and) T cell activation, proliferation, cytokine secretion, and tumor cytotoxicity as determined by flow cytometry were primary readouts.

1. The activity of the targeted BCMA-4-1BBL molecule was assessed in a Jurkat-NFκB-luc2_4-1BB reporter cell assay, where the molecule binds simultaneously to human 4-1BB on Jurkat-NFκB-luc2_4-1BB cells and BCMA expressed on the indicated MM cell lines, leading to 4-1BB superaggregation and thus receptor activation. Subsequent induction of the corresponding signaling pathway leads to activation of NFκB, which induces the expression of firefly luciferase. Activity in luciferase reporter cells is measured by addition of luciferase substrate and luminescent readout.

2. In another readout, the functionality of BCMA-4-1BBL was assessed in a PBMC co-culture assay in the presence of MM cell lines expressing BCMA and a fixed concentration of GPRC5D TCB, providing a synchronous first signal for T cell activation. Alternatively, BCMA-4-1BBL was added to different concentrations of FcRH5-targeted CD3 conjugates (FcRH5 x CD3) at a fixed concentration. FcRH5 x CD3 bispecific antibodies are disclosed, for example, in WO 2016/205520 A1. As indicated, the readouts were upregulation of surface activation markers on T cells and lysis of tumor cells.

3. Using a similar setup, untreated MM patient bone marrow aspirates containing autologous T effector cells and MM target cells expressing BCMA and GPRC5D were used to assess the functional activity of the BCMA-4-1BBL antigen binding molecule in the presence of GPRC5D TCB in vitro . 4.1 T cell activation - Jurkat NFκB/4-1BB reporter cell assay

The Jurkat NFκB reporter cell assay was used to investigate how different BCMA-4-1BBL antigen binding molecules can activate T cells. The assay was performed as described below:

Cell culture: NCI-H929 cells were cultured in RPMI1640 (Gibco) supplemented with 10% FCS (PAN-biotech), 2 mM L-glutamine (Sigma-Aldrich), 1 mM sodium pyruvate (Thermofisher), and 50 μM 2-hydroxyethanol (Thermofisher). Cells were cultured twice a week to maintain a density between 0.5 and 2.0 x10 ⁶ cells/ml. Jurkat IL2 reporter cells and Jurkat NF-κB/4-1BB reporter cell lines were grown in RPMI1640 medium supplemented with 10% FCS, 25 mM HEPES (Thermofisher), 2 mM L-glutamine, 0.1 mM non-essential amino acids (Thermofisher), and 1 mM sodium pyruvate. In addition, for Jurkat NFκB/4-1BB reporter cell lines, the medium was supplemented with 400 µg/ml hygromycin B and 600 µg/mL genomycin (Sigma-Aldrich). Jurkat cells were cultured twice a week to maintain a density between 0.1 and 0.5 x10 ⁶ cells/ml.

Target cells (NCI-H929) and effector cells (Jurkat NFκB/4-1BB reporter cells) were harvested and resuspended in assay medium (Jurkat cell medium without antibiotics) to obtain a cell density of 6 x10 ⁶ cells/ml for target cells and 2 x10 ⁶ cells/ml for effector cells. The cells were then mixed at an E:T ratio of 1:3 and 20 μl of the effector-target cell mixture was plated into each well of a white-walled 384-well flat-bottom plate (353988 Falcon™ 384-Well White Flat-Bottom Tissue Culture Microplate). Next, 20 μl of titrated amounts (600.0 to 0.02 nM) of BCMA-4-1BBL ABM were added to the plates in triplicate to a final volume of 40 μl per well. In addition, two separate control conditions were prepared. Target and effector cells were mixed separately—to indicate a baseline of effector cell-induced NFκB2 signaling in the absence of stimulation. To account for possible non-specific activation of effector cells, a 4-1BBL ABM containing a Fab against an irrelevant target (non-targeted 4-1BBL) was used. The final volume of the control wells (40 μl) was achieved with assay medium. The assay plates were centrifuged at 350 g for 1 min and incubated at 37°C in a humidified CO ₂ incubator for 4 h. The assay plates were incubated at room temperature for 5 min and then 20 μl of ONE-Glo solution (Promega) were added. In addition, the plates were centrifuged at 350 g for 1 min and incubated at room temperature in the dark for 10 min to achieve complete lysis of the cells. Luminescence was measured by (reading: 1 sec per well) using a Tecan Spark10M.

Briefly, the target NCI-H929 cell line expressing high levels of BCMA was mixed at a 1:3 (E:T) ratio with the effector Jurkat NFκB reporter cell line, which induces 4-1BB receptor-mediated signaling, inducing luminescence via the NFκB signaling pathway. Then, titrated (600.0 to 0.02 nM) BCMA-4-1BBL ABMs were added and luminescence was measured after 4 hours. The results showed that all tested BCMA-4-1BBL ABMs induced T cell activation in a dose-dependent manner ( Figure 4A ). Two molecules, P1AG7409 and P1AG7422, outperformed other variants and a molecule containing the BCMA antibody PR (P1AF7814) (Figures A to C ).

The data obtained are shown in Table 18 as mean ± sd of one independent experiment performed in triplicate out of three independent experiments. Table 18 : EC50 values (nM) and Emax (%) for T cell activation as measured in the NFκB/4-1BB reporter cell assay molecular _EC50 (nM) Efficacy (E _max (RLU)) P1AG7409 1.906 863586.6 P1AG7400 4.829 708230.2 P1AG7397 2.148 600361.9 P1AG7422 2.836 802822.6 P1AF7814 1.053 635327.1 4.2 PBMC co-culture assay in the presence of MM cell lines expressing BCMA or CHO-k1 transfectants expressing BCMA

To assess the ability of the BCMA-4-1BBL antigen binding molecule to function in a PBMC co-culture assay in the presence of MM cell lines expressing BCMA, the following method was performed.

Target cells: NCI-H929 and NCI-H929 BCMAko, CHO-k1 transfectants expressing BCMA, or MOLP-2, as indicated, respectively.

NCI-H929 (ATCC® CRL-9068™) is a human multiple myeloma cell line expressing BCMA. To assess the potential target-independent activity of the BCMA-4-1BBL antigen binding molecules, a BCMA knockout variant of the NCI-H929 cell line (generated using CRISPR/Cas9 technology) was also tested. Cells were cultured in RPMI 1640 (Gibco™ 31870074) supplemented with: 10% FCS (Gibco™ 16140-071), 10 mM HEPES (Gibco™ 15630056), 2 mM GlutaMAX-I (Gibco™ 35050-038), 1 mM sodium pyruvate (Gibco™ 11360039) and 50 μM 2-hydroxyethanol (Gibco™ 31350010). Cells were passaged 2 to 3 times per week by adding fresh medium to maintain a density between 0.5x10 ⁶ /ml and 2.5x10 ⁶ /ml. Cells were incubated at 37°C with 5% CO ₂ . On the day of the assay, target cells were harvested, counted, and resuspended in assay medium (RPMI 1640 Gibco™ 31870074 w/HEPES w/GlutaMax plus 10% FCS, Sigma-Aldrich #F4135).

Stable CHO transfectants (parental cell line CHO-k1 ATCC #CCL-61) were cultured in DMEM/F-12 (Gibco, #10565018) supplemented with 10% fetal bovine serum (Gibco, #16140063 or Sigma-Aldrich F4135) and 1% GlutaMAX supplement (Gibco; #31331-028), including 6 μg/ml puromycin (Invivogen; #ant-pr-1). Adherent CHO cells were detached using trypsin (Gibco, TrypLE™ Express Enzyme #2605-010 from ThermoFisher Scientific). The cells were centrifuged at 300 xg for 5 minutes, resuspended in PBS, and counted and viability tested using a Cedex HiRes analyzer. The target cells were centrifuged at 300 xg for 5 minutes, PBS was removed, 1 ml was retained, and the pellet was irradiated for 1.49 minutes (5000 Rad, 50 Gy) using an RS 2000 irradiator (RadSource) without disturbing it. The cells were then resuspended in PBS for subsequent counting using a Cedex HiRes analyzer. For the assay, irradiated target cells were adjusted to 1.2 Mio cells per ml in assay medium (RPMI 1640 Gibco™ 72400-21 w/ HEPES w/GlutaMax plus 10% FCS, Sigma-Aldrich #F4135).

MOLP-2 (DSMZ ACC607) is a human multiple myeloma cell line that expresses, for example, BCMA, GPRC5D, and FcRH5. Cells were cultured in RPMI 1640 containing HEPES and GlutaMax (Gibco™ 72400-21) and supplemented with 20% FCS. Cells were passaged twice a week by adding fresh medium to maintain a density between 0.3x10 ⁶ /ml and 1x10 ⁶ /ml. Cells were incubated at 37°C with 5% CO _2. For this assay, MOLP-2 cells were counted and viability was checked. Cells were washed with D-PBS, centrifuged at 280 xg for 5 minutes, and washed once with PBS. Next, cells were resuspended in PBS to a concentration of 2 Mio cells per ml. Pre-diluted CellTrace Far Red dye (Invitrogen, Thermo Fisher Scientific, #C34564) in PBS was added to the cell suspension at a final concentration of 0.04 μM, and cells were stained at 37°C in a water bath for 15 minutes. Add assay medium (RPMI 1640 Gibco™ 72400-21 w/ HEPES w/GlutaMax plus 10% FCS, Sigma-Aldrich #F4135), centrifuge cells at 280 x g for 5 minutes, count and check viability and adjust to 1.2 Mio cells per ml in assay medium (RPMI 1640 Gibco™ 72400-21 w/ HEPES w/GlutaMax plus 10% FCS, Sigma-Aldrich #F4135).

