CD3 / BCMA / CD38 triple specific antibody

Abstract

The present invention relates to novel trispecific heterodimeric immunoglobulins. More specifically, the present invention relates to trispecific heterodimeric immunoglobulins targeting human CD3 antigen, human BCMA and human CD38 antigen. The present invention also relates to this novel class of trispecific heterodimeric immunoglobulins for use in the treatment of proliferative diseases, particularly cancers such as hematological cancers. The present invention relates to novel trispecific antibodies for use in the treatment of multiple myeloma.

Description

[Technical Field]

[0001] The present invention relates to novel trispecific heterodimeric immunoglobulins. More specifically, the invention relates to trispecific heterodimeric immunoglobulins that target an epitope of the human CD3 antigen, an epitope of human BCMA, and an epitope of the human CD38 antigen. The invention also relates to this novel class of trispecific heterodimeric immunoglobulins for use in the treatment of proliferative diseases, particularly cancers such as hematological cancers. [Background technology]

[0002] Multispecific antibodies targeting one or more immune-related molecules and one or more tumor antigens have been developed over the past few decades for cancer immunotherapy to recruit immune cells, such as T cells, to tumor cells. For example, bispecific antibodies directed against CD3 on T cells and surface target antigens on cancer cells can connect T cells to cancer cells independently of T cell receptor specificity, costimulation, or peptide antigen presentation (WO 2020204708(A1)). Trispecific T cell-engaging antibodies have also shown promising results for the treatment of various cancers, including breast cancer, lymphoma, and multiple myeloma, when two proteins are targeted on T cells and one on tumors (U.S. Patent No. 10,882,922(B2)). Furthermore, tumor cell lysis has been proposed using trispecific binding molecules that engage two tumor-associated antigens expressed on cancer cells in addition to CD3 on T cells or other components of the TCR complex (U.S. Patent Application Publication No. 20210163620(A1)).

[0003] Multispecific immune cell-redirecting antibodies have been shown to mediate T cell redirection in both preclinical and clinical studies (May C et al., (2012) Biochem Pharmacol, 84(9):1105-12; Frankel SR & Baeuerle PA, (2013) Curr Opin Chem Biol, 17(3):385-92; Nie et al., (2020) Antibody Therapeutics, 3(1), 17-62), thus demonstrating their importance for new drug development and the treatment of complex diseases such as inflammatory diseases, autoimmune disorders, and cancers, including hematological cancers, including multiple myeloma (MM), for which curative treatments remain necessary. Multiple myeloma is a neoplastic plasma cell disorder characterized by the clonal proliferation of malignant plasma cells in the bone marrow (BM) microenvironment, monoclonal proteins in the blood or urine, and associated organ failure. Multiple myeloma accounts for 1-2% of all newly diagnosed cancers and approximately 20% of all deaths from hematologic malignancies. The disease is slightly more common in men and African Americans than in the general population. Although multiple myeloma remains an incurable cancer, recent improvements in understanding the pathogenesis of myeloma have led to the development of novel treatments and improved survival rates.

[0004] The diagnosis of multiple myeloma requires the presence of one or more myeloma-defining events (MDEs) plus evidence of either ≥10% clonal plasma cells on BM examination or biopsy-confirmed plasmacytoma. MDEs include the so-called CRAB (hypercalcemia, renal failure, anemia, or lytic bone lesion) features and three specific biomarkers: ≥60% clonal BM plasma cells, a serum free light chain (sFLC) ratio >100 (with associated sFLC levels >100 mg / L), and two or more focal lesions on magnetic resonance imaging. Several genetic abnormalities occurring in tumor plasma cells play important roles in the pathogenesis of myeloma and determine the prognosis of the disease.

[0005] Uncontrolled proliferation of myeloma cells has many consequences, including bone destruction, BM failure, increased plasma volume and viscosity, suppression of normal immunoglobulin production, and kidney damage.

[0006] Symptomatic (active) disease must be treated immediately, whereas asymptomatic (smoldering) myeloma requires only clinical observation, as early treatment with conventional chemotherapy has not yet demonstrated clear benefit. Clinical studies are currently evaluating the ability of immunomodulatory agents to delay progression from asymptomatic to symptomatic myeloma. For active myeloma, current data support the initiation of an induction therapy regimen containing thalidomide, lenalidomide, and / or bortezomib, followed by autologous hematopoietic stem cell transplantation (HSCT) after a major disease response, in patients who can tolerate the conditioning regimen. Consideration of physiological age, which may differ from chronological age, and the presence of comorbidities are factors in determining treatment selection and drug dosage. For example, in patients with significant comorbidities, including cardiopulmonary or hepatic impairment, a reduced intensity approach to treatment is desirable, which reduces treatment-related mortality and reduces the risk of treatment discontinuation.

[0007] Relapsed / refractory multiple myeloma (RRMM) is particularly challenging to treat due to the heterogeneity of the disease at relapse and the lack of clear, biologically based recommendations regarding the selection of salvage therapies at various points in disease progression. With increasing awareness of the inherent clonal heterogeneity and genomic instability of plasma cells, which influence both innate and acquired resistance to therapy, identifying the optimal selection and sequence of treatments has become critical. Novel agents, including proteasome inhibitors (carfilzomib and ixazomib), immunomodulatory agents such as the thalidomide derivatives pomalidomide and lenalidomide, and the histone deacetylase inhibitor panobinostat, have recently been approved by the U.S. Food and Drug Administration (FDA) for relapsed / refractory myeloma. Other molecularly targeted therapies directed against specific cell signaling pathways and survival and growth control, including PI3K / AKT / mTOR inhibitors, Hsp90 inhibitors, cyclin-dependent kinase inhibitors, and kinesin spindle protein inhibitors, are currently in development. Despite advances in the treatment of multiple myeloma, relapse is inevitable in almost all patients. Relapsed myeloma typically becomes more aggressive with each relapse and is associated with shorter survival times. Therefore, additional treatment options are needed.

[0008] In recent years, multiple myeloma patients have benefited from new treatments that target MM-associated antigens such as CD38 and BCMA.

[0009] CD38 (UniProt P28907) was first identified in 1980 as a surface marker (cluster of differentiation) of thymocyte lymphocytes [Lee, H.C., ed. (2002). A Natural History of the Human CD38 Gene. Cyclic ADP-Ribose and NAADP. Springer Publishing]. In 1992, it was further described as a surface marker on B cells, monocytes, and natural killer cells (NK cells). Almost simultaneously, CD38 was discovered to be a marker of B and T cell activation. Daratumumab (Darzalex) and isatuximab (SARCLISA), which target CD38, have been approved for the treatment of multiple myeloma.

[0010] B-cell maturation antigen (BCMA), also known as CD269 and TNFRSF17 (UniProt Q02223), is a member of the tumor necrosis receptor superfamily that is preferentially expressed on differentiated plasma cells [Laabi et al. (1992) EMBO J 11(11):3897-3904; Madry et al. (1998) Int Immunol 10(11):1693-1702]. BCMA is a non-glycosylated type I transmembrane protein involved in B-cell maturation, proliferation, and survival. BCMA is a receptor for two ligands of the TNF superfamily: APRIL (proliferation-inducing ligand, CD256, TNFSF13), a high-affinity ligand for BCMA, and the B-cell activating factor BAFF (THANK, BlyS, B-lymphocyte stimulatory factor, TALL-1, and zTNF4), a low-affinity ligand for BCMA. APRIL and BAFF exhibit structural similarities and overlapping yet distinct receptor binding specificities. The negative regulator TACI also binds to both BAFF and APRIL. Cooperative binding of APRIL and BAFF to BCMA and / or TACI activates the transcription factor NF-KB, increasing the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1) and downregulating the expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thus inhibiting apoptosis and promoting survival. This combined action promotes B cell differentiation, proliferation, survival, and antibody production (reviewed in Rickert RC et al., Immunol Rev (2011) 244(1):115-133). Consistent with this finding, BCMA also supports the proliferation and survival of malignant human B cells, including multiple myeloma cells.

[0011] Bispecific antibodies that bind to CD3 and BCMA for the treatment of MM, such as teclistamab, have been developed to induce T cell-mediated cytotoxicity against BCMA-expressing MM and / or RRMM cells, either alone or in combination with immunotherapeutic drugs such as thalidomide or immunotherapeutic derivatives such as anti-PD-1 and anti-PD-L1 antibodies (U.S. Patent No. 11124577(B2)).

[0012] Exceptional overall response rates have been observed using bispecific antibody or chimeric antigen receptor (CAR) T-cell immunotherapy, in which T lymphocytes are engineered with a synthetic chimeric antigen receptor. Nevertheless, durable responses beyond 2 years remain limited (progression-free survival is less than 40% for treatment with idecbutagene vicleucel (Munshi, NC et al. Idecabtagene vicleucel in Relapsed and Refractory Multiple Myeloma. N. Engl. J. Med. 384, 705-716 (2021)) or teclistamab (Moreau, P. et al. Teclistamab in Relapsed or Refractory Multiple Myeloma. N. Engl. J. Med. NEJMoa2203478 (2022) doi:10.1056 / NEJMoa2203478). It has been proposed that one of the potential reasons for patient relapse could be target downregulation or expansion of a clone lacking sufficient expression of the target (Rodriguez-Lobato, LG, Oliver-Caldes, A., Moreno, DF, Fernandez de Larrea, C. & Blade, J. Why Immunotherapy Fails in Multiple Myeloma. Hemato 2, 1-42 (2020)). This low expression of targets in MM populations has been observed after treatment with CD38-targeted daratumumab (Nijhof, I. et al. CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma. 128, 12 (2016)) and BCMA-specific CAR T cells (Cohen, A. et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J. Clin. Invest. 129, 2210-2221 (2019)).As a result, developing approaches to prevent tumor escape, which is often associated with lower surface expression of targeted tumor-associated antigens, remains a challenge.

[0013] Although promising treatments for hematological cancers such as multiple myeloma are currently available, there remains a need for improved therapies, for example, effector cell-redirecting antibody-based therapies. Indeed, despite the increasing number of available treatments, the prognosis for patients with multiple myeloma and relapsed / refractory multiple myeloma remains poor, given increasing evidence of resistance to currently available treatments and the need to maximize the therapeutic window. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a trispecific antibody or antibody fragment thereof, which comprises at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38.

[0015] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising a common light chain.

[0016] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human CD3 comprises a set of heavy chain CDRs comprising an amino acid sequence selected from the group comprising: SEQ ID NO:181, SEQ ID NO:307, and SEQ ID NO:433; SEQ ID NO:184, SEQ ID NO:310, and SEQ ID NO:436; SEQ ID NO:186, SEQ ID NO:312, and SEQ ID NO:438; SEQ ID NO:188, SEQ ID NO:314, and SEQ ID NO:440; SEQ ID NO:192, SEQ ID NO:318, and SEQ ID NO:444.

[0017] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human BCMA comprises a heavy chain CDR set comprising an amino acid sequence selected from the group comprising: SEQ ID NO:234, SEQ ID NO:360, and SEQ ID NO:486; SEQ ID NO:219, SEQ ID NO:345, and SEQ ID NO:471; SEQ ID NO:227, SEQ ID NO:353, and SEQ ID NO:479; SEQ ID NO:231, SEQ ID NO:357, and SEQ ID NO:483.

[0018] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs comprising amino acid sequences comprising: SEQ ID NO:236, SEQ ID NO:362, and SEQ ID NO:712; SEQ ID NO:239, SEQ ID NO:365, and SEQ ID NO:491; SEQ ID NO:237, SEQ ID NO:363, and SEQ ID NO:700; SEQ ID NO:248, SEQ ID NO:374, and SEQ ID NO:500.

[0019] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 55; 58; 60; 62 and 66.

[0020] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human CD3 comprises a heavy chain variable region comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 592.

[0021] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105 and 108.

[0022] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105 and 108, and wherein the amino acid sequence of SEQ ID NOs: 93, 101, 105 and 108 further comprises the substitution N82aS.

[0023] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human BCMA comprises a heavy chain variable region comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:591.

[0024] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122 and 111.

[0025] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human CD38 comprises a heavy chain variable region comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 593 or 594.

[0026] The present invention also relates to a trispecific antibody or antibody fragment thereof, further comprising a light chain variable region of a light chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0027] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising: SEQ ID NOs: 55; 58; 60; 62 and 66.

[0028] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human BCMA comprises a heavy chain comprising an amino acid sequence selected from the group comprising SEQ ID NOs: 93, 101, 105 and 108; or an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NOs: 93, 101, 105 and 108, wherein the amino acid sequence of SEQ ID NOs: 93, 101, 105 and 108 further comprises the substitution N82aS.

[0029] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising: SEQ ID NOs: 110; 113, 122 and 111.