Effector cells: PBMC

Frozen human peripheral blood mononuclear cells (PBMCs) from human whole blood in CPD were obtained from Cambridge bioscience or Biomex. Alternatively, PBMCs were isolated from chromogenic layers collected from anonymous healthy volunteers through the Zürich blood donation center. PBMCs were isolated from chromogenic layers by diluting the chromogenic layer 2:1 with PBS and pipetting the PBS/chromogenic layer mixture into a Leucosep tube (227290, greiner bio-one) containing 15 ml of Histopaque®-1077 (10771, Sigma) under the barrier. The cells were centrifuged continuously at 450 x g for 30 min at room temperature (density centrifugation). The interphase consisting of PBMCs was collected and washed several times with PBS. PBMCs were resuspended in freezing medium (80023-IBI) at 40 to 67 Mio cells/ml and frozen at -80°C overnight using a cell freezing container (CLS432002 Corning), then transferred to nitrogen vapor phase for storage.

PBMCs were stored in nitrogen vapor phase and thawed on the day of the assay. Cells were counted and then labeled with the cell proliferation dye eFluor™ 450 (65-0842-90, eBioscience™). Briefly, PBMCs were washed once with DPBS (Gibco), the supernatant discarded and the cells resuspended in DPBS to 2 Mio cells per well. 10 ml of the cell suspension was added to a 50 ml-falcon tube, followed by the addition of 10 ml of 10 μM concentrated cell proliferation dye eFluor™ 450 while carefully vortexing (final dye concentration was 5 μM). After incubation at 37°C (water bath) for 10 min, add 30 ml of warm assay medium and incubate the cells at 4°C for 5 min, or add 30 ml of cold medium to stop the labeling reaction. Centrifuge the cells, count and resuspend in assay medium to obtain a cell density of 1.2 x10 ⁶ PBMC/ml.

In another assay, human PBMCs were thawed and washed with PBS, including centrifugation at 300 xg for 15 minutes at room temperature. To label PBMCs with CFSE, CellTrace CFSE dye (Invitrogen from ThermoFischer Scientific, #C34554) was diluted in room temperature PBS (2 mM stock concentration) and cells were incubated at a final concentration of 0.1 μM in a water bath at 37°C for 15 minutes. 20 ml of medium containing 10% FCS was added and the suspension was centrifuged at 300 xg for 15 minutes. Cells were resuspended at a concentration of 1.2 Mio cells per ml in assay medium (RPMI 1640 Gibco™ 72400-2 w/HEPES w/GlutaMax plus 10% FCS, Sigma-Aldrich #F4135).

Preparation of co-cultures

50 μl (0.06x10 ⁶ ) NCI-H929 or NCI-H929 BCMAko cells and 50 μl (0.06 x 10 ⁶ efluor450 labeled PBMCs) were plated in each well of a 96-well round bottom plate (TPP # 92097) to give a 1:1 ratio. Next, 50 μl of titrated amounts (0.12 to 500 nM) of BCMA-4-1BBL antigen binding molecules (0.14 nM to 300 nM) were added to the plate in triplicate. In addition, 50 μl of a fixed concentration (0.32 pM) of GPRC5D-TCB (P1AE6625) was added to the plate to a final volume of 200 μl per well. In addition, three separate control conditions were prepared. Target cells and effector cells with BCMA-4-1BBL antigen binding molecules and without GPRC5D-TCB were added to address potential activation of effector cells by BCMA-4-1BBL molecules in the absence of a first signal. In another control, target cells and effector cells were treated with GPRC5D-TCB alone to indicate a baseline. Finally, a control with target cells and effector cells in which no antibody was added (untreated) was prepared. The control wells were brought to a final volume (200 μl) with assay medium. The assay plates were centrifuged at 350 g for 1 min and incubated at 37°C in a humidified CO ₂ incubator for 4 days.

In another setup, 50 μl (0.06 Mio) of the corresponding CHO-hBCMA variant or parental (BCMA negative) CHO-k1 and 50 μl (0.06 Mio) of eFluor450 labeled PBMC were plated per well in a 96-well flat bottom plate (TPP, #92096) to give a 1:1 effector to target cell ratio. Next, 50 μl of a fixed concentration (final concentration 90 pM) of anti-CD3 IgG (strain SP34-1, BD Biosciences 551916) was added to the plate. In addition, 50 μl of BCMA-4-1BBL antigen binding molecules were added to the plates in triplicate, covering final concentrations of 3.84 pM to 300 nM, with a final volume of 200 μl per well. In addition, the plates contained controls of target and effector cells and anti-CD3 IgG alone to determine the baseline effect induced by the first signal provider alone. Finally, controls with target and effector cells were prepared in which no antibody was added (untreated). The assay medium was used to reach the final volume of the control wells (200 μl). The assay plates were centrifuged at 350 g for 1 min and incubated at 37°C in a humidified _CO2 incubator for 4 days.

Alternatively, 50 μl (0.06 Mio) FarRed-labeled MOLP-2 and 50 μl (0.06 Mio) CFSE-labeled PBMC were plated per well in a 96-well round-bottom plate (TPP, #92097) to give a 1:1 effector to target ratio. Next, 50 μl of different doses of FcRH5 x CD3 were added (final concentrations ranged from 0.03 pM to 32 nM). 50 μl of assay medium was added to some wells, and 50 μl of BCMA-4-1BBL antigen binding molecule at a fixed concentration of 10 nM was added to other wells (final volume was 200 μl per well). In addition, a control containing target and effector cells and 10 nM BCMA-4-1BBL alone was plated. Finally, a control with target and effector cells to which no antibody was added (untreated) was prepared. The assay medium was used to reach the final volume of the control wells (200 μl). The assay plates were centrifuged at 350 g for 1 min and then incubated at 37°C in a humidified _CO2 incubator for 4 days.

FACS staining and readout

After incubation, cells were stained to assess T cell activation and proliferation. Cells were washed once with 200 μL PBS and then stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain (1:500) (ThermoFisher Scientific Catalog No. L34976), FITC anti-human CD4 (strain RPA-T4), BV711 anti-human CD8 (strain RPA-T8), APC anti-human CD25 (strain BC96), and PerCP-Cy5.5 anti-human CD137 (strain 4B4-1) in PBS for 30 min at 4°C. The stain and the strains were from BioLegend. Flow cytometric acquisition was performed on a custom-designed BD Biosciences Fortessa and analyzed using FlowJo software (Tree Star, Ashland, OR) and GraphPad Prism software.

In another experiment, after 4 days of incubation, the plates with co-cultures of PBMCs and variants of CHO-k1 cells were centrifuged at 400 x g for 4 minutes at 4°C. The cells were transferred to a 96-well U-bottom plate (TPP #92097) where they were washed with 150 μL of PBS per well. The cells were centrifuged at 400 x g for 40 minutes at 4°C. This wash step was repeated once. The samples were mixed with an Eppendorf MixMate at 850 rpm for 15 seconds. Afterwards, 25 μL of FACS antibody cocktail (LIVE/DEAD™ Fixable Near-Infrared Dead Cell Stain, 1:1000, ThermoFisher Scientific, #L34976; FITC anti-human CD4, clone RPA-T4; BV711 anti-human CD8, clone RPA-T8; APC anti-human CD25, clone BC96; BUV395 anti-human CD69, clone FN50; PE anti-human CD137, clone 4B4-1, both the stain and the clones were from BioLegend and pre-diluted in PBS) was added to each well, and the cells were incubated at 4°C in the dark for 30 min. Afterwards, cells were washed twice with 150 μl/well of FACS buffer, mixed with Eppendorf MixMate at 850 rpm for 15 seconds, and fixed with 100 μl/well of 1% formaldehyde in PBS at 4°C overnight. The next day, cells were washed twice with 150 μl/well of FACS buffer. Finally, cells were resuspended with 150 μl/well of FACS buffer and 100 μl/sample was acquired using BD FACSymphony™ A5 Cytometer.

In another experiment, the plates with co-cultures of PBMCs and MOLP-2 cells were removed from the incubator after 4 days and centrifuged at 400 xg for 4 minutes at 4°C. The cells were washed once with PBS (200 uL/well) and inserted into a shaker at 850 rpm for 15 seconds to resuspend the cells. PBS including Zombie UV™ Fixable Viability Dye (Biolegend 423108, 1:1000) was added to the wells (50 uL/well) and incubated at room temperature (protected from light) for 20 min. Next, 150 ul of PBS was added to each well, the cells were centrifuged at 400 xg for 4 min, the supernatant was flicked and the cells were resuspended using a shaker as described above. Afterwards, 25 μL of FACS antibody cocktail (BUV737 anti-human CD4, clone SK3 from BD Biosciences; BV785 anti-human CD8, clone SK1; BV605 anti-human CD25, clone BC96; PE anti-human CD137, clone 4B4-1; PE/Dazzle594 anti-human CD3, clone OKT3; except anti-human CD4, all of these clones were from BioLegend and pre-diluted in FACS buffer) was added to each well, and the cells were incubated at 4°C in the dark for 30 min. The cells were washed three times with FACS buffer at 200 μl/well, mixed with Eppendorf MixMate at 850 rpm for 15 seconds, and fixed with 1% formaldehyde in PBS at 50 μl/well for 15 minutes at room temperature in the dark. Next, the cells were washed twice with FACS buffer at 150 μl/well. Finally, the plate was centrifuged, the supernatant was removed and FACS buffer containing counting beads (Life Technologies, C36950) was added to have 5 ul beads (4900 beads per well in 150 μl). The plate was acquired using a BD FACS Fortessa. To determine tumor cell lysis in MOLP-2 cells, set a pre-gate in the FSC-A/SSC-A channel to include larger tumor cells and exclude lymphocytes and counting beads. Next gate on the singletons (FSC-H, FSC-A) and from there gate on FarRed positive/CD3 negative cells. Then gate on Zombie UV positive dead cells. Calculate the dead cell count per μl by dividing the cell count from this gate by the bead count (separate gate in FSC-A/SSC-A) multiplied by the counting bead concentration.

result

As shown in Figures 5A and 5B , all BCMA-4-1BBL antigen binding molecules were able to significantly promote CD8 ⁺ T cell activation (CD25 upregulation) in the presence of BCMA-expressing target cells and the first signal provided by TCB ( Figure 5A ). At 300 nM, none of the BCMA-4-1BBL molecules induced T cell activation in the absence of the first signal/TCB. In addition, there was no promotion of TCB-mediated activation of T cells in the absence of BCMA-expressing targets ( Figure 5B ). Most importantly, these molecules had higher efficacy than the bispecific antibody P1AH1062 (5D5 LM4).

Table 19 summarizes the EC ₅₀ values and Emax values derived from the data shown in Figure 5A and the results obtained with 1 to 3 additional donors. EC ₅₀ values were calculated using GraphPadPrism6. Table 19: EC ₅₀ values (nM) and efficacy of upregulation of CD25 on CD8 T cells molecular _EC50 (nM) Efficacy (Emax(MFI)) Donor 1 Donor 2 Donor 3 (as shown in Figure 5A) Donor 4 Donor 1 Donor 2 Donor 3 (as shown in Figure 5A) Donor 4 P1AG7409 0.980 1.063 3.095 1.053 13532 22866 23932 24010 P1AG7400 1.272 1.477 4.958 2.306 14036 25341 24254 23437 P1AG7422 0.819 1.197 2.315 1.064 14416 26887 20680 22327 P1AG7397 0.481 0.889 1.240 1.036 13585 25095 19191 19788 P1AH1062 Not tested Not tested 1.324 0.498 Not tested Not tested 9254 9094

In another co-culture assay, the functional activity of the bispecific BCMA-4-1BBL molecules P1AG7397 and P1AG7400 was tested in combination with anti-CD3 IgG as a primary signal provider and in the presence of CHO transfectants expressing wild-type or mutant forms of human BCMA (see also Example 3, binding to transfectants).

Consistent with the retained binding of both BCMA-4-1BBL molecules to various point mutations in the ECD of human BCMA compared to wild-type human BCMA (Example 3), both molecules exhibited similar concentration-dependent promotion of T cell activation (as indicated by upregulation of CD25 on CD8 T cells) beyond CD3 IgG-mediated T cell activation in the presence of mutant or wild-type BCMA expressed on CHO transfectants ( Figures 5C to 5H , Table 19A ). The difference in maximal activity of wild-type and mutant BCMA transfectants is due to the different expression levels of human BCMA wild-type and variants on the transfectants. This data supports the broader use of BCMA-4-1BBL in patients harboring one of the tested BCMA point mutations without any significant loss of activity being expected. Table 19A: EC ₅₀ values (nM) for T cell activation as measured by flow cytometry and detection of CD25 on CD8 T cells molecular _EC50 (nM) CHO-K1_BCMA (wild type) CHO-K1_BCMA R27P_ strain_12 CHO-K1_BCMA P34del_ clone_C10 CHO-K1_BCMA P33S_Strain_D5 CHO-K1_BCMA S30del_Strain_H1 P1AG7400 1.02 0.09 0.98 1.24 1.89 P1AG7397 0.83 0.33 2.5 1.85 0.95

To test the combination with another CD3 bispecific molecule in the treatment of multiple myeloma, another series of assays were performed to test the promotion of T cell activation and tumor cell lysis on CD3 binders targeting FcRH5 (FcRH5 x CD3). 10 nM BCMA-4-1BBL further enhanced FcRH5 x CD3-induced T cell activation and lysis of MOLP-2 tumor cells over a broad range of FcRH5 x CD3 concentrations, i.e., 32 pM to 32 nM for T cell activation (frequency of CD137-positive CD8 T cells, Figures 5I to 5L ) and 320 pM to 32 nM for tumor cell lysis ( Figures 5M to 5P ). These data support the ability of BCMA-4-1BBL to further enhance T cell activation and tumor cell lysis induced by various T cell engagers. 4.3 In vitro functional characterization of BCMA - targeting 4-1BBL agonist antigen binding molecules

The therapeutic efficacy of the bispecific BCMA-4-1BBL antigen binding molecule in combination with GPRC5D-TCB was tested in heparinized primary MM patient bone marrow aspirate (BMA) samples obtained within 48 hours of extraction.

The cell amount at baseline, its viability and phenotype, and the percentage of pathological MM plasma cells were analyzed by flow cytometry. Next, at least 100,000 MM plasma cells (MM PCs) were seeded in 24- or 48-well plates. A fixed concentration of the GPRC5D-CD3 bispecific molecule (GPRC5D TCB, 1 or 10 nM as indicated) was tested alone or in combination with increasing doses of different BCMA-4-1BBL antigen binding molecules. As a negative reference, TCB isotypes were added, respectively non-targeting 4-1BBL molecules. After 96 hours, cells were transferred to 15 ml falcon tubes. Sterile PBS was added to 1 mL. According to standard methods, cells were centrifuged at 540 g for 5 min and red blood cells were lysed for 15 min. Cells were centrifuged at 800 g for 10 min, washed once with sterile PBS, and incubated with maleimide for 20 min at room temperature to stain dead and live cells. Cells were washed with 12 ml PBS (including 0.09% NaN ₃ and 0.5% BSA) to remove unbound maleimide. Thereafter, the cell pellet was resuspended in 500 μL PBS and filtered through a 5 ml polystyrene round-bottom tube with a cell filter cap to discard cell clumps and remove potential membrane aggregates. The cells were centrifuged again at 540 g for 5 min. Finally, the cells were stained with the indicated antibodies against different surface markers for 20 min at room temperature in the dark ( Table 20 and Table 21 ), washed as described above and resuspended in sterile PBS. The total sample was obtained by flow cytometry. Table 20 : Antibody combinations used to analyze pre-treated samples BB515 BB700 PE APC APCR700 APC/Cy7 BV421 BV480 CD38ME CYT-38F/CYT-38F2 Cytognos CD138 clone MI15 BD Bioscienes BCMA strain 19F2 Biolegend GPRC5D strain from Roche CD56 clone N901 (NKH-1) BC CD45 clone 2D1 Biolegend CD137 strain 4B4-1 Biolegend CD3 clone SK7 BD Biosciences BV605 BV650 BUV395 BUV496 4-1BBL strain C65-485 BD Biosciences CD28 clone CD28.2 BD Biosciences HLADR strain Tu39 BD Biosciences CD8 clone HIT8a BD Biosciences Table 21 : Antibody combinations used to determine MM-PC lysis and effector cell activation BB515 BB700 PE PE-CF594 APC PC7 APCR700 APC/Cy7 CD38ME CYT-38F/CYT-38F2, Cytognos CD138, strain MI15, BD Bioscienes CD127, strain A019D5, Biolegend CD57, clone NK-1, BD Biosciences CD25, clone 2A4, BD Biosciences CD13, clone L138, BD Biosciences CD56 clone N901 (NKH-1), BC CD45, clone 2D1, Biolegend BV421 BV480 BV605 BV650 BUV395 BUV496 CD4 B49197 CD16, clone 3G8, BD Biosciences CD3 clone SK7, BD Biosciences PD1 strain EH12, BD Biosciences HLADR strain Tu39, BD Biosciences CD14 clone MɸP9, BD Biosciences

In another experiment, frozen and thawed primary MM bone marrow mononuclear cells (BMMNCs) were used to test the therapeutic efficacy of BCMA-4-1BBL antigen binding molecules and GPRC5D-TCB in combination therapy, as described below.

On the first day, samples were thawed and resuspended in StemSpan SFEMII (StemCell) medium containing 20% human serum, 55 μM β-hydroxyethanol (Gibco catalog number 11528926), and 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, 100x stock solution). 3 Mio cells were plated per well of a 12-well plate containing 100 ng/mL recombinant human IL-6 and kept in an incubator at 37°C with 5% _CO2 for 24 hours.

Afterwards, samples were harvested and 0.1 Mio cells were used for baseline characterization. The remaining cells were washed twice with sterile PBS and stained with NIR live/dead stain and sorted for viability using a FACS BD Aria sorter. Viable cells were resuspended in culture medium and 0.1 Mio cells were plated per well of a 96-well plate. Cells were incubated in a total volume of 150 uL per well at 37°C with 5% CO ₂ for 96 hours in the absence or presence of the indicated antibodies. Samples were washed twice with sterile PBS and stained as follows: First, live/dead staining was performed using NIR dye (ThermoFisher) at room temperature in the dark for 20 minutes. After a wash step, TruStain Fcx Blocking Human was added in FACS buffer (PBS containing 2% fetal bovine serum, 1% 0.5M EDTA pH 8, 0.25% NaN ₃ -Na 2 O) and the cells were incubated for another 10 min at room temperature (RT) in the dark before adding the corresponding antibody cocktail ( see Table 25 ).

Surface staining was performed for 20 minutes at room temperature in the dark. Cells were washed once with FACS buffer and fixed in PBS containing 2% paraformaldehyde (PFA) for 10 minutes at room temperature in the dark. Samples were washed with FACS buffer, resuspended in FACS buffer, and analyzed by flow cytometry after adding counting beads to determine the absolute number of cells. Table 22 : Antibody combinations used for baseline characterization and effector cell activation Baseline group: Mark Fluorescent dye hCD45 BUV395 T cells CD3 BV510 CD8 BUV737 CD4 PerCPCy5.5 B cells CD19 FITC Activated cells/bone marrow cells HLA-DR BV786 Activate T cells 4-1BB PE-Cy5 CD25 Pe-Dazzle594 CD28 BV650 CD69 PE-Cy7 Multiple Myeloma (+ Malignancy) CD138 BV421 CD38 BV711 BCMA PE GPR5CD AF647 NK cells/malignant cells CD56 BV605 Live/Dead NIR Same type group Mark Fluorescent dye hCD45 BUV395 CD3 BV510 CD8 BUV737 CD4 PerCPCy5.5 CD19 FITC Same type BV786 Same type PE-Cy5 Same type Pe-Dazzle594 Same type BV650 Same type PE-Cy7 CD138 BV421 CD38 BV711 Same type PE Same type AF647/APC CD56 BV605 Live/Dead NIR Activation Group: Mark Fluorescent dye hCD45 BUV395 CD3 BV510 T cells CD8 BUV737 CD4 PerCPCy5.5 B cells CD19 FITC Activated cells/bone marrow cells HLA-DR BV786 Activate T cells 4-1BB PE-Cy5 CD25 Pe-Dazzle594 CD28 BV650 CD69 PE-Cy7 PD-1 PE CD39 APC TIM-3 BUV615 Multiple Myeloma (+ Malignancy) CD138 BV421 CD38 BV711 NK cells/malignant cells CD56 BV605 Live/Dead NIR

FACS surface antibodies were purchased from BD Biosciences, Miltenyi, or LuBiosciences and used according to the manufacturer's recommendations. The detection reagent antibody against human GPRC5D was produced by Roche.