[0030] The present invention also relates to a trispecific antibody or antibody fragment thereof, which further comprises a light chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0031] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a set of heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723, and ... the at least one binding moiety that binds to human CD38 comprises a heavy chain CDR set comprising the amino acid sequences of SEQ ID NOs: 239, 365, and 491, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

[0032] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein at least one binding moiety that binds to human CD3 comprises a set of heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 181, 307, and 433, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723; at least one binding moiety that binds to BCMA comprises a set of heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 234, 360, and 486, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723; and at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs comprising the amino acid sequences of SEQ ID NOs: 236, 362, and 712, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

[0033] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the at least one binding moiety that binds human CD3 and / or the at least one binding moiety that binds human BCMA and / or the at least one binding moiety that binds human CD38 is a Fab fragment.

[0034] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the at least one binding moiety that binds human CD3 and the at least one binding moiety that binds human BCMA, or the at least one binding moiety that binds human CD3 and the at least one binding moiety that binds human CD38, or the at least one binding moiety that binds human BCMA and the at least one binding moiety that binds human CD38, are fused to one another.

[0035] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the at least one binding moiety that binds human BCMA is fused N-terminally to the C-terminus of the at least one binding moiety that binds human CD3, or the at least one binding moiety that binds human BCMA is fused N-terminally to the C-terminus of the at least one binding moiety that binds human CD38.

[0036] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the at least one binding moiety that binds human BCMA is fused C-terminally to the N-terminus of the at least one binding moiety that binds human CD3, or the at least one binding moiety that binds human BCMA is fused C-terminally to the N-terminus of the at least one binding moiety that binds human CD38.

[0037] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the at least one binding moiety that binds human CD3 and the at least one binding moiety that binds human BCMA, or the at least one binding moiety that binds human CD3 and the at least one binding moiety that binds human CD38, or the at least one binding moiety that binds human BCMA and the at least one binding moiety that binds human CD38, are fused to each other via a peptide linker.

[0038] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the trispecific antibody or antibody fragment thereof comprises a non-naturally occurring Fc domain.

[0039] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising a set of three amino acid chains comprising amino acid sequences selected from the group comprising SEQ ID NO:522, SEQ ID NO:523 and SEQ ID NO:1; SEQ ID NO:530, SEQ ID NO:531 and SEQ ID NO:1; SEQ ID NO:532, SEQ ID NO:533 and SEQ ID NO:1; SEQ ID NO:534, SEQ ID NO:535 and SEQ ID NO:1; SEQ ID NO:536, SEQ ID NO:537 and SEQ ID NO:1; SEQ ID NO:538, SEQ ID NO:539 and SEQ ID NO:1; SEQ ID NO:540, SEQ ID NO:541 and SEQ ID NO:1; SEQ ID NO:542, SEQ ID NO:543 and SEQ ID NO:1; SEQ ID NO:544, SEQ ID NO:545 and SEQ ID NO:1; SEQ ID NO:546, SEQ ID NO:547, and SEQ ID NO:1; and SEQ ID NO:548, SEQ ID NO:549 and SEQ ID NO:1.

[0040] Preferably, the present invention relates to a trispecific antibody or antibody fragment thereof comprising three amino acid chains having amino acid sequences selected from the group comprising SEQ ID NO:546, SEQ ID NO:547 and SEQ ID NO:1; and SEQ ID NO:548, SEQ ID NO:549 and SEQ ID NO:1.

[0041] More preferably, the trispecific antibody or antibody fragment thereof disclosed herein comprises three amino acid chains having the amino acid sequences of SEQ ID NO:546, SEQ ID NO:547 and SEQ ID NO:1.

[0042] The present invention also relates to a trispecific antibody or antibody fragment thereof, which is a heterodimeric immunoglobulin comprising a first engineered immunoglobulin chain comprising said first engineered domain and a second engineered immunoglobulin chain comprising a second engineered domain, wherein said heterodimeric immunoglobulin heterodimerizes via said first and second engineered domains.

[0043] In particular, the non-naturally occurring Fc domain comprises a first engineered CH3 domain and a second engineered CH3 domain.

[0044] More specifically, the first engineered CH3 domain comprises substitutions from the group including: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering) and the second engineered CH3 domain comprises substitutions from the group including: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering).

[0045] The present invention further relates to a trispecific antibody or an antibody fragment thereof for use as a medicament.

[0046] In particular, for use in the treatment of multiple myeloma, relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, active multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, cervical cancer, germinal center B-cell lymphoma or B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), high-grade serous ovarian cancer, peritoneal cancer.

[0047] The present invention also relates to the epitopes on the extracellular domain of human CD38 bound by the trispecific antibodies or antibody fragments thereof disclosed herein.

[0048] More specifically, the present invention also relates to an epitope on the extracellular domain of human CD38, comprising residues Glu103, Gln107, Thr114, Thr116, Arg194, Arg195, Glu198, Ala199, Asp202, Ser224, His228, Asn229, Gln231, Pro232, Glu233, Lys234, Val235, Gln236, Ile265, Ser267, Lys268, Arg269, and Asn270, as detected by X-ray crystallography. In particular, the X-ray crystallography has a resolution of at least 5 Å, preferably at least Å, and even more preferably at least 3.5 Å. In a most preferred example, the resolution is about 3.4 Å.

[0049] More specifically, the present invention also relates to an epitope on the extracellular domain of human CD38 that is bound by an antibody according to any one of the preceding claims and that includes residues Glu103, Gln107, Thr114, Thr116, Arg194, Arg195, Glu198, Ala199, Asp202, Ser224, His228, Asn229, Gln231, Pro232, Glu233, Lys234, Val235, Gln236, Ile265, Ser267, Lys268, Arg269, and Asn270, as detected by X-ray crystallography. In particular, the X-ray crystallography has a resolution of at least 5 Å, preferably at least Å, and even more preferably at least 3.5 Å. In the most preferred example, the resolution is about 3.4 Å.

[0050] The present invention also relates to epitopes on the human BCMA extracellular domain that are bound by the trispecific antibodies or antibody fragments thereof disclosed herein.

[0051] The present invention also relates to epitopes on the human CD3 extracellular domain bound by the trispecific antibodies or antibody fragments thereof disclosed herein.

[0052] The present invention also relates to an antibody or antibody fragment thereof, or antigen-binding fragment thereof, that binds to the same epitope on CD3, and / or BCMA, and / or CD38 as a reference antibody or antibody fragment thereof, wherein the reference antibody or antibody fragment thereof is a trispecific antibody or antibody fragment thereof, and wherein the at least one binding moiety that binds to human CD3 comprises a set of heavy chain CDRs comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; the at least one binding moiety that binds to BCMA comprises a set of heavy chain CDRs comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and the at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs comprising SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0053] The present invention also relates to an antibody or antigen-binding fragment that binds to the same epitope on CD3 as a reference antibody, wherein the reference antibody is a trispecific antibody, and wherein the at least one binding moiety that binds to human CD3, and / or BCMA, and / or CD38 comprises a set of heavy chain CDRs comprising SEQ ID NOs: 181, 307, and 433, and a set of light chain CDRs comprising SEQ ID NOs: 721, 722, and 723; the at least one binding moiety that binds to BCMA comprises a set of heavy chain CDRs comprising SEQ ID NOs: 234, 360, and 486, and a set of light chain CDRs comprising SEQ ID NOs: 721, 722, and 723; and the at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs comprising SEQ ID NOs: 236, 362, and 712, and a set of light chain CDRs comprising SEQ ID NOs: 721, 722, and 723.

[0054] The present invention also relates to an antibody or antigen-binding fragment that binds to an epitope on human CD3, wherein the epitope on CD3 comprises the amino acid sequence of SEQ ID NO:726.

[0055] The present invention also relates to an antibody or antigen-binding fragment that binds to an epitope on human CD38, the epitope on CD38 comprising residues Glu103, Gln107, Thr114, Thr116, Arg194, Arg195, Glu198, Ala199, Asp202, Ser224, His228, Asn229, Gln231, Pro232, Glu233, Lys234, Val235, Gln236, Ile265, Ser267, Lys268, Arg269, and Asn270 as detected by X-ray crystallography. In particular, the X-ray crystallography has a resolution of at least 5 Å, preferably at least Å, and even more preferably at least 3.5 Å. In a most preferred embodiment, the resolution is about 3.4 Å.

[0056] The present invention also relates to isolated nucleic acids encoding the trispecific antibodies disclosed herein.

[0057] The present invention also relates to host cells containing the isolated nucleic acids.

[0058] The present invention further relates to an antibody or antibody fragment thereof that binds to human CD3, comprising a set of heavy chain CDRs comprising amino acid sequences selected from the group consisting of SEQ ID NO:181, SEQ ID NO:307, and SEQ ID NO:433; SEQ ID NO:184, SEQ ID NO:310, and SEQ ID NO:436; SEQ ID NO:186, SEQ ID NO:312, and SEQ ID NO:438; SEQ ID NO:188, SEQ ID NO:314, and SEQ ID NO:440; SEQ ID NO:192, SEQ ID NO:318, and SEQ ID NO:444, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NO:721, SEQ ID NO:722, and SEQ ID NO:723.

[0059] The present invention further relates to an antibody or antibody fragment thereof that binds to human BCMA, comprising a set of heavy chain CDRs comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 234, 360, and 486; SEQ ID NOs: 219, 345, and 471; SEQ ID NOs: 227, 353, and 479; SEQ ID NO: 231, 357, and 483; and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

[0060] The present invention further relates to a trispecific heterodimeric antibody comprising a first and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises one or more substitutions selected from the group comprising Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises one or more substitutions selected from the group comprising Q347E, Y349A, L351F, S364T, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering). and wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via the first and second engineered CH3 domains, and wherein the trispecific heterodimeric antibody comprises at least three binding moieties, each binding moiety binding to a different antigen.

[0061] In particular, the present invention relates to trispecific heterodimeric antibodies comprising first and second engineered CH3 domains, wherein the first engineered CH3 domain comprises substitutions from the group comprising Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises substitutions from the group comprising Q347E, Y349A, L351F, S364T, T366V, K370T , T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via the first and second engineered CH3 domains, and wherein the trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding moieties, each binding moiety binding to a different antigen.

[0062] Provided herein are antibodies and antigen-binding fragments thereof that immunospecifically bind to human BCMA, CD38, and CD3. Related polynucleotides capable of encoding the provided BCMA-, CD38-, and CD3-specific antibodies and antigen-binding fragments, cells expressing the provided antibodies and antigen-binding fragments, and related vectors and detectably labeled antibodies and antigen-binding fragments are also described. Additionally, methods of using the provided antibodies and antigen-binding fragments are described. For example, BCMA-specific and / or CD38-specific antibodies and antigen-binding fragments can be used to diagnose or monitor the progression, regression, or stability of a BCMA- and / or CD38-expressing cancer, to determine whether a patient should be treated for cancer, or to determine whether a subject has a BCMA- and / or CD38-expressing cancer and therefore may be suitable for treatment with a BCMA-specific anti-cancer therapy and / or a CD38-expressing cancer, such as a multispecific antibody against BCMA, CD38, and CD3 described herein.

[0063] Further provided herein are multispecific antibodies that immunospecifically bind to BCMA, CD38, and CD3, as well as multispecific antigen-binding fragments thereof. Related polynucleotides capable of encoding the provided BCMAxCD38xCD3 multispecific antibodies, cells expressing the provided antibodies, and related vectors and detectably labeled multispecific antibodies are also described. Additionally, methods of using the provided multispecific antibodies are described. For example, BCMAxCD38xCD3 multispecific antibodies can be used to diagnose or monitor the progression, regression, or stability of BCMA-expressing and / or CD38-expressing cancers to determine whether a patient should undergo cancer treatment, or to determine whether a subject has a BCMA-expressing and / or CD38-expressing cancer and therefore may be suitable for treatment with a BCMA-specific anti-cancer therapeutic and / or a CD38-specific anti-cancer therapeutic, such as the BCMAxCD38xCD3 multispecific antibodies described herein.

[0064] As utilized in accordance with this disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated: Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

[0065] It will be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" embodiments and embodiments.

[0066] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprised in a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoramidate. The term "polynucleotide" specifically encompasses single- and double-stranded forms of DNA.

[0067] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that (1) is not associated with all or a portion of a polynucleotide with which it is found in nature, (2) is linked to a polynucleotide with which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0068] An "isolated polypeptide" is one that (1) is free of at least some other polypeptides with which it is normally found; (2) is essentially free of other polypeptides from the same source, e.g., the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) is not associated (by covalent or noncovalent interactions) with portions of polypeptides with which it is naturally associated; (6) is operably associated (by covalent or noncovalent interactions) with polypeptides with which it is not naturally associated; or (7) is not naturally occurring. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA, or other RNA, can be of synthetic origin, or any combination thereof. Preferably, an isolated polypeptide is found in its natural environment and is substantially free of polypeptides or other contaminants that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0069] The present invention relates to multispecific binding molecules, e.g., multispecific binding proteins such as antibodies or antibody fragments thereof, comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38.