Results: Figures 6A to 6E and Table 23 show that the BCMA-4-1BBL antigen binding molecule P1AG7422 was able to significantly promote the activation of CD8 ⁺ and CD4 ⁺ T cells (CD25 upregulation) and increase the level of the degranulation marker CD107a, exceeding the activation mediated by the GPRC5D-targeted CD3 binder (GPRC5D-TCB) and induction of degranulation. In the presence of 200 or 53 nM of non-targeted 4-1BBL, there was no induction of activation over that of TCB, indicating that the 4-1BBL molecule requires cross-linking via the tumor antigen targeting moiety to be active. Table 23 : Percentage of tumor cell lysis and corresponding upregulation of the activation marker CD25 or the degranulation marker CD107a on CD8 ⁺ or CD4 ⁺ T cells after 96 hours of incubation of primary MM BM aspirates with the indicated molecules as assessed by flow cytometry treatment Tumor cell lysis % CD25 ⁺ CD8 ⁺ T cells % CD25 ⁺ CD4 ⁺ T cells % CD107a ⁺ CD8 ⁺ T cells % CD107a ⁺ CD4 ⁺ T cells % Untreated 0 0.03 0.91 0.72 0.78 TCB homologous 3.15 0.09 1.5 0.98 1 GPRC5D-TCB only 46.65 36.5 42.57 18.83 17.63 Non-targeted GPRC5D-TCB + 53.18 nM 4-1BBL 44.96 38.25 44.48 21.71 19.64 Non-targeted GPRC5D-TCB + 200 nM 4-1BBL 44.84 39.34 42.85 22.61 19.34 GPRC5D-TCB + 1 nM BCMA-4-1BBL 45.24 36.45 42.18 21.09 19.53 GPRC5D-TCB + 3.7 nM BCMA-4-1BBL 49.89 45.78 46.43 27.42 21.06 GPRC5D-TCB + 14.14 nM BCMA-4-1BBL 59.56 48.55 46.62 30.44 23.55 GPRC5D-TCB + 53.18 nM BCMA-4-1BBL 54.54 51.06 50.04 32.28 24.1 GPRC5D-TCB + 200 nM BCMA-4-1BBL 54.99 52 51.82 31.84 25.07

This was also confirmed with another primary MM patient BM aspirate sample, as depicted in Figures 7A to 7E and Table 24. Here, in the presence of BCMA-4-1BBL molecules P1AG7409 and P1AG7400 at 4.4 nM and higher concentrations, promotion of TCB-mediated lysis of primary malignant MM plasma cells or T cell activation (CD25 upregulation) was observed, but no significant lysis was observed in the presence of non-targeted 4-1BBL at concentrations even as high as 40 and 120 nM. Table 24 : Percentage of tumor cell lysis and corresponding upregulation of the activation marker CD25 on CD8 ⁺ or CD4 ⁺ T cells after 96 hours of incubation of primary MM BM aspirates with the indicated molecules as assessed by flow cytometry treatment Tumor cell lysis % %CD25+ CD8+ T cells %CD25+CD4+ T cells Untreated 0 7.03 7.93 TCB homologous -29.44 8.67 8.53 GPRC5D-TCB only 32.13 33.58 33.8 Non-targeted GPRC5D-TCB + 200 nM 4-1BBL 19.93 43.2 39.72 GPRC5D-TCB + 1 nM BCMA-4-1BBL 7400 33.2 46.62 40.1 GPRC5D-TCB + 3.7 nM BCMA-4-1BBL 7400 40.92 61.98 47.79 GPRC5D-TCB + 14.14 nM BCMA-4-1BBL 7400 45.39 60.71 46.78 GPRC5D-TCB + 53.18 nM BCMA-4-1BBL 7400 41.61 59.07 45.56 GPRC5D-TCB + 200 nM BCMA-4-1BBL 7400 29.56 47.2 39.52 GPRC5D-TCB + 1 nM BCMA-4-1BBL 7409 44.34 47.41 40.19 GPRC5D-TCB + 3.7 nM BCMA-4-1BBL 7409 42.86 58.2 46.99 GPRC5D-TCB + 14.14 nM BCMA-4-1BBL 7409 49.24 61.35 48.45 GPRC5D-TCB + 53.18 nM BCMA-4-1BBL 7409 48.89 60.42 48.5 GPRC5D-TCB + 200 nM BCMA-4-1BBL 39.74 49.57 43.61

Another series of experiments was performed using thawed BM MNCs from primary MM patients. As indicated in Figures 8A to 8D and Tables 25 and 26, 40 nM BCMA-4-1BBL (P1AG7400) induced significant lysis of malignant MM plasma cells in all four samples evaluated, exceeding GPRC5D-targeted TCB. The maximal effects induced by TCB and by the combination of TCB and BCMA-4-1BBL varied between MM patient samples, suggesting that immune cell composition, effector cell to target cell ratio, and T cell fitness may affect the overall results. Table 25 : Percentage of tumor cell lysis and corresponding upregulation of the activation marker CD25 on CD8 ⁺ or CD4 ⁺ T cells after 96 hours of incubation of primary MM BM aspirates with the indicated molecules as assessed by flow cytometry treatment Tumor cell lysis % CD25 MFI on CD8+ T cells CD25 MFI on CD4+ T cells Untreated 0.00 650.17 732.45 TCB homologous -32.93 541.47 700.76 GPRC5D-TCB only 77.57 15197.3 19857.16 Non-targeted GPRC5D-TCB + 40 nM 4-1BBL 80.57 17703.97 20753.89 Non-targeted GPRC5D-TCB + 120 nM 4-1BBL 81.89 21333.67 23928.77 GPRC5D-TCB + 1.5 nM BCMA-4-1BBL 7400 82.3 19415.77 22953.07 GPRC5D-TCB + 4.44 nM BCMA-4-1BBL 7400 86.35 26367.28 25230.39 GPRC5D-TCB + 13.3 nM BCMA-4-1BBL 7400 87.29 25549.06 25802.42 GPRC5D-TCB + 40 nM BCMA-4-1BBL 7400 85.17 24739.75 24742.11 Table 26 : Increased lysis of primary malignant MM plasma cells after incubation of primary MM BM MNCs with BCMA-4-1BBL (P1AG7400) for 96 hours as assessed by flow cytometry MM BM MNC, donor 1 MM BM MNC, donor 2 MM BM MNC, donor 3 MM BM MNC, donor 4 treatment Live MM cells% Live MM cells% Live MM cells% Live MM cells% Untreated 1 1 1 1 Only 0.025 nM GPRC5D-TCB 0.510519 1.090909 0.428571 0.387435 0.025 nM GPRC5D-TCB + P1AG7400 0.382889 0.636364 0.351351 0.240838

Samples were normalized to untreated cells (untreated cells = 1) Example 5 In vivo functional characterization of 4-1BBL agonist antigen binding molecules targeting BCMA

The efficacy study described here was designed to investigate the efficacy of the combination of a BCMA-4-1BBL antigen binding molecule and an anti-GPRC5D/anti-CD3 bispecific antibody (GPRC5D x CD3).

Cell culture: Human NCI-H929 cells (obtained from Roche Nutley) were cultured in RPMI1640 high glucose medium containing 10% FCS, 2 mM L-glutamine, 10 mM HEPES and 1 mM sodium pyruvate (37°C, 5% CO ₂ ). To generate tumor-bearing mice, 50 μl of cell suspension (2.5 x10 ⁶ cells) was co-injected with 50 μl of matrix gel subcutaneously into the right abdomen of anesthetized humanized NSG mice.

Mouse Model: Humanized NSG mice were provided by Jackson Laboratories, Sacramento USA. For implantation, animals were irradiated (140 cGy) and injected with CD34 ⁺ cord blood cells (9x10 ⁴ cells) from healthy donors (hematopoietic stem cells; HSC). After receipt, animals were housed for one week to acclimate to the new environment and observed. Mice were maintained under specific pathogen-free conditions using a 12 h light/12 h dark day cycle according to agreed guidelines (GV-Solas; Felasa; TierschG). Ongoing health status monitoring was performed regularly. The experimental research protocol has been reviewed and approved by the local government (ROB-55.2-2532.Vet_03-20-170).

Randomization and Treatment: When the tumor volume of subcutaneous tumors reached 200 mm³, humanized mice were randomized into different treatment groups (n=10/group) based on tumor volume and body weight. All antibodies and vehicle (histidine buffer) were administered intravenously (iv) once a week. Co-stimulatory molecules were injected 48 hours ( Figures 9A to 9H ) or 24 hours ( Figures 10A to 10H ) after GPRC5D x CD3 treatment.

Animal monitoring: Animals were monitored daily for clinical symptom control and adverse effects. Body weight and tumor growth were monitored twice a week (caliper measurements). Animals were killed according to termination criteria or at the end of the experiment.

Statistics: Graphs were generated using GraphPad prism software. Time-to-event analysis was performed using the in-house tool DOPSa (based on software R). The critical tumor volume was chosen as the maximum observed baseline tumor volume plus the standard deviation of the baseline tumor volume (rounded to the nearest ten). Study groups were compared using the log-rank test, and p values were corrected for multiple testing using the Bonferroni-Holm method. Significant changes with p<0.05 compared to the control group (GPRC5D x CD3) are indicated with asterisks (* p<0.05, ** p<0.01, *** p<0.001).