[0070] As used herein, the term "binding protein" or "binding molecule" refers to a non-naturally occurring or recombinant or engineered molecule, e.g., a non-naturally occurring or recombinant or engineered antibody, that specifically binds to at least one target antigen, e.g., a CD38 polypeptide, a BCMA polypeptide, or a CD3 polypeptide of the disclosure.

[0071] A "recombinant" molecule is a molecule prepared, expressed, produced, or isolated by recombinant means.

[0072] The terms "antibody" and "immunoglobulin" as used herein include whole antibodies and any antigen-binding fragments or single chains thereof. Naturally occurring antibodies typically comprise tetramers. Each such tetramer typically comprises two pairs of identical polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 amino acids or more, typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide comprises a variable domain (VH) and three constant domains (CH1, CH2, and CH3), where the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), where the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[0073] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 or more amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd edition, 1989) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. Both light and heavy chain variable domains typically comprise, from the amino terminus to the carboxyl terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0074] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) have described the Kabat CDRs as follows: It has been discovered that certain subportions within the CDRs adopt nearly identical peptide backbone conformations despite the large diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan, 1995, FASEB J. 9:133-39; MacCallum, 1996, J. Mol. Biol. 262(5):732-45, and Lefranc, 2003, Dev. Comp. Immunol. 27:55-77. Still other CDR boundary definitions may not strictly adhere to one of the above systems; they may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding, yet still overlap with the Kabat CDRs. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia.Identification of predicted CDRs using amino acid sequences is well known in the art, for example, see Martin, AC, "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2, Kontermann R., Dubel S., eds. Springer-Verlag, Berlin, pp. 33-51 (2010). Other conventional methods may involve examining the amino acid sequence of a heavy chain variable domain and / or a light chain variable domain to identify CDR sequences by, for example, comparing known amino acid sequences of other heavy and light chain variable domains to determine regions of sequence hypervariability. Numbered sequences may be aligned visually or by using an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are traditionally used to accurately delineate framework and CDR regions, thereby correcting sequence-based assignments. All such alternative definitions are encompassed by the present invention, and the sequences provided herein are not intended to exclude alternatively defined CDR sequences that may include only a portion of the CDR sequences provided in the Sequence Listing. In a preferred embodiment of the invention, CDRH1 corresponds to Kabat positions 26-35, CDRH2 corresponds to Kabat positions 50-58, and CDRH3 corresponds to Kabat positions 93-102. In another preferred embodiment, CDRL1 corresponds to Kabat positions 24-34, CDRL2 corresponds to Kabat positions 50-56, and CDRL3 corresponds to Kabat positions 89-97.

[0075] The term "antibody fragment" as used herein includes a portion of a full-length antibody. Non-limiting examples of antibody fragments include: (i) a fragment crystallizable (Fc) consisting of two constant heavy chain fragments, consisting of the CH2 and CH3 domains in IgA, IgD, and IgG, and the CH2, CH3, and CH4 domains in IgE and IgM, paired by disulfide bonds and non-covalent interactions; (ii) an antigen-binding fragment (Fab) consisting of VL, CL, VH, and CH1 linked by disulfide bonds; (iii) Fab' consisting of VL, CL, VH, CH1, and one or more cysteine ​​residues from the hinge region linked by disulfide bonds; (iv) Fab'-SH, a Fab' fragment in which the cysteine ​​residue contains a free sulfhydryl group; (v) F(ab')2 consisting of two Fab fragments linked at the hinge region by disulfide bonds; (vi) (xii) single-domain antibodies (dAbs) consisting of a VH or VL domain; (xiii) diabodies consisting of two scFv fragments in which the VH and VL domains are connected by a short peptide that prevents their pairing in the same chain and allows non-covalent dimerization of the two scFvs; (xiv) trivalent triabodies in which three scFvs with VH and VL domains linked by a short peptide form a trimer; and the like.

[0076] As used herein, the term "Fc" refers to a molecule comprising the sequence of a non-antigen-binding fragment obtained by digestion of an antibody or generated by other means, regardless of whether it is in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of a native Fc is preferably human and may be any immunoglobulin. Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from one to four, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4). One example of an Fc is a disulfide-linked dimer resulting from papain digestion of IgG. As used herein, the term "native Fc" refers collectively to the monomeric, dimeric, and multimeric forms.

[0077] An F(ab) fragment typically contains one light chain and one heavy chain VH and CH1 domain, where the VH-CH1 heavy chain portion of the F(ab) fragment cannot form disulfide bonds with another heavy chain polypeptide. As used herein, an F(ab) fragment may contain one light chain containing two variable domains separated by an amino acid linker, and one heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain.

[0078] An F(ab') fragment typically contains one light chain and a portion of one heavy chain, including an additional portion of the constant region (between the CH1 and CH2 domains), so that interchain disulfide bonds can form between the two heavy chains to form an F(ab')2 molecule.

[0079] One embodiment of the present disclosure provides binding proteins with biological and immunological specificity for one to three target antigens.

[0080] As used herein, the term "antigen" or "target antigen" or "antigen target" refers to a molecule or portion of a molecule that can bind to a binding protein and / or can be used to generate antibodies in an animal that can specifically bind to an epitope of that antigen. A target antigen can have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein can compete with intact antibodies and / or antibody fragments thereof that recognize the target antigen. Antigens are bound by binding proteins, such as antibodies, through an antigen-binding site, also referred to herein as a "binding moiety" or "binding domain."

[0081] In certain embodiments of the present invention, one or more binding moieties of an antibody disclosed herein are antibody fragments selected from the non-limiting examples of antibody fragments listed above. In certain embodiments, one or more binding moieties of an antibody disclosed herein are Fab fragments. In more specific embodiments, all binding moieties of an antibody of the present invention are Fab fragments.

[0082] Based on the number of antigen-binding sites, binding proteins such as antibodies or antibody fragments can be classified as monovalent or multivalent. The term "valency" is used herein to indicate the number of binding sites. The term "monovalent binding protein" refers to a binding protein having one antigen-binding site. For example, a monovalent antibody is an antibody having one antigen-binding site. The term "monovalent binding protein" refers to a binding protein having two or more antigen-binding sites. For example, a multivalent antibody is one having two or more antigen-binding sites. Non-limiting examples of multivalent binding proteins are bivalent and trivalent. The term "bivalent binding protein" refers to a binding protein having two antigen-binding sites. For example, a bivalent antibody is an antibody having two antigen-binding sites. The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. For example, a trivalent antibody is an antibody having three antigen-binding sites.

[0083] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0084] As used herein, the term "multispecific binding protein" refers to any binding protein, including an antibody or antibody fragment thereof, that has two or more binding sites and binds to different epitopes of the same antigen or different antigen targets.

[0085] The term "bispecific binding protein" refers to a binding protein, including an antibody or antibody fragment thereof, that specifically binds to two different antigen targets. In some embodiments, a bispecific binding protein, such as an antibody or antibody fragment thereof, binds to two different antigens. In some embodiments, a bispecific binding protein binds to two different epitopes on the same antigen.

[0086] In some embodiments, the trispecific binding protein, e.g., an antibody or antibody fragment thereof, binds to three different antigens, hi some embodiments, the trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0087] In some embodiments, the trivalent binding protein may bind to one antigen target (i.e., a monospecific trivalent binding protein). In other embodiments, the trivalent binding protein may bind to two antigen targets (i.e., a bispecific trivalent binding protein). In other embodiments, the trivalent binding protein may bind to three antigen targets (i.e., a trispecific trivalent binding protein).

[0088] The term "epitope" encompasses any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, a binding protein has an equilibrium dissociation constant (K D ) is, for example, <10 -8 M, more preferably the equilibrium dissociation constant is <10 -9 M, most preferably the dissociation constant is <10 -10 When it is M, it is said to specifically bind to the antigen.

[0089] In preferred embodiments of the invention, the antibodies disclosed herein are trivalent, preferably trispecific, more preferably trispecific antibodies comprising at least three binding moieties, and in certain embodiments, antibodies of the invention are trispecific antibodies or antibody fragments thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38.

[0090] "CD3" is cluster of differentiation 3, a multimeric protein complex composed of four polypeptide chains: epsilon (ε), gamma (γ), delta (δ), and zeta (ζ), which assemble and function as three pairs of dimers (εγ, εδ, ζζ). In some embodiments, the binding proteins of the disclosure bind to the extracellular domain of one or more of the CD3 polypeptide chains, i.e., CD3ε, CD3γ, CD3δ, and CD3ζ. Exemplary CD3 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD3ε (e.g., as set forth in SEQ ID NO: 610) and the extracellular domain of cynomolgus monkey CD3ε (e.g., as set forth in SEQ ID NO: 614); the extracellular domain of human CD3δ (e.g., as set forth in SEQ ID NO: 611) and the extracellular domain of cynomolgus monkey CD3δ (e.g., as set forth in SEQ ID NO: 613); the extracellular domain of human CD3γ (e.g., as represented by SEQ ID NO: 612) and the extracellular domain of cynomolgus monkey CD3γ; the extracellular domain of human CD3ζ and the extracellular domain of cynomolgus monkey CD3ζ.

[0091] "BCMA" is a B-cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17; BCMA is expressed by mature B lymphocytes and overexpressed in malignant plasma cells. In some embodiments, the binding proteins of the disclosure bind the extracellular domain of one or more BCMA polypeptides. Exemplary BCMA extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human BCMA (e.g., as set forth in SEQ ID NO: 615) and the extracellular domain of cynomolgus monkey BCMA (e.g., as set forth in SEQ ID NO: 616).

[0092] "CD38" refers to the Cluster of Differentiation 38 polypeptide, a glycoprotein found on the surface of many immune cells. In some embodiments, the binding proteins of the present disclosure bind the extracellular domain of one or more CD38 polypeptides. Exemplary CD38 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD38 (e.g., as set forth in SEQ ID NO: 617) and the extracellular domain of cynomolgus monkey CD38 (e.g., as set forth in SEQ ID NO: 618).

[0093] The term "T cell engager" refers to a binding protein directed against the host's immune system, more specifically the cytotoxic activity of T cells, as well as against tumor target proteins.

[0094] In a preferred embodiment, the present invention provides a trispecific antibody or antibody fragment thereof that binds to human CD3 and human BCMA and human CD38. In a preferred embodiment of the invention, the antibody is monoclonal.

[0095] In certain embodiments, the trispecific antibodies of the present disclosure comprise a common light chain.

[0096] An "isolated" binding protein is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with diagnostic or therapeutic uses of the binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified (1) to greater than 95% by weight, and most preferably greater than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding protein includes the binding protein in situ within a recombinant cell, since at least one component of the binding protein's natural environment will not be present.

[0097] The terms "substantially pure" or "substantially purified," as used herein, refer to a compound or chemical species that is the predominant chemical species present (i.e., more abundant, on a molar basis, than any other individual chemical species in a composition). In some embodiments, a substantially purified fraction is a composition in which the chemical species constitutes at least about 50 percent (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition constitutes greater than about 80%, about 85%, about 90%, about 95%, or about 99% of all macromolecular species present in the composition. In still other embodiments, the chemical species is purified to near homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0098] A nucleic acid is "isolated" or "substantially pure" when it has been purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See, e.g., F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids of the invention can be, for example, DNA or RNA, and may or may not contain intron sequences.

[0099] Dissociation constant (K D ) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (GE). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). As used herein, "K" refers to a surface plasmon resonance (SPR) assay. D The term "dissociation constant" refers to the dissociation constant of the interaction between a particular binding protein or binding portion of a binding protein, or an antibody or antibody fragment thereof, and a target antigen and / or antigenic epitope.

[0100] As used herein, the term "binds to" with respect to a binding protein means that the binding protein or antigen-binding fragment thereof binds to at least about 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 M or less K D"A" refers to the ability to bind to an antigen containing an epitope with an affinity at least two-fold higher than its affinity for a non-specific antigen.

[0101] As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient flexibility for the light and heavy chain domains to fold into a cross-over dual variable region immunoglobulin. Linkers are inserted at the sequence level at the transition between variable domains or between variable and constant domains, respectively. Because the approximate sizes of immunoglobulin domains are well understood, transitions between domains can be identified. The exact location of a domain transition can be determined by locating peptide stretches that do not form secondary structure elements such as β-sheets or α-helices, as indicated by experimental data or as can be assumed by modeling or secondary structure prediction techniques.

[0102] In certain embodiments, the present disclosure provides nucleic acid molecules comprising nucleotide sequences encoding the polypeptide chains that form the binding proteins of the present invention. Another embodiment of the present disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding the polypeptide chains that form such binding proteins. Yet another embodiment of the present disclosure provides host cells that express such binding proteins (i.e., comprise nucleic acid molecules or vectors encoding the polypeptide chains that form such binding proteins).

[0103] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid) or other means (e.g., virus) used to transfer or provide coding information to a host cell. The term "vector" encompasses a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA fragments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome, particularly nucleic acids that facilitate viral assembly combined with coding information to facilitate transfer into host cells by virus-like particles.