Combination of GPRC5D x CD3 and BCMA-4-1BBL

To compare GPRC5D x CD3 monotherapy and combination with BCMA-4-1BBL molecules, NCI-H929 tumor-bearing animals were treated weekly and monitored for tumor growth inhibition. BCMA-4-1BBL co-stimulatory molecules P1AG7422 and P1AG7409 were administered 48 hours after GPRC5D x CD3 treatment (P1AE3357). Overall, GPRC5D x CD3 monotherapy (1 mg/kg) induced moderate tumor growth inhibition. The combination with P1AG7422 induced significantly higher antitumor efficacy at both concentrations (10 and 3 mg/kg) compared to GPRC5D x CD3 monotherapy ( Figures 9A to 9H ). The GPRC5D x CD3 and P1AG7422 treatment group had the highest number of responders (10 mg/kg; 8 responders) and showed the lowest tumor volume at termination.

In follow-up studies, P1AG7397 and P1AG7400 were tested as combination partners of GPRC5D x CD3 ((P1AE3357)) at two different doses (10 and 20 mg/kg; 24 hours after GPRC5D x CD3) ( Figures 10A to 10H ). All combinations induced significantly higher tumor growth inhibition compared to GPRC5D x CD3 monotherapy (Table 28). P1AG7397 induced slightly more number of responders (n=4) compared to the combination of P1AG7400 and GPRC5D x CD3 (n=3). Most homogeneous responses were induced by the combination of GPRC5D x CD3 and 10 mg/kg P1AG7400. This is also shown in the tumor weight at termination ( Figure 10H ), indicating more heterogeneous tumor weight and size after GPRC5D x CD3 combined with 10 and 20 mg/kg P1AG7397 or 20 mg/kg P1AG7400. The final measured tumor volume is shown in Table 27. Table 27 : Tumor Volume at Termination (mm ³ ) Treatment group GPRC5D x CD3 (1 mg/kg) + P1AG7397 (10 mg/kg) GPRC5D x CD3 (1 mg/kg) + P1AG7397 20 mg/kg) GPRC5D x CD3 (1 mg/kg) + P1AG7400 (10 mg/kg) GPRC5D x CD3 (1 mg/kg) + P1AG7400 (20 mg/kg) Research Day Day 57 Day 41 Day 54 Day 57 Day 41 Day 54 Day 57 Day 41 Day 54 Day 57 Animal X01 twenty one 10.99 178.88 12.89 Animals X02 114.71 1026.7 1280.5 270.71 Animals X03 21.09 1354.8 10.08 1735.4 Animals X04 1330.1 1375.5 3.99 1877.2 Animals X05 2146.5 999.46 754.4 1698.3 Animals X06 292.27 15.5 616.85 390.99 Animals X07 28.82 751.22 7.72 Animals X08 1475.6 14.74 19.9 9.4 Animals X09 1495.2 1100.9 225.75 1650.8 Animals X10 1228.6 9.16 1442 2147.9 Table 28 : P values of the combination group compared with GPRC5D x CD3 Comparison with GPRC5D x CD3 P-value Significance P1AG7397 (10 mg/kg) 0.0004 *** P1AG7397 (20 mg/kg) 0.0053 ** P1AG7400 (10 mg/kg) 0.0043 ** P1AG7400 (20 mg/kg) 0.0203 *

Log-rank test p＞0.05*, p＞0.01**, p＞0.001***

In the third experiment, P1AG7400 was tested in combination with GPRC5D x CD3 (0.05 mg/kg P1AE6625) at different doses (40, 4 and 0.4 mg/kg) and compared with GPRC5D x CD3 monotherapy. P1AG7400 was injected 24 hours after GPRC5D x CD3. GPRC5D x CD3 monotherapy induced strong tumor growth inhibition in the NCI-H929 model ( Figures 11A to 11F ). However, tumors began to recur after the 4th treatment cycle (Table 29). All three doses of P1AG7400 (40 and 10 mg/kg) inhibited tumor regrowth. At the end of the experiment (day 64), there were 5, 7 and 4 responders remaining in the P1AG7400 40, 4 and 0.4 mg/kg combination groups, respectively. In the GPRC5D x CD3 monotherapy group, there were no responders left. Table 29 : Time points of tumor escape Group No response After the 4th cycle After the 5th cycle After the 6th cycle After the 7th cycle Relievers GPRC5D x CD3 (0.05 mg/kg) 4 1 5 3 2 0 GPRC5D x CD3 (0.05 mg/kg) + P1AG7400 (40 mg/kg) 0 5 1 4 5 GPRC5D x CD3 (0.05 mg/kg) + P1AG7400 (4 mg/kg) 0 5 3 2 5 GPRC5D x CD3 (0.05 mg/kg) + P1AG7400 (0.4 mg/kg) 2 5 4 4

These results demonstrate that GPRC5D x CD3-induced tumor growth inhibition in the NCI-H929 tumor model is promoted by BCMA-41BBL. Example 6 Pharmacological properties of BCMA - targeted 4-1BBL agonist antigen binding molecules 6.1 PK properties of huFcRn gene-transfected mice

Transgenic mice carrying human FcRn instead of mouse FcRn are thought to be more predictive of human clearance than wild-type mice (C57/Bl6). Female huFcRn Tg32 homozygous SCID mice (n = 3 or 4) received a single IV bolus dose of 5 mg/kg of P1AG740 or P1AG7397. Female huFcRn Tg32 homozygous BL6 mice (n = 3 or 4) received a single IV bolus dose of 5 mg/kg of P1AG7409 or P1AG7422, respectively. Blood samples were collected at 0.17, 7, 24, 48, 72, 168, 336, 408, and 504 hours after dosing and processed to serum prior to bioanalysis. Serum samples were analyzed by immunoassay using anti-human FCpan (CH2) or kCH capture and detection reagents. As shown in Figure 12 and Table 30 , all tested BCMA-4-1BBL antigen binding molecules had similar plasma concentration-time curves and clearance values ranging from 4 to 7 mL/kg/day in these mouse strains. Table 30 : Pharmacokinetic data of hu FcRn transgenic mice molecular Cmax (μg/mL) Tmax (hours) AUClast (day*μg/mL) P1AG7400 76.3 0.167 350 P1AG7397 142 0.167 652 P1AG7409 112 0.167 118 P1AG7422 153 0.167 546 6.2 Off-target binding assessment assay

Cell microarray technology (Charles River Laboratories) was used to screen for potential off-target binding interactions. BCMA-4-1BBL agonistic antigen binding molecules were screened for binding to fixed HEK293 cells expressing 6019 individual full-length human plasma membrane proteins and cell surface-tethered human secreted proteins, as well as an additional 397 human heterodimers. Fluorescent images of spotted cells were analyzed using ImageQuant software (GE Healthcare, version 8.2).

Table 31 reports the results of the screening after visual inspection of spot intensity as strong, moderate, weak, or no interaction. Table 31 : Results of Cell Microarray Assays TAPIR ID BCMA interaction 4-1BB Interaction LDLR Interaction P1AE7397 Strong Strong no P1AG7409 Strong Strong Weak/Medium P1AG7422 Strong Strong no

As a conclusion, all antigen binding molecules showed strong interactions with their main targets, namely BCMA and 4-1BB. P1AG7409 showed weak or moderate interactions with LDLR (low-density lipoprotein receptor). For P1AG7397 and P1AG7422, no other interactions were detected, indicating that these molecules are highly specific for their main targets. 6.3 Immunogenicity Tests 6.3.1 MAPPs Assay

The MAPPs assay was used to identify potential T cell epitopes from four BCMA-4-1BBL antigen-binding molecules: P1AG7409, P1AG7422, P1AG7400, and P1AG7397 ( Figures 13A to 13D ). Major histocompatibility complex II (MHC-II) associated peptide proteomics (MAPP) is a mass spectrometry-based method used to identify and relatively quantify naturally processed and presented MHC-II associated peptides that have the potential to activate T cells and contribute to the immunogenicity of drugs.

Generation and Assay of Human Monocyte-Derived DCs Procedure: Peripheral blood mononuclear cells (PBMCs) were isolated from the chromophore layer of healthy donors by gradient density centrifugation using Ficoll-Paque PLUS (GE Healthcare Europe GmbH, Glattbrugg, Switzerland). Monocytes were isolated by positive immunoselection using anti-CD14-coated microbeads and a magnetic separator (MACS, Miltenyi Biotech, Bergisch Gladbach, Germany). CD14 ⁺ cells were then cultured at a concentration of 0.3 × ¹⁰⁶ cells/mL in 100 mm ultra-low attachment dishes (Corning Inc., Corning, NY, USA) in serum-free Cellgro medium containing 1% GlutaMAX, 1% penicillin/streptomycin. Monocytes were differentiated into immature DCs with 50 ng/mL GM-CSF and 5 ng/mL IL-4 for 5 days at 37°C with 5% _CO2 and then challenged with 50 μg/mL of the test protein in the presence of 1 μg/mL lipopolysaccharide (LPS) from Salmonella abortus (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) for 24 h. Mature DCs were harvested, washed with phosphate-buffered saline (PBS), and the cell pellets were frozen at -80°C before subsequent immunoprecipitation.

Isolation of HLA-DR presented peptides: Cell pellets were lysed in 20 mM Tris buffer, pH 7.8, containing 1% (v/v) Triton X-100 and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany) on a ThermoMixer at 1100 rpm at 4°C for 1 h. HLA-DR immune complexes were isolated by immunoprecipitation using biotin-conjugated anti-human HLA-DR antibodies (strain L243, RayBiotech). Lysates were incubated with antibodies overnight at 4°C on a rotator. The samples were washed five times with a buffer containing 20 mM N- (2-hydroxyethyl)piperidinium- N'- 2-ethanesulfonic acid-NaOH (pH 7.9), 150 mM KCl, 1 mM MgCl ₂ , 0.2 mM CaCl ₂ , 0.2 mM EDTA, 10% (v/v) glycerol, and 0.1% (v/v) digitonin, and five times with purified water. MHC-II peptides were eluted from HLA-DR molecules twice by adding 18 μL of 0.1% trifluoroacetic acid. The eluate was collected and stored at 4°C before mass spectrometry analysis.

Data acquisition: Data acquisition and data processing were performed essentially as described: Steiner et al. J. Proteome Res. 2020, 19, 3792-3806. MHC-II peptide preparations obtained from approximately 3 × ¹⁰⁶ human monocyte-derived DCs (moDCs) per sample were separated by electrospray ionization (LC-ESI-MS/MS) on a nanocapillary liquid chromatography system (UltiMate 3000 RSLC, Thermo Scientific, CA, USA) using a self-packed fused silica C18 reversed phase column (75 μm × 170 mm, i.d., ReproSil-Pur C18-AQ, 3 μm, Dr. Maisch GmbH) connected to a Q-Exactive HFX Orbitrap mass spectrometer (Thermo Scientific). The sample (15 μL volume dissolved in 0.5% (v/v) formic acid in 2% (v/v) acetonitrile/water) was loaded at 8 μL/min for 2 to 3 min onto an Acclaim PepMap C18 capture column (100 μm id × 20 mm, Thermo Scientific) using a vented tee design. The peptides were then eluted using a nonlinear 39 min gradient from 2% to 45% B at a flow rate of 250 nL/min, followed by an 11 min column wash and 10 min re-equilibration [buffer A: 0.1% (v/v) formic acid in 2% (v/v) acetonitrile/water; buffer B: 0.1% (v/v) formic acid in acetonitrile]. MHC-II peptides were analyzed by tandem MS using standard operating parameters. Check scans were recorded at a resolution of 60,000 in the Orbitrap mass analyzer with the lock mass option activated (scan range m/z 400 to 1650). Data-dependent MS/MS spectra of the 18 most abundant ions from the check scans were recorded in the Orbitrap cell at a resolution of 15,000. Target ions selected for MS/MS were dynamically excluded for 7 s. Peptides were identified using the latest PEAKS Studio version available at the time (X Pro version, Bioinformatics Solutions Inc., ON, Canada). Raw MS data were searched against the human protein database UniProtKB (http://www.uniprot.org, 2015_10, approximately 88,500 TrEMBL and SwissProt entries containing amino acid sequences of test therapeutic proteins) with a mass tolerance of ±10 ppm for precursor ions and ±0.025 Da for fragment ions. Met-sulfoxide, Asn/Gln deamidation and N-terminal pyroglutamylation were considered as differential modifications. Data were searched without enzyme specificity and peptide results were reported with a 1% specFDR cutoff. All LC-MS/MS runs for a given donor were batch processed and the area under the curve of the identified features (2 min retention time shift tolerance; feature reporting option: All) were exported to a column-delimited table without further normalization.

Statistical analysis: DataMAPPs is a data analysis tool created by Roche to help visualize otherwise complex mass spectrometry-derived data in the form of heatmaps. The program is publicly available at https://www.R-project.org/ and can be executed using a standard R installation. See the reference (Steiner et al., 2020) for a detailed description of the package. DataMAPPs is called after the LC-MS/MS data have been processed with PEAKS software and the results have been exported in a suitable table format. In brief, the dataMAPPs processing pipeline consists of the following: (1) Data import and consistency check: Sample annotation and peptide quantitation (PEAKS output) files are read into memory and checked for consistency. Only peptides with a length of 10 to 30 amino acids are considered for analysis and mapped against the sequence of the biological agent being studied. Further filtering is performed to retain only antibody-related signals that are specific to the molecule being studied in a given LC-MS/MS analysis. A final step eliminates duplicate entries and sums the signals from peptides identified with different charge states. Peak areas are converted to the log2 scale prior to processing; (2) Data QC: The pipeline flags samples with a low number of identified peptides (the cutoff is typically set to half the median peptide count of the corresponding donor) or low similarity (median Pearson correlation <0.8 with all other samples from the same donor). The user can modify the cutoff value after reviewing a series of QC plots, which allows for appropriate, data set-specific processing while being able to rerun the program in a reproducible manner; (3) Data normalization: Peptide abundances (based on peak area) are normalized based on an adapted version of the GRSN (Global Rank Invariant Set Normalization) procedure. (4) Replicate aggregation: Technical replicates (i.e., samples run from the same donor, same treatment, same dose) can be averaged to retain information about all peptides detected in at least one of the replicates; (5) Peptide mapping: Antibody-associated peptides are mapped to the protein amino acid sequence of the tested biological product. In addition, peptides with adjacent amino acid positions are binned into different epitope clusters (hotspots), whose abundance reflects the total intensity of each of the assembled MHC-II peptides; (6) Data export and visualization: The dataMAPPs workflow includes standard functionality to generate several heatmaps (epitope cluster or single peptide level, per antibody or global experimental summary) to enable rapid visualization and comparison of results.

Results: Analysis of the BCMA-4-1BBL antigen-binding molecule P1AG7409 revealed two potential T-cell epitopes in the non-germline regions (clusters C1 and C3). However, for these clusters the frequency appeared to be low (1/10 donors).

Analysis of P1AG7422 revealed one binding cluster containing any non-germline residues. A total of 3/10 donors (35%) presented potentially immunogenic T cell epitopes that could contribute to the immunogenicity of the compounds.

Analysis of P1AG7400 did not reveal any binding clusters in the non-germline region. Therefore, none of the donors present potentially immunogenic T cell epitopes that could contribute to the immunogenicity of the compound.

Analysis of P1AG7422 revealed two binding clusters. Of these, two clusters (C1 and C2) contained non-germline residues at frequencies of 1/10 and 3/10, respectively. A total of 4/10 donors (40%) presented potentially immunogenic T cell epitopes that could contribute to the immunogenicity of the compound. 6.3.2 DC:T cell assay ( Epibase® DC:CD4 restimulation assay developed by Lonza )

T cell activation is an important component of the immune response to therapeutic proteins and is often required for clinical development of anti-drug antibodies. The DC-T cell assay was used to evaluate the ability of four BCMA-4-1BBL antigen binding molecules: P1AG7409, P1AG7400, P1AG7397, and P1AG7422 to induce CD4 ⁺ T cells following presentation of the T cell epitope by APCs.

Materials: Donors were recruited in a Phase I clinical trial unit in the UK. All samples were collected under ethical protocols approved by the local REC (Research Ethics Committee) and written informed consent was obtained from each donor before sample donation. All samples were stored under the terms of Lonza's HTA (Human Tissue Authority) license for the use of samples in research. PBMCs from healthy donors were prepared from whole blood within six hours of blood draw. Cells were stored frozen in vapor nitrogen until used for assays. The quality and functionality of each PBMC preparation was analyzed by activation for seven days with positive controls such as KLH to assess natural T cell responses.

Keyhole limpet hemocyanin (KLH) was used as a technical control and was reconstituted aseptically in single-use aliquots and stored at -80°C according to the manufacturer's recommendations. In addition, bevacizumab (Avastin®) was included as a positive standard protein. All samples were tested at a final concentration of 0.3 μM in both the DC stimulation phase and the APC restimulation phase.

Methods: Mononuclear spheres were isolated from frozen PBMC samples by magnetic bead selection and differentiated into immature DCs (iDCs) using GM-CSF and IL-4. iDCs were then harvested, washed, and loaded with each individual test protein/peptide for 4 hours at 37°C. DC maturation cocktail containing TNFα and IL-1β was then added for an additional 40 to 42 hours to activate/mature DCs (mDCs). Expression of key DC surface markers (CD11c, CD14, CD40, CD80, CD83, CD86, CD209, and HLA-DR) was assessed at both the immature and mature stages by flow cytometry to ensure DC activation prior to T cell interaction. mDCs were then co-cultured with autologous CD4 ⁺ T cells (isolated by magnetic bead selection) for 6 days at 37°C with 5% _CO2 in a humidified atmosphere. On day 6, autologous monocytes were isolated from PBMCs using magnetic bead selection and loaded with the selected protein/peptide used to initially load DCs. After incubation for 4 hours at 37°C with 5% _CO2 in a humidified atmosphere, monocytes were added to anti-IFNγ pre-coated FluoroSpot plates (Mabtech) and DC:CD4 co-cultures were performed in quadruplicate. FluoroSpot plates were incubated at 37°C with 5% _CO2 in a humidified atmosphere for 40 to 42 hours. After incubation, FluoroSpot plates were developed using an in-house protocol and the number of spot forming cells (SFC) per well was assessed for each cytokine under each test condition.

Surface marker QC checks on monocyte-derived DCs at both the immature and mature stages are performed to assess any possible effects of the test product on DC differentiation and allow assessment of DC quality prior to subsequent co-culture with CD4+ T cells. Surface markers are determined by flow cytometry using fluorescently labeled antibodies and a Guava® easyCyte™ 8HT flow cytometer.

Data analysis: Data management and statistical analysis were performed using the R programming language (https://www.R-project.org/, v. 3.6.1). Data were converted to log2 scale, and generalized linear models (GLM) were applied to quantify SI (fold change and 95% CI). The data set was adjusted (adjusted exponential heteroskedasticity, Gaussian noise injection at the low end of the SFU scale, linear regression of each SI and extrapolation to the blank value of 0) and QC plots were generated (DC differentiation markers, reproducibility of compound and donor levels, relative stimulation of donors).

To aid in immunogenicity risk assessment, the stimulation index (SI) (ratio between SFU/well and matched blank) was calculated for each tested condition for each donor. A positive donor response was counted if at least a 2-fold change in SI was established and p<0.05 (unadjusted p-value for GLM). The number of positive donor responses to a treatment in a cohort of 30 healthy donors gives the response rate relative to this treatment.