[0104] Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As plasmids are the most commonly used form of vector, the terms "plasmid" and "vector" can be used interchangeably herein. However, the present disclosure is intended to encompass other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).

[0105] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cells are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain modifications may occur in subsequent generations due to either mutation or environmental influences, but such cells are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express binding proteins, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). To recombinantly express a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the polypeptide chain of the binding protein, such that the polypeptide chain is expressed in the host cell and, preferably, secreted into the medium in which the host cell is cultured and from which the binding protein can be recovered.

[0106] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell; a cell has been transformed if it has been modified to contain new DNA. For example, a cell has been transformed if it has been genetically altered from its original state. After transformation, the transforming DNA may recombine with the cell's DNA by being physically integrated into the cell's chromosome, or it may be maintained transiently as an episomal element without replication, or it may replicate independently as a plasmid. A cell is considered stably transformed if the DNA is replicated as the cell divides. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA by a cell; a cell has been "transfected" if the exogenous DNA has been introduced inside the cell membrane. Numerous transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into a suitable host cell.

[0107] As used herein and applied to an object, the term "naturally occurring" refers to the fact that an object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man is naturally occurring. Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or that has been structurally modified or synthesized by man.

[0108] As used herein, the 20 common amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 common amino acids (e.g., D-amino acids); unnatural amino acids and analogs, such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids, may also be suitable components of the polypeptide chain of a binding protein. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0109] Naturally occurring residues can be divided into the following classes based on common side chain properties: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues affecting chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0110] Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class, while non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class.

[0111] Those skilled in the art will be able to determine suitable variants of the polypeptide chain of a binding protein using well-known techniques. For example, those skilled in the art can identify suitable regions of the polypeptide chain that can be modified without destroying activity by targeting regions that are not believed to be important for activity. Alternatively, those skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In addition, even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions that do not destroy biological activity or adversely affect the polypeptide structure.

[0112] The term "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 60% identity, e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity) when compared and aligned for maximum correspondence over a specified region, or, if not specified, over the entire sequence. For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. Methods for aligning sequences for comparison are well known to those skilled in the art. Non-limiting examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402; and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information; the Clustal Omega algorithm (Sievers F, Higgins DG. Clustal Omega for accurate alignment of many protein sequences. Protein Sci. 2018 Jan;27(1):135-145.); the MUSCULE algorithm (Edgar, R.C. MUSCLE: a method for multiple sequence alignment with reduced temporal and spatial complexity. BMC Bioinformatics 5,113(2004). https: / / doi.org / 10.1186 / 1471-2105-5-113).

[0113] The terms "patient" and "subject" are used interchangeably herein. As used herein, the term "patient" encompasses human and animal subjects. Animal subjects include all vertebrates, e.g., mammals and non-mammals (e.g., non-human primates, sheep, dogs, cows, chickens).

[0114] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include those with a disorder, as well as those susceptible to the disorder, or those in whom the disorder is to be prevented. In certain embodiments of the invention, the disorder is characterized by BCMA expression, e.g., overexpression, and / or CD38 expression, e.g., overexpression, including cancer and non-cancer diseases such as gastrointestinal, nervous, and pulmonary disorders (Szlasa W at al., Targeting CD38 in Neoplasms and Non-Cancer Diseases. Cancers. 2022 Aug 28;14(17):4169). For example, antibodies or antibody fragments thereof according to the invention can be used to treat B-cell malignancies and autoimmune disorders. In other specific embodiments, antibodies or antibody fragments thereof of the invention can be used to treat humans with or susceptible to cancer, or to ameliorate cancer in a human subject. The antibodies can also be used to prevent cancer in a human patient. In more specific embodiments, the antibodies of the present invention or antibody fragments thereof are used to treat cancer (e.g., hematological malignancies). In preferred embodiments, the cancer is a BCMA-expressing cancer and / or a CD38-expressing cancer. Non-limiting examples of cancers treated by the antibodies of the present invention or antibody fragments thereof include leukemias such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myeloblastic leukemia, acute monocytic leukemia and acute megakaryoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, and leukemia. Includes multiple myeloma, active multiple myeloma, plasma cell leukemia, lymphoma, germinal center B-cell lymphoma or B-cell acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, myelodysplastic syndromes (MDS), breast cancer including Her2+ breast cancer, prostate cancer, cervical cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), high-grade serous ovarian cancer, peritoneal cancer, smoldering myeloma, and glioma.

[0115] The present invention also relates to trispecific antibodies or antibody fragments thereof for use as a medicament, in particular for use in the treatment of the above-mentioned disorders, in particular for use in the treatment of cancer and autoimmune disorders, and more particularly for use in the treatment of hematological cancers, including but not limited to those mentioned above. In a preferred embodiment, the trispecific antibodies of the present invention are for use in the treatment of multiple myeloma, including relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, and active multiple myeloma.

[0116] The present invention also relates to the use of the trispecific antibodies of the present invention or antibody fragments thereof for the treatment of the aforementioned disorders, in particular for the treatment of cancer and autoimmune disorders, and more particularly for the treatment of hematological cancers, including but not limited to those mentioned above. In a preferred embodiment, the present invention relates to the use of the trispecific antibodies disclosed herein for the treatment of multiple myeloma, including relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, active multiple myeloma.

[0117] The present invention also relates to methods for treating cancer and autoimmune disorders, comprising administering a therapeutically effective amount of the trispecific antibody of the present invention to a patient in need thereof. In particular, the present invention also relates to methods for treating hematological cancer, comprising administering a therapeutically effective amount of the trispecific antibody of the present invention or an antibody fragment thereof to a patient in need thereof. More particularly, the hematological cancer is selected from the list including, but not limited to, those mentioned above. In a preferred embodiment, the present invention relates to a method for treating multiple myeloma, including relapsed multiple myeloma, treatment-resistant multiple myeloma, relapsed / treatment-resistant multiple myeloma, smoldering multiple myeloma, and active multiple myeloma, comprising administering to a patient in need thereof a therapeutically effective amount of the trispecific antibody or antibody fragment thereof disclosed herein.

[0118] The present invention also relates to pharmaceutical compositions comprising the trispecific antibodies, e.g., containing one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical or sterile composition comprising a trispecific antibody or antibody fragment thereof of the present disclosure, the MBM preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0119] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition that is capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0120] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier," as used herein, refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

[0121] The terms "effective amount" and "therapeutically effective amount," when used in reference to pharmaceutical compositions containing one or more binding proteins, refer to an amount or dosage sufficient to produce a desired therapeutic outcome. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated for a certain period of time. The effective amount may vary depending on the specific binding protein used and also depends on various factors and conditions related to the patient being treated and the severity of the disorder. For example, when a binding protein is administered in vivo, factors such as the patient's age, weight, and health status, as well as dose-response curves and toxicity data obtained from preclinical animal studies, will be considered. Determining the effective or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of one of ordinary skill in the art.

[0122] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the binding protein.

[0123] Anti-CD3 binding protein Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to CD3 polypeptides (e.g., human and cynomolgus CD3 polypeptides). In some embodiments, the antigen-binding domains and / or binding proteins of the present disclosure are "cross-reactive" with human and cynomolgus CD3 polypeptides.

[0124] In some embodiments, the binding proteins disclosed herein are monospecific, or bispecific, or trispecific, or multispecific and / or monovalent, bivalent, trivalent, or multivalent, and comprise a binding moiety that binds to CD3, e.g., human CD3.

[0125] In certain embodiments, the antibodies or antibody fragments of the invention are selected from the group consisting of SEQ ID NO:160, SEQ ID NO:286 and SEQ ID NO:412; SEQ ID NO:176, SEQ ID NO:302 and SEQ ID NO:428; SEQ ID NO:177, SEQ ID NO:303 and SEQ ID NO:429; SEQ ID NO:178, SEQ ID NO:304 and SEQ ID NO:430; SEQ ID NO:179, SEQ ID NO:305 and SEQ ID NO:431; SEQ ID NO:180, SEQ ID NO:306 and SEQ ID NO:432; SEQ ID NO:181, SEQ ID NO:307 and SEQ ID NO:433; SEQ ID NO:182, SEQ ID NO:308 and SEQ ID NO:434; SEQ ID NO:183, SEQ ID NO:309 and SEQ ID NO:435; SEQ ID NO:184, SEQ ID NO:310 and SEQ ID NO:436; The present invention also provides a binding moiety that binds to human CD3, comprising a heavy chain CDR set comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:185, SEQ ID NO:311 and SEQ ID NO:437; SEQ ID NO:186, SEQ ID NO:312 and SEQ ID NO:438; SEQ ID NO:187, SEQ ID NO:313 and SEQ ID NO:439; SEQ ID NO:188, SEQ ID NO:314 and SEQ ID NO:440; SEQ ID NO:189, SEQ ID NO:315 and SEQ ID NO:441; SEQ ID NO:190, SEQ ID NO:316 and SEQ ID NO:442; SEQ ID NO:191, SEQ ID NO:317 and SEQ ID NO:443; SEQ ID NO:192, SEQ ID NO:318 and SEQ ID NO:444; and SEQ ID NO:193, SEQ ID NO:319 and SEQ ID NO:445. In a more particular embodiment, the binding moiety that binds to human CD3 also comprises a consensus light chain CDR set comprising the amino acid sequence of SEQ ID NO:1. More specifically, the light chain CDR set comprises the amino acid sequences of SEQ ID NO:721, SEQ ID NO:722 and SEQ ID NO:723.

[0126] Preferably, the binding moiety that binds to human CD3 comprises a heavy chain CDR set comprising amino acid sequences selected from the group consisting of SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; SEQ ID NO: 184, SEQ ID NO: 310, and SEQ ID NO: 436; SEQ ID NO: 186, SEQ ID NO: 312, and SEQ ID NO: 438; SEQ ID NO: 188, SEQ ID NO: 314, and SEQ ID NO: 440; SEQ ID NO: 192, SEQ ID NO: 318, and SEQ ID NO: 444. More preferably, the binding moiety that binds to human CD3 comprises a CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433. In a more particular embodiment, the binding moiety that binds to human CD3 also comprises a consensus light chain CDR set comprising the amino acid sequence of SEQ ID NO: 1. More particularly, the light chain CDR set comprises the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723. More preferably, the binding moiety that binds to human CD3 comprises a set of heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0127] In other embodiments, the binding moiety that binds human CD3 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 50-67. In more specific embodiments, the binding moiety that binds human CD3 also comprises a light chain variable region of a light chain comprising an amino acid sequence at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0128] Preferably, the binding moiety that binds human CD3 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 58, 60, 62, and 66. In a more specific embodiment, the binding moiety that binds human CD3 also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0129] In one embodiment, the binding moiety that binds to human CD3 comprises a heavy chain variable region comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:592.

[0130] In other embodiments, the binding moiety that binds human CD3 comprises a heavy chain comprising an amino acid sequence at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 50-67. In more specific embodiments, the binding moiety that binds human CD3 also comprises a light chain at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0131] Preferably, the binding moiety that binds to human CD3 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 58, 60, 62, and 66, and a light chain that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0132] The present invention relates to multispecific, e.g., bispecific or trispecific, and multivalent, e.g., bivalent or trivalent, antibodies or antibody fragments thereof comprising any of the binding moieties that bind to human CD3 described above. In any of the bispecific or trispecific binding proteins described above, the target antigen other than CD3 can be any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BIYS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as MIP-1b), CCL6 (also known as MIP-1c), CCL7 (also known as MIP-1d), CCL8 (also known as MIP-1e), CCL9 (also known as MIP-1f), CCL10 (also known as MIP-1f), CCL11 (also known as MIP-1f), CCL12 (also known as MIP-1a), CCL13 (also known as MIP-1f), CCL14 (also known as MIP-1f), CCL15 (also known as MIP-1f), CCL16 (also known as MIP-1f), CCL17 (also known as MIP-1f), CCL18 (also known as MIP-1f), CCL19 (also known as MIP-1f), CCL210 (also known as MIP-1f), CCL2210 (also known as MIP-1f), CCL23 (also known as MIP-1f), CCL24 (also known as MIP-1f), CCL25 (also b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CCR7, CD19, CD20, CD23 (also known as FCER2, the receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD47, CD48, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD123, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L) also known as PD-L2), CD274 (also known as PD-L1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF),CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IF Na, IgE, IGF1R, IL2Rβ, IL1, IL1RAP, ILILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rlL10, rILI0, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as the IL25 receptor), IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36, IL37, IL38, IL39, IL40, IL41, IL42, IL43, IL44, IL45, IL46, IL47, IL48, IL49, IL50, IL51, IL52, IL53, IL54, IL55, IL56, IL57, IL58, IL59, IL59Rb (also known as the IL5 receptor), ... L22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, KG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), S TEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1), XCL1, and XCL2. In some embodiments, one or more of the above antigen targets are human antigen targets. In preferred embodiments, the antibody or antibody fragment thereof is a trivalent bispecific antibody comprising at least two binding moieties, at least one of which binds human CD3 and at least one of which binds BCMA, preferably at least two of which bind BCMA. In a more preferred embodiment, the antibody of the present invention is a trivalent trispecific antibody comprising at least three binding moieties, at least one of which binds human CD3 and at least one of which binds BCMA.At least one binds to CD38.