Results: The DCs used in this assay were of high quality and expressed high levels of T cell co-stimulatory molecules required for T cell activation. Based on the readout of the DC-T cell assay, the BCMA-4-1BBL antigen binding molecules P1AG7400 and P1AG739 were associated with a low risk of sequence-related immunogenicity for CD4 ⁺ T cell responses (below the 10% threshold described in Siegel et al., Pharmaceutics 2022, 14(12), 2672), while P1AG740 and P1AG7422 showed an incidence of T cell activation (IFN-γ release) above the 10% threshold. 6.3.3 Overall Risk of Immunogenicity

Administration of therapeutic antibodies may result in the formation of anti-drug antibodies (ADA), which may negatively impact the safety of treatment outcomes (e.g., allergic reactions, immune complex-mediated diseases). The risk of inducing unexpected immune responses and the consequences of such responses were assessed for four BCMA-4-1BBL antigen binding molecules, P1AG7409, P1AG7400, P1AG7397, and P1AG7422, using a combination of nonclinical assays (MAPPs and DC-T cell assays) as previously described herein. Overall, the BCMA-4-1BBL antigen binding molecules can be ranked from lowest to highest risk of immunogenicity as follows: P1AG7400, P1AG7397, P1AG7409, and P1AG7422. ***

In Figures 1A and 1B , schematic diagrams of exemplary molecules as described herein are shown. Figure 1A shows a schematic diagram of a BCMA antibody variant that is a monovalent hu IgG1 PGLALA isotype ("Fc silent"). Figure 1B schematically illustrates the structure of a monovalent BCMA-targeting split-trimeric 4-1BBL Fc (kih) P329G LALA fusion antigen-binding molecule, which comprises a CH1-CL exchange in the arm to which two and one 4-1BBL are fused, respectively. The thick black dots represent knob-and-hole modifications. Certain amino acids (so-called charged variants) are included in the CH1 and CL domains of the Fab molecule containing the BCMA antigen-binding domain to allow better pairing with the light chain. Figures 2A and 2B show the dose-dependent binding of various BCMA-4-1BBL antigen binding molecules and the comparator molecule P1AH1062 to cells expressing human BCMA ( Figure 2A ) or human 4-1BB ( Figure 2B ). Non-targeted 4-1BBL molecules only bind to cells expressing 4-1BB, not to cells expressing BCMA. Figures 3A and 3B show that all exemplified BCMA-4-1BBL antigen binding molecules have similar binding behavior to CHO cells expressing BCMA (CHO-huBCMA cells) and to Jurkat reporter cells expressing human 4-1BB, while the comparator molecule P1AH1062 binds much less. Figures 3C to 3H show the binding of two BCMA-4-1BBL antigen binding molecules, P1AG7400 and P1AG7397, and the BCMA-targeted CD3 T cell engager, Alnuctamab, to CHO cells expressing human BCMA with the indicated point mutations. Shown are binding in the absence of BCMA ( Figure 3C ) and to human wt BCMA ( Figure 3D ) and to BCMA variants human BCMA P33S ( Figure 3E ), human BCMA P34del ( Figure 3F ), human BCMA R27P ( Figure 3G ), and human BCMA S30del ( Figure 3H ). Another BCMA-targeted CD3 T cell binder, teclistamab, did not bind to two mutant BCMA variants, R27P ( Fig. 3G ) and S30del ( Fig. 3H ), while elranatamab only weakly bound to BCMA with the R27P mutation at high concentrations. None of the molecules tested bound to BCMA-negative CHOk1 cells ( Fig. 3C ). Figs . 4A to 4C show the dose-dependent activation of NFκB in the Jurkat reporter cell assay when titrated amounts (600.0 to 0.02 nM) of BCMA-4-1BBL antigen-binding molecules were added and target cells were bridged to effector cells. The data obtained are shown as the mean ± sd of one independent experiment performed in triplicate out of three independent experiments. In Figure 4A , the concentration of the BCMA-4-1BBL molecule or its control is plotted against the light release units (RLU), and Figures 4B and 4C provide a graphical summary of the EC ₅₀ values and Emax values calculated from three independent experiments performed in triplicate and shown together as the mean ± sd. Figures 5A and 5B show that dose-dependent activation of CD8 ⁺ T cells by the BCMA-4-1BBL antigen binding molecule occurs only in the presence of the first signal (TCB) and BCMA expression. The MM cell line NCI-H929 expressing BCMA ( Figure 5A ) was co-cultured with healthy donor PBMC (ratio 1:1). Alternatively, NCI-H929 BCMAko cells (knockout cells that do not express BCMA) were used as target cells ( Figure 5B ). Co-cultures were treated with GPRC5D-TCB (providing the first signal) alone or in combination with BCMA-4-1BBL or non-targeting 4-1BBL (negative reference) titrated from 500 nM to 0.14 nM (1:3 dilution steps) and incubated for 4 days. Controls were left untreated or treated with the BCMA-4-1BBL molecule alone (3 highest concentrations). Data are shown as mean ± sd of triplicates from one donor (representing 2 to 4 donors tested; BCMA ko N=1). Figures 5C to 5H show the binding of two BCMA-4-1BBL antigen binding molecules, P1AG7400 and P1AG7397, to CHO cells expressing human BCMA with the indicated point mutations. Shown are binding in the absence of BCMA ( Figure 5C ) and to human wt BCMA ( Figure 5D ) and to BCMA variants human BCMA R27P ( Figure 5E ), human BCMA P34del ( Figure 5F ), human BCMA P33S ( Figure 5G ), human BCMA S30del ( Figure 5H ). In the presence of mutant or wild-type BCMA expressed on CHO transfectants, both molecules demonstrated similar concentration-dependent promotion of T cell activation beyond CD3 IgG-mediated T cell activation (as indicated by upregulation of CD25 on CD8 T cells). Figures 5I to 5P show that BCMA-4-1BBL (P1AG7400) is able to promote T cell activation and tumor cell lysis beyond that of the anti-FcRH5/anti-CD3 bispecific antibody (FcRH5 x CD3). BCMA-4-1BBL was added at a fixed concentration to varying concentrations of FcRH5 x CD3 in a co-culture assay with healthy donor PBMCs in the presence of MM cell lines expressing BCMA. Figures 5I , 5J , 5K , and 5L show the increase in the frequency of CD137-positive CD8 T cells in 4 donors when BCMA-4-1BBL was added, respectively. Tumor cell lysis is shown in Figures 5M , 5N , 5O , and 5P , respectively. The combination significantly promoted T cell activation and tumor cell lysis. Figures 6A to 6E show the results of exemplary ex vivo tests using bone marrow samples from primary MM patients. Tumor cell lysis (TCL) was determined by flow cytometry, and maleimide-positive cells with low FSC were defined as dead cells ( Figure 6A ). T cell activation was determined by flow cytometry, assessing upregulation of CD25 on CD8 ( Fig. 6B ) or CD4 (Fig. 6C ) T cells after incubation for 96 hours with 1 nM GPRC5D-TCB in the absence or presence of the indicated concentrations of BCMA-4-1BBL (P1AG7422) or a negative reference molecule. Degranulation was determined by flow cytometry, assessing upregulation of CD107a on CD8 ( Fig. 6D ) or CD4 ( Fig. 6E ) T cells after incubation for 96 hours with 1 nM GPRC5D-TCB in the absence or presence of the indicated concentrations of BCMA-4-1BBL or a negative reference molecule. All data shown refer to single tube measurements for each condition. Figures 7A to 7E show the results of another exemplary ex vivo assay using bone marrow samples from primary MM patients. Tumor cell lysis (TCL) was determined by flow cytometry, and maleimide-positive cells with low FSC were defined as dead cells ( Figure 7A ). T cell activation was determined by flow cytometry, assessing upregulation of CD25 on CD8 (Figure 7B and Figure 7C) or CD4 ( Figure 7D and Figure 7E ) T cells after 96 hours of incubation with 1 nM GPRC5D-TCB in the absence or presence of the indicated concentrations of BCMA-4-1BBL ( P1AG7400 or P1AG7409 ) or negative reference molecules. All data shown refer to single tube measurements for each condition. Figures 8A to 8D show the results of ex vivo assays using BM MNCs from bone marrow samples of primary MM patients. Tumor cell lysis was determined by flow cytometry, assessing the percentage of live MM PCs after 96 hours of incubation with 0.025 nM GPRC5D-TCB in the absence or presence of 40 nM BCMA-4-1BBL (P1AG7400) or reference molecules. All data shown refer to single tube measurements for each condition. Figures 9A to 9H depict results from in vivo experiments testing the efficacy of GPRC5D x CD3 monotherapy and its combination with BCMA-4-1BBL antigen binding molecules (10 mg/kg and 3 mg/kg P1AG7409 and 10 mg/kg and 3 mg/kg P1AG7422) in the NCI-H929 tumor model. Figures 9A to 9F show tumor growth inhibition in single animals, and Figure 9G shows tumor volume as the median (+/- IQR) for each treatment group. Animals with a final tumor burden less than the size at the start of treatment were defined as responders. Tumor volumes at termination (study day 41) are shown for the GPRC5D x CD3 monotherapy groups and combinations as median (+/- IQR) in Figure 9H . Figures 10A to 10H show results from in vivo experiments testing the efficacy of GPRC5D x CD3 monotherapy and its combination with BCMA-4-1BBL antigen binding molecules P1AG7397 (10 and 20 mg/kg) and P1AG7400 (10 and 20 mg/kg) in the NCI-H929 tumor model. Tumor growth inhibition in single animals is shown ( Figures 10A to 10F ) and as the median (+/- IQR) for each treatment group ( Figure 10G ). Animals with a final tumor burden less than the size at the start of treatment were defined as responders. Tumor volume at termination (study day 57) is shown as the median (+/- IQR) for the GPRC5D x CD3 monotherapy and combination groups ( Figure 10H ). Figures 11A to 11F show the results of in vivo experiments testing the efficacy of GPRC5D x CD3 monotherapy (0.05 mg/kg) and the BCMA-4-1BBL antigen binding molecule P1AG7400 (40, 4 and 0.4 mg/kg) in the NCI-H929 tumor model. Figures 16A to 16E show tumor growth inhibition in single animals. Figure 11A shows tumor volume as the median (+/- IQR) for each treatment group. Figures 11B to 11F show tumor growth inhibition in single animals. Animals with a final tumor burden less than the size at the start of treatment were defined as responders. Figure 12 shows a comparison of plasma concentration-time curves of BCMA-4-1BBL antigen binding molecules P1AG7400, P1AG7397, P1AG7409 and P1AG7422 as measured in HuFcRN transgenic mice (PK study). Figures 13A to 13D show heatMAPPS representations of the results observed in the MAPPs assay (Example 6.3.1) of bispecific antibodies P1AG7409 (Figure 13A), P1AG7422 (Figure 13B), P1AG7400 (Figure 13C) and P1AG7397 (Figure 13D). Clusters found in each domain are highlighted.