[0133] The present invention also relates to monospecific antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind to CD3, eg, human CD3.

[0134] In certain aspects, the present invention provides SEQ ID NO:160, SEQ ID NO:286 and SEQ ID NO:412; SEQ ID NO:176, SEQ ID NO:302 and SEQ ID NO:428; SEQ ID NO:177, SEQ ID NO:303 and SEQ ID NO:429; SEQ ID NO:178, SEQ ID NO:304 and SEQ ID NO:430; SEQ ID NO:179, SEQ ID NO:305 and SEQ ID NO:431; SEQ ID NO:180, SEQ ID NO:306 and SEQ ID NO:432; SEQ ID NO:181, SEQ ID NO:307 and SEQ ID NO:433; SEQ ID NO:182, SEQ ID NO:308 and SEQ ID NO:434; SEQ ID NO:183, SEQ ID NO:309 and SEQ ID NO:435; SEQ ID NO:184, SEQ ID NO:310 and SEQ ID NO:436; SEQ ID NO:185, SEQ ID NO:311 and SEQ ID NO:437; SEQ ID NO:186, SEQ ID NO:312 and SEQ ID NO:438 and a light chain CDR set of a common light chain comprising the amino acid sequence of SEQ ID NO: 1, in particular a light chain CDR set comprising the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0135] In a preferred embodiment, the antibody or antibody fragment that binds to human CD3 comprises a heavy chain CDR set comprising amino acid sequences selected from the group consisting of SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; SEQ ID NO: 184, SEQ ID NO: 310, and SEQ ID NO: 436; SEQ ID NO: 186, SEQ ID NO: 312, and SEQ ID NO: 438; SEQ ID NO: 188, SEQ ID NO: 314, and SEQ ID NO: 440; SEQ ID NO: 192, SEQ ID NO: 318, and SEQ ID NO: 444, and a light chain CDR set comprising the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0136] In a preferred embodiment, the antibodies disclosed herein that comprise a binding moiety that binds to human CD3 as described above are monoclonal.

[0137] Anti-BCMA binding proteins Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to BCMA polypeptides (e.g., human and cynomolgus BCMA polypeptides). In some embodiments, the antigen-binding domains and / or binding proteins of the disclosure are "cross-reactive" with human and cynomolgus BCMA polypeptides.

[0138] In some embodiments, the binding proteins disclosed herein are monospecific, or bispecific, or trispecific, or multispecific and / or monovalent, bivalent, trivalent, or multivalent, and comprise binding moieties that bind to BCMA, e.g., human BCMA.

[0139] In certain embodiments, the antibodies or antibody fragments of the invention are selected from the group consisting of SEQ ID NO:209, SEQ ID NO:335, and SEQ ID NO:461; SEQ ID NO:210, SEQ ID NO:336, and SEQ ID NO:462; SEQ ID NO:211, SEQ ID NO:337, and SEQ ID NO:463; SEQ ID NO:212, SEQ ID NO:338, and SEQ ID NO:464; SEQ ID NO:213, SEQ ID NO:339, and SEQ ID NO:465; SEQ ID NO:214, SEQ ID NO:340, and SEQ ID NO:466; SEQ ID NO:215, SEQ ID NO:341, and SEQ ID NO:467; SEQ ID NO:216, SEQ ID NO:342, and SEQ ID NO:468; SEQ ID NO:217, SEQ ID NO:343, and SEQ ID NO:469; SEQ ID NO:218, SEQ ID NO:344, and SEQ ID NO:470; SEQ ID NO:219, SEQ ID NO:345, and SEQ ID NO:471; SEQ ID NO:220, SEQ ID NO:346, and SEQ ID NO:472; SEQ ID NO:221, SEQ ID NO:347, and SEQ ID NO:473; SEQ ID NO:222, SEQ ID NO:348, and SEQ ID NO:474; and a binding moiety that binds to human BCMA, comprising a heavy chain CDR set comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:223, SEQ ID NO:349, and SEQ ID NO:475; SEQ ID NO:224, SEQ ID NO:350, and SEQ ID NO:476; SEQ ID NO:225, SEQ ID NO:351, and SEQ ID NO:477; SEQ ID NO:226, SEQ ID NO:352, and SEQ ID NO:478; SEQ ID NO:227, SEQ ID NO:353, and SEQ ID NO:479; SEQ ID NO:228, SEQ ID NO:354, and SEQ ID NO:480; SEQ ID NO:229, SEQ ID NO:355, and SEQ ID NO:481; SEQ ID NO:230, SEQ ID NO:356, and SEQ ID NO:482; SEQ ID NO:231, SEQ ID NO:357, and SEQ ID NO:483; SEQ ID NO:232, SEQ ID NO:358, and SEQ ID NO:484; SEQ ID NO:233, SEQ ID NO:359, and SEQ ID NO:485; SEQ ID NO:234, SEQ ID NO:360, and SEQ ID NO:486; and SEQ ID NO:235, SEQ ID NO:361, and SEQ ID NO:487. In a more particular embodiment, the binding moiety that binds to human BCMA also comprises a consensus light chain CDR set comprising the amino acid sequence of SEQ ID NO: 1. More particularly, the light chain CDR set comprises the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0140] In more specific embodiments, the binding moiety that binds human BCMA comprises a heavy chain CDR set comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 234, 360, and 486; SEQ ID NOs: 219, 345, and 471; SEQ ID NOs: 227, 353, and 479; and SEQ ID NOs: 231, 357, and 483. Preferably, the binding moiety that binds human BCMA comprises the CDR set of SEQ ID NOs: 234, 360, and 486. In more specific embodiments, the binding moiety that binds human CD3 also comprises a consensus light chain CDR set comprising the amino acid sequence of SEQ ID NO: 1. More specifically, the light chain CDR set comprises the amino acid sequences of SEQ ID NOs: 721, 722, and 723. Preferably, the binding moiety that binds human BCMA comprises a heavy chain CDR set comprising the amino acid sequences of SEQ ID NOs: 234, 360, and 486, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

[0141] In other embodiments, the binding moiety that binds to human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-109. In more specific embodiments, the binding moiety that binds to human BCMA also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0142] In other embodiments, the binding moiety that binds to human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-109, wherein the amino acid sequence of SEQ ID NOs: 83-109 further comprises the mutation N82aS. In more specific embodiments, the binding moiety that binds to human BCMA also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0143] Preferably, the binding moiety that binds human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105, and 108. In a more particular embodiment, the binding moiety that binds human BCMA also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0144] Preferably, the binding moiety that binds to human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105, and 108, wherein the amino acid sequence of SEQ ID NOs: 93, 101, 105, and 108 further comprises the substitution N82aS. In more specific embodiments, the binding moiety that binds to human BCMA also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0145] In more specific embodiments, the binding moiety that binds to human BCMA comprises a heavy chain variable region comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:591.

[0146] In other embodiments, the binding moiety that binds human BCMA comprises a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-109. In more specific embodiments, the binding moiety that binds human BCMA also comprises a light chain that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0147] In other embodiments, the binding moiety that binds human BCMA comprises a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-109, wherein the amino acid sequence of SEQ ID NOs: 83-109 further comprises the mutation N82aS. In more specific embodiments, the binding moiety that binds human BCMA also comprises a light chain that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0148] Preferably, the binding moiety that binds to human BCMA comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105, and 108, and a light chain that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0149] Preferably, the binding moiety that binds to human BCMA is a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105, 108. and a light chain that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0150] The present invention relates to multispecific, e.g., bispecific or trispecific, and multivalent, e.g., bivalent or trivalent, antibodies or antibody fragments thereof comprising any of the binding moieties that bind to human BCMA described above. In any of the bispecific or trispecific binding proteins described above, the target antigen other than BCMA can be any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BIYS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1a), CCL5 (also known as MIP-1a), CCL6 (also known as MIP-1a), CCL7 (also known as MIP-1a), CCL8 (also known as MIP-1a), CCL9 (also known as MIP-1a), CCL10 (also known as MIP-1a), CCL11 (also known as MIP-1a), CCL12 (also known as MIP-1a), CCL13 (also known as MIP-1a), CCL14 (also known as MIP-1a), CCL15 (also known as MIP-1a), CCL16 (also known as MIP-1a), CCL17 (also known as MIP-1a), CCL18 (also known as MIP-1a), CCL19 (also known as MIP-1a), CCL20 (also known as MIP-1a), CCL21 (also known as MIP-1a), CCL22 (also known as MIP-1a), CCL23 (also known as MIP-1a), CCL24 (also known CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL3 (also known as TECK), CCL4 (also known as TECK), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL36 (also known as TECK), CCL40 (also known as TECK), CCL41 (also known as TECK), CCL42 (also known as TECK), CCL43 (also known as TECK), CCL44 (also known as TECK), CCL45 (also known as TECK), CCL46 (also known as TECK), CCL47 (also known as TECK), CCL48 (also known as TECK), CCL49 (also known as TECK), CCL50 (also known as TECK), CCL51 (also known as TECK), CCL52 (al CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CCR7, CD19, CD20, CD23 (also known as FCER2, the receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD47, CD48, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L) known as PD-L2), CD274 (known as PD-L1), CD275 (known as B7H2), CD276 (known as B7H3), CD278 (known as ICOS), CD279 (known as PD-1), CDH1 (known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (known as M-CSF), CSF-2 (known as GM-CSF), CSF-3 (known as GCSF),CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IF Na, IgE, IGF1R, IL2Rβ, IL1, IL1RAP, ILILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rlL10, rILI0, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as the IL25 receptor), IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36, IL37, IL38, IL39, IL40, IL41, IL42, IL43, IL44, IL45, IL46, IL47, IL48, IL49, IL50, IL51, IL52, IL53, IL54, IL55, IL56, IL57, IL58, IL59, IL59Rb (also known as the IL5 receptor), ... L22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, KG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), S TEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1), XCL1, and XCL2. In some embodiments, one or more of the above antigen targets are human antigen targets. In preferred embodiments, the antibody or antibody fragment thereof is a trivalent bispecific antibody comprising at least two binding moieties, at least one of which binds human CD3 and at least one of which binds BCMA, preferably at least two of which bind BCMA. In a more preferred embodiment, the antibody of the present invention is a trivalent trispecific antibody comprising at least three binding moieties, at least one of which binds human CD3 and at least one of which binds BCMA.At least one binds to CD38.

[0151] The present invention also relates to monospecific antibodies or antibody fragments thereof comprising any of the above-mentioned binding moieties that bind to BCMA, e.g., human BCMA.

[0152] In certain aspects, the present invention provides SEQ ID NO:209, SEQ ID NO:335, and SEQ ID NO:461; SEQ ID NO:210, SEQ ID NO:336, and SEQ ID NO:462; SEQ ID NO:211, SEQ ID NO:337, and SEQ ID NO:463; SEQ ID NO:212, SEQ ID NO:338, and SEQ ID NO:464; SEQ ID NO:213, SEQ ID NO:339, and SEQ ID NO:465; SEQ ID NO:214, SEQ ID NO:340, and SEQ ID NO:466; SEQ ID NO:215, SEQ ID NO:341, and SEQ ID NO:467; SEQ ID NO:216, SEQ ID NO:342, and SEQ ID NO:469. Sequence number 342, and sequence number 468; sequence number 217, sequence number 343, and sequence number 469; sequence number 218, sequence number 344, and sequence number 470; sequence number 219, sequence number 345, and sequence number 471; sequence number 220, sequence number 346, and sequence number 472; sequence number 221, sequence number 347, and sequence number 473; sequence number 222, sequence number 348, and sequence number 474; sequence number 223, sequence number 349, and sequence number 475; sequence number 22 and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs:721, 722 and 723.

[0153] In a more particular aspect, the present invention also relates to an antibody or antibody fragment thereof that binds to human BCMA, comprising a set of heavy chain CDRs comprising amino acid sequences selected from the group consisting of SEQ ID NO:234, SEQ ID NO:360, and SEQ ID NO:486; SEQ ID NO:219, SEQ ID NO:345, and SEQ ID NO:471; SEQ ID NO:227, SEQ ID NO:353, and SEQ ID NO:479; SEQ ID NO:231, SEQ ID NO:357, and SEQ ID NO:483, and a set of light chain CDRs comprising the amino acid sequences of SEQ ID NO:721, SEQ ID NO:722, and SEQ ID NO:723.

[0154] In a preferred embodiment, the antibodies disclosed herein that comprise a binding moiety that binds to human BCMA as described above are monoclonal.

[0155] Anti-CD38 binding protein Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to a CD38 polypeptide (e.g., a human or cynomolgus CD38 polypeptide). In some embodiments, the antigen-binding domains and / or binding proteins of the present disclosure are "cross-reactive" with human and cynomolgus CD38 polypeptides.

[0156] In some embodiments, the binding proteins disclosed herein are monospecific, or bispecific, or trispecific, or multispecific and / or monovalent, bivalent, trivalent, or multivalent, and comprise a binding moiety that binds to CD38, e.g., human CD38.

[0157] In certain embodiments, the antibodies or antibody fragments of the invention are selected from the group consisting of SEQ ID NO:238, SEQ ID NO:364, and SEQ ID NO:490; SEQ ID NO:239, SEQ ID NO:365, and SEQ ID NO:491; SEQ ID NO:240, SEQ ID NO:366, and SEQ ID NO:492; SEQ ID NO:241, SEQ ID NO:367, and SEQ ID NO:493; SEQ ID NO:242, SEQ ID NO:368, and SEQ ID NO:494; SEQ ID NO:243, SEQ ID NO:369, and SEQ ID NO:495; SEQ ID NO:244, SEQ ID NO:370, and SEQ ID NO:496; SEQ ID NO:245, SEQ ID NO:371, and SEQ ID NO:497; SEQ ID NO:246, SEQ ID NO:372, and SEQ ID NO:498; SEQ ID NO:247, SEQ ID NO:37 and SEQ ID NO: 237, SEQ ID NO: 363, and SEQ ID NO: 700. In a more particular embodiment, the binding moiety that binds to human CD38 also comprises a set of heavy chain CDRs comprising the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 499; SEQ ID NO: 248, SEQ ID NO: 374, and SEQ ID NO: 500; SEQ ID NO: 249, SEQ ID NO: 375, and SEQ ID NO: 501; SEQ ID NO: 250, SEQ ID NO: 376, and SEQ ID NO: 502; SEQ ID NO: 251, SEQ ID NO: 377, and SEQ ID NO: 503; SEQ ID NO: 252, SEQ ID NO: 378, and SEQ ID NO: 504; SEQ ID NO: 253, SEQ ID NO: 379, and SEQ ID NO: 505; SEQ ID NO: 236, SEQ ID NO: 362, and SEQ ID NO: 712; and SEQ ID NO: 237, SEQ ID NO: 363, and SEQ ID NO: 700. In a more particular embodiment, the binding moiety that binds to human CD38 also comprises a set of consensus light chain CDRs comprising the amino acid sequence of SEQ ID NO: 1. More specifically, the CDR set comprises the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0158] In a more specific embodiment, the binding moiety that binds human CD38 comprises a heavy chain CDR set comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 236, 362, and 712; SEQ ID NOs: 239, 365, and 491; SEQ ID NOs: 237, 363, and 700; SEQ ID NOs: 248, 374, and 500. Preferably, the binding moiety that binds human CD38 comprises a CDR set comprising SEQ ID NOs: 236, 362, and 712 or SEQ ID NOs: 239, 365, and 491. In a more specific embodiment, the binding moiety that binds human CD3 also comprises a consensus light chain CDR set comprising the amino acid sequence of SEQ ID NO: 1. More specifically, the light chain CDR set comprises the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

[0159] In other embodiments, the binding moiety that binds human CD38 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-127. In more specific embodiments, the binding moiety that binds human CD38 also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0160] Preferably, the binding moiety that binds human CD38 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 111, 113, and 122. In a more specific embodiment, the binding moiety that binds human CD38 also comprises a light chain variable region of a light chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1. The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein the at least one binding moiety that binds human CD38 comprises a heavy chain variable region comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 593 or 594.

[0161] In other embodiments, the binding moiety that binds human CD38 comprises a heavy chain comprising an amino acid sequence at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-127. In more specific embodiments, the binding moiety that binds human BCMA also comprises a light chain at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0162] Preferably, the binding moiety that binds to human CD38 comprises a heavy chain variable region of a heavy chain comprising an amino acid sequence at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 111, 113, and 122, and a light chain at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1.

[0163] The present invention relates to multispecific, e.g., bispecific or trispecific, and multivalent, e.g., bivalent or trivalent, antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind to human CD38.

[0164] In any of the above-mentioned or trispecific proteins, the target antigen other than CD38 can be any of the following exemplary antigen targets: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BIYS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 ( CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CCR7, CD19, CD20, CD2 3 (also known as FCER2, the receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD47, CD48, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PD-L2), CD274 (also known as PD-L1), CD275 (also known as B7H2), CD276 (B7 H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A,FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rβ, IL1, IL1RAP, ILILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rILL10, rILI0, IL12, IL13, IL13RaI, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), I TK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, KG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFα, TFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1), XCL1, and XCL2. In some embodiments, one or more of the above antigen targets is a human antigen target. In a preferred embodiment, the antibody of the invention is a trivalent trispecific antibody comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to BCMA, and at least one of which binds to CD38.

[0165] The present invention also relates to monospecific antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind to CD38, eg, human CD38.

[0166] In certain aspects, the present invention provides a method for the preparation of a nucleotide sequence of SEQ ID NO:238, SEQ ID NO:364, and SEQ ID NO:490; SEQ ID NO:239, SEQ ID NO:365, and SEQ ID NO:491; SEQ ID NO:240, SEQ ID NO:366, and SEQ ID NO:492; SEQ ID NO:241, SEQ ID NO:367, and SEQ ID NO:493; SEQ ID NO:242, SEQ ID NO:368, and SEQ ID NO:494; SEQ ID NO:243, SEQ ID NO:369, and SEQ ID NO:495; SEQ ID NO:244, SEQ ID NO:370, and SEQ ID NO:496; SEQ ID NO:245, SEQ ID NO:371, and SEQ ID NO:497; SEQ ID NO:246, SEQ ID NO:372, and SEQ ID NO:498; SEQ ID NO:247, SEQ ID NO:373, and SEQ ID NO:499; SEQ ID NO:248, SEQ ID NO:374, and SEQ ID NO: No. 500; SEQ ID NO: 249, SEQ ID NO: 375, and SEQ ID NO: 501; SEQ ID NO: 250, SEQ ID NO: 376, and SEQ ID NO: 502; SEQ ID NO: 251, SEQ ID NO: 377, and SEQ ID NO: 503; SEQ ID NO: 252, SEQ ID NO: 378, and SEQ ID NO: 504; SEQ ID NO: 253, SEQ ID NO: 379, and SEQ ID NO: 505; SEQ ID NO: 236, SEQ ID NO: 362, and SEQ ID NO: 712; and SEQ ID NO: 237, SEQ ID NO: 363, and SEQ ID NO: 700; and a light chain CDR set comprising the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0167] In a more particular aspect, the present invention relates to an antibody or antibody fragment thereof that binds to human CD38, comprising a set of heavy chain CDRs comprising amino acid sequences selected from the group consisting of SEQ ID NO:236, SEQ ID NO:362, and SEQ ID NO:712; SEQ ID NO:239, SEQ ID NO:365, and SEQ ID NO:491; SEQ ID NO:237, SEQ ID NO:363, and SEQ ID NO:700; SEQ ID NO:248, SEQ ID NO:374, and SEQ ID NO:500.

[0168] In a preferred embodiment, the antibodies disclosed herein that comprise a binding moiety that binds to human CD38 as described above are monoclonal.

[0169] Trivalent bispecific anti-CD3 and anti-BCMA antibodies In one particular aspect, the present invention relates to a trivalent bispecific antibody or antibody fragment thereof comprising at least two binding moieties, at least one of which binds human CD3 and at least one of which binds BCMA, preferably at least two of which bind BCMA.

[0170] In certain embodiments of the invention, the trivalent bispecific anti-CD3 and anti-BCMA antibody is a heterodimeric immunoglobulin, in particular constructed using the previously described BEAT® heavy chain (He) heterodimerization technology (Skegro et al., (2017) J Biol Chem 292(23):9745-9759 and Stutz et al., (2020) J Biol Chem 295(28):9392-9408, WO 2012131555), and comprising a BEAT (A) chain, also referred to herein as BTA, and a BEAT (B) chain, also referred to herein as BTB. More specifically, a trivalent bispecific anti-CD3 and anti-BCMA antibody is designed by combining CD3 binders as Fab and BCMA dual dAb from the state of the art on a silenced BEAT Fc backbone (LALA mutation, Hezareh et al., 2001, J Virol, 75(24):12161-12168).

[0171] In certain embodiments of the invention, the trivalent bispecific anti-CD3 and anti-BCMA comprises a BTA chain comprising the amino acid sequence of SEQ ID NO: 515, a BTB chain comprising the amino acid sequence of SEQ ID NO: 516, and a common light chain comprising the amino acid sequence of SEQ ID NO: 1; or a BTA chain comprising the amino acid sequence of SEQ ID NO: 509, a BTB chain comprising the amino acid sequence of SEQ ID NO: 510, and a common light chain comprising the amino acid sequence of SEQ ID NO: 1; or a BTA chain comprising the amino acid sequence of SEQ ID NO: 513, a BTB chain comprising the amino acid sequence of SEQ ID NO: 514, and a common light chain comprising the amino acid sequence of SEQ ID NO: 1; or a BTA chain comprising the amino acid sequence of SEQ ID NO: 511, a BTB chain comprising the amino acid sequence of SEQ ID NO: 512, and a common light chain comprising the amino acid sequence of SEQ ID NO: 1; or a BTA chain comprising the amino acid sequence of SEQ ID NO: 517, a BTB chain comprising the amino acid sequence of SEQ ID NO: 518, and a common light chain comprising the amino acid sequence of SEQ ID NO: 1. The present invention also discloses trivalent bispecific anti-CD3 and anti-BCMA antibodies comprising a BTA chain and / or a BTB chain and / or a common light chain that are at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99% or 100% identical to the amino acid sequences set forth above.

[0172] Trispecific anti-CD3, anti-BCMA and anti-CD38 protein The present invention relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, also referred to herein as a CD3 / BCMA / CD38 antibody. In a preferred embodiment, the present invention provides a trispecific antibody or antibody fragment thereof that binds to human CD3, human BCMA, and human CD38. In a preferred embodiment of the present invention, the antibody is monoclonal. Binding moieties that bind to CD3, BCMA, or CD38 according to the present invention are described above.

[0173] Trispecific antibodies are a group of engineered antibody derivatives that recognize three different target antigens. In a specific embodiment of the present invention, trispecific anti-CD3, anti-BCMA, and anti-CD38 antibodies are heterodimeric immunoglobulins, specifically constructed using TREAT® trispecific antibody technology based on the BEAT® platform. The BEAT® platform is based on replacing the protein-protein interface of the CH3 domain pair of an antibody Fc region with the protein-protein interface of the T cell receptor (TCR) α-β constant region. The resulting BEAT® interface drives the preferential formation of heterodimeric bispecific or trispecific antibodies (bsAbs or TriAbs) over homodimeric contaminants. Furthermore, CD3 / BCMA / CD38 antibodies utilize a Fab CD38-binding arm, a Fab BCMA-binding arm, and a Fab CD3-binding arm as Fab units, all of which utilize a common light chain to prevent light chain mispairing, a common issue associated with bsAb and even TriAbs platforms. Further manipulations have been performed to allow for an easier and faster purification process, retaining the native conformation of the antibody structure, stability, and binding to the neonatal Fc receptor (FcRn), and maximizing the serum half-life of CD3 / BCMA / CD38 antibodies (Skegro et al., J Biol Chem, 2017, 292(23):9745-9759; Stutz et al., J Biol Chem, 2020, 295(28):9392-9408).

[0174] A binding moiety that binds to CD3, BCMA or CD38 according to the present invention may be any of the binding moieties that bind to CD3, BCMA or CD38 described above.

[0175] In certain particular embodiments, at least one binding moiety that binds human CD3 comprises a set of heavy chain CDRs selected from the group comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; SEQ ID NO: 184, SEQ ID NO: 310, and SEQ ID NO: 436; SEQ ID NO: 186, SEQ ID NO: 312, and SEQ ID NO: 438; SEQ ID NO: 188, SEQ ID NO: 314, and SEQ ID NO: 440; SEQ ID NO: 192, SEQ ID NO: 318, and SEQ ID NO: 444, and at least one that binds BCMA comprises a set of heavy chain CDRs selected from the group comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486; ... and wherein the at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs selected from the group comprising: SEQ ID NO:219, SEQ ID NO:345, and SEQ ID NO:471; SEQ ID NO:227, SEQ ID NO:353, and SEQ ID NO:479; SEQ ID NO:231, SEQ ID NO:357 and SEQ ID NO:483, and the at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs that includes: SEQ ID NO:236, SEQ ID NO:362 and SEQ ID NO:712; SEQ ID NO:239, SEQ ID NO:365, and SEQ ID NO:491; SEQ ID NO:237, SEQ ID NO:363, and SEQ ID NO:700; SEQ ID NO:248, SEQ ID NO:374, and SEQ ID NO:500.