TW202502811A_113119807_SEQL.xml

Claims (42)

An immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) a Fab molecule comprising (i) a heavy chain variable region ( _VH BCMA) comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 1 (GYTFTNYWMH), a CDR-H2 of SEQ ID NO: 2 (IIHPNSGSTNYNEKFQG), and a CDR-H3 of SEQ ID NO: 3 (GIYDYPFAY); and (ii) a light chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 5 (AASSLQS); and CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); and (b) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 7 (AASNLES), and a CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); and (c) a VL comprising a light chain complementary determining region CDR-L1 of SEQ ID NO: 4 (RASESVSIHGTHLMH), a CDR-L2 of SEQ ID NO: 8 (AASNLQS), and a CDR-L3 of SEQ ID NO: 6 (QQSIEDPYT); (b) a first polypeptide and a second polypeptide, which are linked to each other by a disulfide bond, The antigen-binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL connected to each other by a peptide linker, each extracellular domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, and the second polypeptide comprises one extracellular domain of 4-1BBL, the extracellular domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; and (c) an IgG Fc domain. An immunostimulatory antigen-binding molecule as claimed in claim 1, wherein the immunostimulatory antigen-binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:189 and SEQ ID NO:190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. The immunostimulatory antigen-binding molecule of claim 1 or 2, wherein the Fab molecule comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:16. An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 3, wherein the IgG Fc domain is an IgG1 Fc domain. An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 4, wherein the Fc comprises a modification that promotes the binding of the first unit and the second unit of the Fc domain. The immunostimulatory antigen-binding molecule of claim 5, wherein the first unit of the IgG Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second unit of the IgG Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (according to the Kabat EU index numbering). An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 6, wherein the IgG Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. An immunostimulatory antigen-binding molecule according to any one of claims 1 to 7, wherein the IgG Fc domain is of the human IgG1 subclass and comprises the amino acid mutations L234A, L235A and P329G (according to the Kabat EU index number). An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 8, comprising (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CL domain; (aiii) the CL domain, fused at the C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit); and (aiv) one of the subunits of the Fc domain (e.g., the first subunit); (b) a second polypeptide comprising: (bi) a third extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CH1 domain; and (bii) the CH1 domain; (c) a third polypeptide comprising: (ci) the heavy chain of the Fab molecule fused at the C-terminus to the N-terminus of another of the subunits of the Fc domain (e.g., the second subunit); and (cii) another of the subunits of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule. The immunostimulatory antigen-binding molecule of claim 9, wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 10, comprising (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO:21, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:23, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:25. An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 10, comprising (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:208, a second polypeptide comprising the amino acid sequence of SEQ ID NO:22, a third polypeptide comprising the amino acid sequence of SEQ ID NO:207, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO:24. An immunostimulatory antigen-binding molecule that specifically binds to a B cell maturation agent (BCMA), comprising (a) a Fab molecule comprising (i) a heavy chain variable region ( _VL BCMA) selected from the group consisting of: (a) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 27 (QITAKSNNYATYYADSVKG), and a CDR-H3 of SEQ ID NO: 28 (DGYH); and (b) a VH comprising a heavy chain complementary determining region CDR-H1 of SEQ ID NO: 26 (GFTFSNAWMD), a CDR-H2 of SEQ ID NO: 29 (QITAKSNNYATYYAAPVKG), and a CDR-H3 of SEQ ID NO: 28 (DGYH); and (ii) a light chain variable region (V _L BCMA) comprising a light chain complementation determining region CDR-L1 of SEQ ID NO: 30 (RASEDIRNGLA), a CDR-L2 of SEQ ID NO: 31 (NANSLHT) and a CDR-L3 of SEQ ID NO: 32 (EDTSKYPYT); (b) a first polypeptide and a second polypeptide linked to each other by a disulfide bond, wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two extracellular domains of 4-1BBL linked to each other by a peptide linker, each extracellular domain comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, and characterized in that the second polypeptide comprises an extracellular domain of 4-1BBL, the extracellular domain comprising SEQ ID NO: 9 or SEQ ID NO: 10. and (c) IgG Fc domain. An immunostimulatory antigen-binding molecule as claimed in claim 13, wherein the immunostimulatory antigen-binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:189 and SEQ ID NO:190, and characterized in that the second polypeptide comprises an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. The immunostimulatory antigen-binding molecule of claim 13 or 14, wherein the antigen-binding domain comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 (VH2a) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34 (VL2a), or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 (VH1b) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:36 (VL1a). An immunostimulatory antigen-binding molecule as claimed in any one of claims 13 to 15, wherein the IgG Fc domain is an IgG1 Fc domain. An immunostimulatory antigen-binding molecule as claimed in any one of claims 13 to 16, wherein the Fc domain is a human Fc domain. An immunostimulatory antigen-binding molecule as claimed in any one of claims 13 to 17, wherein the IgG Fc domain comprises a modification that promotes the association of the first unit with the second unit of the Fc domain. The immunostimulatory antigen-binding molecule of claim 18, wherein the first subunit of the IgG Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the IgG Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (according to the Kabat EU index numbering). An immunostimulatory antigen-binding molecule as claimed in any one of claims 13 to 19, wherein the IgG Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. An immunostimulatory antigen-binding molecule according to any one of claims 13 to 20, wherein the IgG Fc domain is of the human IgG1 subclass and comprises the amino acid mutations L234A, L235A and P329G (according to the Kabat EU index number). An immunostimulatory antigen-binding molecule as claimed in any one of claims 13 to 21, comprising (a) a first polypeptide comprising: (ai) a first extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a second extracellular domain of 4-1BBL or a fragment thereof; (aii) a second extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CL domain; (aiii) the CL domain, fused at the C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit); and (aiv) one of the subunits of the Fc domain (e.g., the first subunit); (b) a second polypeptide comprising: (bi) a third extracellular domain of 4-1BBL or a fragment thereof, fused at the C-terminus to the N-terminus of a CH1 domain; and (bii) the CH1 domain; (c) a third polypeptide comprising: (ci) the heavy chain of the Fab molecule fused at the C-terminus to the N-terminus of another of the subunits of the Fc domain (e.g., the second subunit); and (cii) another of the subunits of the Fc domain (e.g., the second subunit); and (d) a fourth polypeptide comprising the light chain of the Fab molecule. The immunostimulatory antigen-binding molecule of claim 22, wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). An immunostimulatory antigen-binding molecule as claimed in any one of claims 13 to 23, comprising (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 37, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 38; or (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 39, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 40. An antibody that specifically binds to BCMA, wherein the antibody comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:15, or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:13 and _VL BCMA comprising the amino acid sequence of SEQ ID NO:16. An antibody that specifically binds to BCMA, wherein the antibody comprises (a) _VH BCMA comprising the amino acid sequence of SEQ ID NO:33 (VH2a) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:34 (VL2a), or (b) _VH BCMA comprising the amino acid sequence of SEQ ID NO:35 (VH1b) and _VL BCMA comprising the amino acid sequence of SEQ ID NO:36 (VL1a). One or more isolated polynucleotides encoding an immunostimulatory antigen-binding molecule of any one of claims 1 to 26 or an antibody of claim 25 or 26. One or more vectors, in particular expression vectors, comprising the polynucleotide of claim 27. A host cell comprising the polynucleotide of claim 27 or the vector of claim 28. A method for producing an immunostimulatory antigen-binding molecule that specifically binds to BCMA, comprising the steps of: a) culturing the host cell of claim 28 under conditions suitable for expressing the immunostimulatory antigen-binding molecule, and optionally b) recovering the immunostimulatory antigen-binding molecule. An immunostimulatory antigen-binding molecule that specifically binds to BCMA, produced by the method of claim 30. A pharmaceutical composition comprising the immunostimulatory antigen-binding molecule of any one of claims 1 to 24 or 31 and at least one pharmaceutically acceptable excipient. An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 24 or 31 or a pharmaceutical composition as claimed in claim 31, which is used as a medicine. An immunostimulatory antigen-binding molecule as claimed in any one of claims 1 to 24 or 31 or a pharmaceutical composition as claimed in claim 32, for use in enhancing (a) T cell activation or (b) T cell effector function. An immunostimulatory antigen binding molecule as claimed in any one of claims 1 to 24 or 31 or a pharmaceutical composition as claimed in claim 32, for use in treating a disease. An immunostimulatory antigen binding molecule or pharmaceutical composition for use as claimed in claim 35, wherein the disease is cancer. An immunostimulatory antigen binding molecule as claimed in any one of claims 1 to 24 or 31 or a pharmaceutical composition as claimed in claim 32 for use in the treatment of cancer, wherein the use is for administration in combination with chemotherapy, radiation therapy and/or other agents used in cancer immunotherapy. An immunostimulatory antigen binding molecule according to any one of claims 1 to 24 or 31 or a pharmaceutical composition according to claim 32 for use in treating cancer, wherein the use is for administration in combination with a T cell activating anti-CD3 bispecific antibody selected from anti-GPRC5D/anti-CD3 antibody, anti-BCMA/anti-CD3 antibody or anti-FcRH5/anti-CD3 antibody. An immunostimulatory antigen-binding molecule or pharmaceutical composition for use as claimed in claim 38, wherein the T cell-activating anti-CD3 bispecific antibody is an anti-GPRC5D/anti-CD3 antibody. Use of an immunostimulatory antigen binding molecule as claimed in any one of claims 1 to 24 or 31 or a pharmaceutical composition as claimed in claim 32 in the manufacture of a medicament for treating a disease, in particular for treating cancer. A method for treating a disease, particularly cancer, in an individual, comprising administering to the individual an effective amount of the immunostimulatory antigen binding molecule of claim 1 to 24 or 31 or the pharmaceutical composition of claim 32. The method of claim 41, further comprising administering in combination with chemotherapy, radiation therapy and/or other agents used in cancer immunotherapy, specifically in combination with a T cell activating anti-CD3 bispecific antibody selected from anti-GPRC5D/anti-CD3 antibody, anti-BCMA/anti-CD3 antibody or anti-FcRH5/anti-CD3 antibody.