[0176] More specifically, the trispecific antibodies of the invention comprise at least one binding moiety that binds human CD3 comprising a set of heavy chain CDRs comprising SEQ ID NO: 181, SEQ ID NO: 307, SEQ ID NO: 433; at least one binding moiety that binds BCMA comprising a set of heavy chain CDRs comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486; and at least one binding moiety that binds human CD38 comprising a set of heavy chain CDRs comprising SEQ ID NO: 236, SEQ ID NO: 362, and SEQ ID NO: 712, or SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491. In a particularly preferred embodiment, the at least one binding moiety that binds human CD38 comprises a set of heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491.

[0177] In certain embodiments, the trispecific antibodies or antibody fragments thereof disclosed herein further comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO:1.

[0178] Even more specifically, the present invention relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein said at least one binding moiety that binds human CD3 comprises a set of heavy chain CDRs comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; said at least one binding moiety that binds BCMA comprises a set of heavy chain CDRs comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and said at least one binding moiety that binds human CD38 comprises a set of heavy chain CDRs comprising SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0179] Even more specifically, the present invention relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds human CD3, at least one of which binds human BCMA, and at least one of which binds human CD38, wherein said at least one binding moiety that binds human CD3 comprises a set of heavy chain CDRs comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; said at least one binding moiety that binds BCMA comprises a set of heavy chain CDRs comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and said at least one binding moiety that binds human CD38 comprises a set of heavy chain CDRs comprising SEQ ID NO: 236, SEQ ID NO: 362, and SEQ ID NO: 712, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0180] The present invention also relates to an antibody or antibody fragment or antigen-binding fragment thereof that binds to the same epitope on CD3, and / or BCMA, and / or CD38 as a reference antibody, wherein the reference antibody is a trispecific antibody comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a heavy chain CDR set comprising SEQ ID NO: 721. , SEQ ID NO: 722, and SEQ ID NO: 723, and wherein the at least one binding moiety that binds to BCMA comprises a heavy chain CDR set comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723, and the at least one binding moiety that binds to human CD38 comprises a heavy chain CDR set comprising SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491, and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0181] The present invention also relates to an antibody or antibody fragment or antigen-binding fragment thereof that binds to the same epitope on CD3, and / or BCMA, and / or CD38 as a reference antibody, wherein the reference antibody is a trispecific antibody comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a heavy chain CDR set comprising SEQ ID NO: 721. , SEQ ID NO: 722, and SEQ ID NO: 723, and wherein the at least one binding moiety that binds to BCMA comprises a heavy chain CDR set comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723, and the at least one binding moiety that binds to human CD38 comprises a heavy chain CDR set comprising SEQ ID NO: 236, SEQ ID NO: 362, and SEQ ID NO: 712, and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0182] In certain embodiments of the invention, the trispecific antibody comprises at least one binding moiety that binds to human CD3 comprising a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62 and 66; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122 and 111. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region of a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0183] In certain embodiments of the invention, the trispecific antibody comprises at least one binding moiety that binds to human CD3 comprising a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62 and 66; and at least one binding moiety that binds to human BCMA, comprising a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122 and 111. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region of a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0184] In certain embodiments of the invention, the trispecific antibody comprises at least one binding moiety that binds to human CD3 comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62 and 66; 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122 and 111. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0185] In certain embodiments of the invention, the trispecific antibody comprises at least one binding moiety that binds to human CD3 comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 5558; 60; 62 and 66; at least one binding moiety that binds to human BCMA comprising a heavy chain comprising an amino acid sequence that is 99% or 100% identical, wherein the amino acid sequences of SEQ ID NOs: 93, 101, 105, and 108 further comprise the substitution N82aS; and at least one binding moiety that binds to human CD38 comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122, and 111. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0186] More specifically, the trispecific antibody or antibody fragment thereof of the present invention comprises at least one binding moiety that binds to human CD3, the heavy chain comprising a heavy chain variable region comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; at least one binding moiety that binds to human BCMA, comprising a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NOs: 110 and 113. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region of a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0187] More specifically, the trispecific antibodies or antibody fragments thereof of the present invention comprise at least one binding moiety that binds to human CD3, the heavy chain comprising a heavy chain variable region comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; at least one binding moiety that binds to human BCMA, comprising a heavy chain variable region of a heavy chain comprising an amino acid sequence that is 9% or 100% identical, wherein the amino acid sequence of SEQ ID NO: 108 further comprises the mutation N82aS; and at least one binding moiety that binds to human CD38, comprising a heavy chain variable region of a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110 and 113. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region of a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0188] Even more specifically, the trispecific antibody or antibody fragment thereof of the invention comprises at least one binding moiety that binds to human CD3 comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and at least one binding moiety that binds to human BCMA, comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NO: 110 and 113, preferably SEQ ID NO: 113. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0189] Even more specifically, the trispecific antibody or antibody fragment thereof of the invention comprises at least one binding moiety that binds to human CD3 comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; or at least one binding moiety that binds to human BCMA comprising a heavy chain comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NO: 110 and 113, preferably SEQ ID NO: 113. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0190] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the invention comprises at least one binding moiety that binds to human CD3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding moiety that binds to human BCMA, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 108; at least one binding moiety that binds to human CD38, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 113; and a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0191] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the invention comprises at least one binding moiety that binds to human CD3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding moiety that binds to human BCMA, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 108, wherein the amino acid sequence of SEQ ID NO: 108 further comprises the mutation N82aS; and at least one binding moiety that binds to human CD38, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 113; and a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0192] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the invention comprises at least one binding moiety that binds to human CD3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding moiety that binds to human BCMA, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 108; and at least one binding moiety that binds to human CD38, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 110; and a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0193] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the invention comprises at least one binding moiety that binds to human CD3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding moiety that binds to human BCMA, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 108, wherein the amino acid sequence of SEQ ID NO: 108 further comprises the mutation N82aS; and at least one binding moiety that binds to human CD38, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 110; and a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0194] In a more particular embodiment of the invention, the trispecific antibody comprises at least one binding moiety that binds to human CD3 comprising a heavy chain variable region comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 592; at least one binding moiety that binds to human BCMA comprising a heavy chain variable region comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NO: 593 and 594, preferably SEQ ID NO: 594. In a more particular embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region of a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0195] The present invention also relates to an antibody or antigen-binding fragment that binds to an epitope on human CD3, wherein the epitope on CD3 comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:726.

[0196] In particular, the present invention also relates to an antibody or antibody fragment thereof or antigen-binding fragment that binds to the same epitope on human CD3 as the reference antibody or antibody fragment thereof, comprising at least one binding moiety that binds to human CD3, one binding moiety that binds to human BCMA, and one binding moiety that binds to CD38, while the antibody binds to human CD3, human BCMA, and human CD38 and the reference antibody is a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, at least one of which binds to human CD38, and at least one of which binds to human CD3. the at least one binding moiety that binds to BCMA comprises a set of heavy chain CDRs comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and the at least one binding moiety that binds to human CD38 comprises a set of heavy chain CDRs comprising SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491, and a set of light chain CDRs comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0197] The present invention also relates to an epitope on human CD3 comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 726.

[0198] The present invention also relates to an antibody or antigen-binding fragment that binds to an epitope on human CD38, the epitope on CD38 comprising residues Glu103, Gln107, Thr114, Thr116, Arg194, Arg195, Glu198, Ala199, Asp202, Ser224, His228, Asn229, Gln231, Pro232, Glu233, Lys234, Val235, Gln236, Ile265, Ser267, Lys268, Arg269, and Asn270 as detected by X-ray crystallography. In particular, the resolution as detected by X-ray crystallography is at least 5 Å, preferably at least Å, and even more preferably at least 3.5 Å. In a most preferred example, the resolution is about 3.4 Å.

[0199] The present invention also relates to an epitope on human CD38, comprising residues Glu103, Gln107, Thr114, Thr116, Arg194, Arg195, Glu198, Ala199, Asp202, Ser224, His228, Asn229, Gln231, Pro232, Glu233, Lys234, Val235, Gln236, Ile265, Ser267, Lys268, Arg269, and Asn270, as detected by X-ray crystallography. In particular, the resolution as detected by X-ray crystallography is at least 5 Å, preferably at least Å, and even more preferably at least 3.5 Å. In a most preferred example, the resolution is about 3.4 Å.

[0200] The present invention also provides full-length antibodies, as well as antibody fragments and / or binding portions that bind to human CD3 and / or human BCMA and / or human CD38. Antibody fragments include, but are not limited to, (i) Fab fragments, including Fab' and Fab'-SH, consisting of the VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of the VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable domain (Ward ES et al., (1989) Nature, 341:544-546); (v) F(ab')2 fragments, which are bivalent fragments comprising two linked Fab fragments; and (vi) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that enables the two domains to bind to form an antigen-binding site (Bird RE et al., (1988) Science, 242:423-426; Huston JS et al., (1989) Science, 242:423-426). al., (1988) Proc. Natl. Acad. Sci. USA, 85:5879-83), (vii) bispecific single-chain Fv dimers (International Application PCT / US92 / 09965), (viii) "diabodies" or "triabodies," which are multivalent or multispecific fragments constructed by genetic fusion (Tomlinson I & Hollinger P (2000) Methods Enzymol. 326:461-79, WO 94 / 13804, Holliger P et al., (1993) Proc. Natl. Acad. Sci. USA, 90:6444-48), and (ix) scFvs genetically fused to the same or different antibodies (Coloma MJ & Morrison SL (1997) Nature Biotechnology, 15(2):159-163).

[0201] In certain embodiments of the invention, the trispecific antibody is a full-length antibody or antibody fragment thereof, wherein at least one binding moiety that binds human CD3 and / or at least one binding moiety that binds human BCMA and / or at least one binding moiety that binds human CD38 is an antibody fragment, such as a Fab region. As used herein, the term "Fab" or "Fab region" or "Fab domain" encompasses a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to this region alone or to this region in a full-length antibody or antibody fragment.

[0202] In certain embodiments of the invention, the trispecific antibody comprises at least one binding moiety that binds human CD3 and at least one binding moiety that binds human BCMA fused to each other, or at least one binding moiety that binds human CD3 and at least one binding moiety that binds human CD38 fused to each other, or at least one binding moiety that binds human BCMA and at least one binding moiety that binds human CD38 fused to each other.

[0203] In a specific embodiment, the trispecific antibody has at least one binding moiety that binds human BCMA fused N-terminally to the C-terminus of at least one binding moiety that binds human CD3. In another embodiment, the trispecific antibody has at least one binding moiety that binds human BCMA fused N-terminally to the C-terminus, and said at least one binding moiety binds human CD38.

[0204] In another embodiment, a trispecific antibody has at least one binding moiety that binds human BCMA fused at its C-terminus to the N-terminus of at least one binding moiety that binds human CD3. In a further embodiment, a trispecific antibody of the invention has at least one binding moiety that binds human BCMA fused at its C-terminus to the N-terminus, and at least one binding moiety that binds human CD38.

[0205] In another embodiment, a trispecific antibody has at least one binding moiety that binds human CD3 fused C-terminally to the N-terminus of at least one binding moiety that binds human CD38. In a further embodiment, a trispecific antibody of the invention has at least one binding moiety that binds human CD3 fused N-terminally to the C-terminus, said at least one binding moiety that binds human CD38.

[0206] In certain embodiments, the binding moieties are fused to one another via a linker, such as a peptide linker.

[0207] In a particular embodiment, the binding portion of the trispecific antibody of the invention is a Fab fragment.

[0208] In an even more specific embodiment, an antibody of the invention has three binding moieties that are Fab fragments, and the binding moiety that binds human BCMA is fused at its C-terminus to the N-terminus of the Fab heavy or light chain of the binding moiety that binds human CD3, or the binding moiety that binds human BCMA is fused at its C-terminus to the N-terminus of the Fab heavy or light chain of the binding moiety that binds human CD38.

[0209] In an even more specific embodiment, an antibody of the invention has three binding moieties that are Fab fragments, and the binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of the Fab heavy or light chain of the binding moiety that binds human CD3, or the binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of the Fab heavy or light chain of the binding moiety that binds human CD38.

[0210] In a particular embodiment of the invention, the binding moieties of the antibodies fused as described above are fused via a peptide linker. Preferably, the linker has a sequence selected from SEQ ID NOs: 604 and 605. Other preferred peptide linkers are listed in the section "Linkers" below.

[0211] Preferably, the trispecific antibody according to the present invention is selected from the group consisting of SEQ ID NO:522, SEQ ID NO:523 and SEQ ID NO:1; SEQ ID NO:530, SEQ ID NO:531 and SEQ ID NO:1; SEQ ID NO:532, SEQ ID NO:533 and SEQ ID NO:1; SEQ ID NO:534, SEQ ID NO:535 and SEQ ID NO:1; SEQ ID NO:536, SEQ ID NO:537 and SEQ ID NO:1; SEQ ID NO:538, SEQ ID NO:539 and SEQ ID NO:1; SEQ ID NO:540, SEQ ID NO:541 and SEQ ID NO:1; SEQ ID NO:542, SEQ ID NO:543 and SEQ ID NO:1 1; SEQ ID NO:544, SEQ ID NO:545 and SEQ ID NO:1; SEQ ID NO:546, SEQ ID NO:547 and SEQ ID NO:1; and SEQ ID NO:548, SEQ ID NO:549 and SEQ ID NO:1.

[0212] Preferably, the present invention relates to a trispecific antibody comprising a set of three amino acid chains that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising: SEQ ID NO:546, SEQ ID NO:547 and SEQ ID NO:1; and SEQ ID NO:548, SEQ ID NO:549 and SEQ ID NO:1.

[0213] In a more preferred embodiment, the trispecific antibody or antibody fragment thereof according to the invention comprises a set of three amino acid chains of the amino acid sequences of SEQ ID NO: 546, SEQ ID NO: 547 and SEQ ID NO: 1.

[0214] nucleic acid Standard recombinant DNA methods are used to construct polynucleotides encoding polypeptides that form binding proteins, to incorporate these polynucleotides into recombinant expression vectors, and to introduce such vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the terms used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Similarly, standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0215] Another aspect of the present disclosure relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecule comprises a sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence.

[0216] Certain aspects of the present disclosure relate to kits of polynucleotides. In some embodiments, one or more of the polynucleotides is a vector (e.g., an expression vector). The kit may be useful, inter alia, for producing one or more of the binding proteins described herein, e.g., a bispecific or trispecific binding protein of the present disclosure. In some embodiments, the kit includes one, two, three, or four polynucleotides.

[0217] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to induce transcription of the nucleic acid sequence encoding the binding protein. A promoter can refer to a nucleic acid control sequence that induces transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence. Examples of promoters include promoters derived from the genomes of viruses (e.g., polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, polyomavirus, e.g., simian virus 40 (SV40)), heterologous eukaryotic promoters (e.g., actin promoter, immunoglobulin promoter, heat shock promoter), cytomegalovirus immediate early enhancer / chicken β-actin CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK) promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), the lac system, the tip system, the tac system, the trc system, promoters from the major operator and promoter regions of λ phage, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast α-mating factor. A polynucleotide encoding a binding protein of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein, or other suitable promoters readily recognized by one of ordinary skill in the art.

[0218] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. An expression vector can include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" encompasses promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences derived from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin, etc.) and enhancer sequences derived from eukaryotic viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral vectors, vaccinia viral vectors, Sindbis viral vectors, measles viral vectors, herpes viral vectors, lentiviral vectors, retroviral vectors, adeno-associated viral vectors, etc.). Expression vectors may be used to transfect host cells such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and may be used to transfect any cell of interest.

[0219] Another aspect of the present disclosure relates to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system can be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors.

[0220] Isolated host cells Another aspect of the present disclosure relates to an isolated host cell comprising one or more isolated polynucleotides, polynucleotide kits, vectors, and / or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells can include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture, and their mutant 293T), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary carcinoma cells (e.g., MMT 060562, ATCC CCL51), TR1 cells, MRC5 cells, FS4 cells, human liver cancer lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0221] Another aspect of the present disclosure relates to methods of producing any of the binding proteins described herein. In some embodiments, the methods include a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any of the isolated nucleic acids, vectors, and / or vector systems described herein) under conditions such that the host cell expresses the binding protein, and b) isolating the binding protein from the host cell.

[0222] Methods for culturing host cells under conditions for protein expression are well known to those skilled in the art. Cell culture is the process of growing cells under controlled conditions in an artificial environment. The terms "cell culture" or "culture" or "host cell culture" refer to the growth and / or propagation and / or maintenance of cells under controlled artificial conditions. Optimal culture conditions are obtained by controlling and adjusting several parameters, including the composition of the cell culture medium, the operating parameters of the bioreactor, the nutrient supply mode, and the culture duration. The composition of the culture medium is optimized for favorable cell vitality and regeneration, and examples of cell culture medium components include, but are not limited to, essential amino acids, salts, glucose, growth factors, and antibiotics. Important bioreactor operating parameters are temperature, pH, agitation rate, oxygenation, and carbon dioxide levels. Nutrients can be supplied in various ways. In batch mode culture, all necessary nutrients are present in an initial basal medium and are used until exhausted while waste products accumulate; in fed-batch culture, additional feed medium is supplied to prevent nutrient depletion and extend the culture; alternatively, in perfusion mode, cells in culture are continuously replenished with fresh medium containing nutrients that flows through the bioreactor, removing cellular waste products. Those skilled in the art will understand that the culture period is important as it must be long enough to allow the cells to produce consistent amounts of product, but cannot be too long so as to compromise cell viability.

[0223] Methods for isolating proteins from cultured host cells are well known to those skilled in the art and include, for example, chromatography techniques. The term "chromatography" refers to the separation of compounds of a mixture based on their ability to interact with the stationary phase of a chromatography resin, from which they can be retained or eluted. Chromatography techniques are known in the art; for example, ion exchange chromatography separates ions and polar molecules based on charge differences; examples of ion exchange chromatography techniques are cation exchange chromatography and anion exchange chromatography. Affinity chromatography relies on the specific interaction of a protein with an immobilized ligand. Non-limiting examples of ligands useful for purifying antibodies by affinity chromatography are Protein A and Protein G. Protein A is a cell wall protein isolated from Staphylococcus aureus and has the property of binding to immunoglobulin Fc (but not to the antigen-binding site). As used herein, the term "protein A chromatography" refers to affinity chromatography in which the ligand that binds to an antibody is protein A, and the term "protein A" includes native protein A, recombinant protein A, and analogs or derivatives thereof. Both protein analogs and derivatives retain their binding activity for antibodies (e.g., IgG Fc). Protein G is a cell wall protein isolated from group G Streptococcus, whose N-terminal portion is an albumin-binding domain and whose C-terminal portion is an IgG-binding domain and a cell wall-binding domain. As used herein, the term "protein G chromatography" refers to affinity chromatography in which the ligand that binds to an antibody is protein G, and the term "protein G" includes native protein G, recombinant protein G, and analogs or derivatives thereof. Methods for isolating proteins from cultured host cells can include an affinity chromatography step (e.g., protein A affinity chromatography) followed by one or more steps of ion exchange chromatography and / or size exclusion chromatography.

[0224] antibody structure In certain embodiments of the invention, the binding moieties of a trispecific antibody or antibody fragment thereof are fused to one another (i.e., connected together). In certain embodiments, at least one binding moiety that binds to human CD3 and at least one binding moiety that binds to human BCMA, or at least one binding moiety that binds to human CD3 and at least one binding moiety that binds to human CD38, or at least one binding moiety that binds to human BCMA and at least one binding moiety that binds to human CD38 are fused to one another. In more particular embodiments, at least one binding moiety that binds to human CD3 and at least one binding moiety that binds to human BCMA, or at least one binding moiety that binds to human CD38, or at least one binding moiety that binds to human BCMA and at least one binding moiety that binds to human CD38 are fused to one another via a linker, e.g., a peptide linker.

[0225] In certain embodiments, the binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of the binding moiety that binds human CD3. In other embodiments, the binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of the binding moiety that binds human CD38. In other embodiments, the binding moiety that binds human BCMA is fused at its C-terminus to the N-terminus of the binding moiety that binds human CD3 ...8.

[0226] In certain embodiments, the binding moiety that binds human CD3 is fused at its N-terminus to the C-terminus of the binding moiety that binds human BCMA. In other embodiments, the binding moiety that binds human CD3 is fused at its N-terminus to the C-terminus of the binding moiety that binds human CD38. In other embodiments, the binding moiety that binds human CD3 is fused at its C-terminus to the N-terminus of the binding moiety that binds human BCMA. In other embodiments, the binding moiety that binds human CD3 is fused at its C-terminus to the N-terminus of the binding moiety that binds human CD38.

[0227] In certain embodiments, the binding moiety that binds human CD38 is fused at its N-terminus to the C-terminus of the binding moiety that binds human BCMA. In other embodiments, the binding moiety that binds human CD38 is fused at its N-terminus to the C-terminus of the binding moiety that binds human CD3. In other embodiments, the binding moiety that binds human CD38 is fused at its C-terminus to the N-terminus of the binding moiety that binds human BCMA. In other embodiments, the binding moiety that binds human CD38 is fused at its C-terminus to the N-terminus of the binding moiety that binds human CD3.

[0228] In preferred embodiments, a binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of a binding moiety that binds human CD3, or a binding moiety that binds human BCMA is fused at its C-terminus to the N-terminus of a binding moiety that binds human CD38.

[0229] In a preferred embodiment, the binding moieties linked as described above are fused to each other via a linker, eg, a peptide linker.

[0230] In preferred embodiments, the binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of the binding moiety that binds human CD3 via a peptide linker, or the binding moiety that binds human BCMA is fused at its C-terminus to the N-terminus of the binding moiety that binds human CD38 via a peptide linker.

[0231] In an even more preferred embodiment, the binding moiety that binds human BCMA is fused at its N-terminus to the C-terminus of the binding moiety that binds human CD3 via the peptide linker of SEQ ID NO: 605, or the binding moiety that binds human BCMA is fused at its C-terminus to the N-terminus of the binding moiety that binds human CD38 via the peptide linker of SEQ ID NO: 604.

[0232] In certain embodiments of the invention, the binding portion of the antibodies disclosed herein is an antibody fragment selected from the non-limiting list of "antibody fragments" above. In more particular embodiments, the binding portion that binds to human CD3 and / or the binding portion that binds to human BCMA and / or the binding portion that binds to human CD38 is a Fab domain.

[0233] In certain embodiments, antibodies of the invention comprise an Fc domain and at least three binding moieties. In more particular embodiments, at least a first binding moiety is fused at its N-terminus to the C-terminus of a second binding moiety. In certain aspects, a first binding moiety, e.g., a Fab domain, located between the antibody Fc domain and a second binding moiety, e.g., a second Fab domain, is referred to as "Fc-proximal," and the second binding moiety is referred to as "Fc-distal."

[0234] In certain embodiments, the second binding moiety, eg, an Fc distal binding moiety, is fused to the N-terminus of the VH (variable heavy chain) domain of the first binding moiety, eg, an Fc proximal binding moiety.

[0235] In preferred embodiments, the binding moiety that binds CD38 is Fc-proximal and the binding moiety that binds CD3 or BCMA is Fc-distal, preferably the binding moiety that binds CD38 is proximal and the binding moiety that binds BCMA is Fc-distal. In preferred embodiments, the binding moiety that binds BCMA is Fc-proximal and the binding moiety that binds CD3 or the binding moiety that binds CD38 is Fc-distal, more preferably the binding moiety that binds BCMA is Fc-proximal and the binding moiety that binds CD3 is Fc-distal.

[0236] In a specific embodiment of the present invention, a trispecific antibody or antibody fragment thereof of the present invention comprises three binding moieties, one that binds to human CD3, one that binds to human BCMA, and one that binds to human CD38, wherein the three binding moieties are Fab fragments, and the antibody is constructed using the TREAT® trispecific antibody technology based on the BEAT® platform described above. In a specific embodiment, the binding moiety that binds to human BCMA and the binding moiety that binds to human CD38 are located on the same arm of the BEAT antibody, more specifically, the binding moiety that binds to the human BCMA binding arm is in the Fc-distal position, and the binding moiety that binds to human CD38 is in the Fc-proximal position. In a more specific embodiment, the binding moiety that binds to human BCMA and the binding moiety that binds to human CD38 are fused via a flexible linker, and preferably comprise the amino acid sequence of SEQ ID NO: 604. In a preferred specific embodiment, the binding moiety that binds to human CD3 and the binding moiety that binds to human BCMA are located on the same arm of the BEAT antibody, more specifically, the binding moiety that binds to the human CD3 binding arm is in the Fc-distal position, and the binding moiety that binds to human BCMA is in the Fc-proximal position. In a more particular embodiment, the binding moiety that binds human CD3 and the binding moiety that binds human BCMA are fused via a flexible linker, and preferably comprise the amino acid sequence of SEQ ID NO: 605.

[0237] Linker In some embodiments, linkers of the invention, such as L1, L2, L3, and L4, range in length from zero amino acids (length = 0) to about 100 amino acids, or up to 100, 50, 40, 30, 20, or 15 amino acids. Linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. L1, L2, L3, and L4 in a single binding protein can all have the same amino acid sequence, or they can all have different amino acid sequences.

[0238] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues; a peptide with five glycine residues; a peptide with six glycine residues; a peptide with seven glycine residues; and a peptide with eight glycine residues. Other combinations of amino acid residues can be used, such as the peptides GGGT or GGGGS, or repeats of the above peptides, such as the peptide GGGGS GGGGS GGGGS. In a preferred embodiment, the linker of the invention connecting different binding moieties has the amino acid sequence of SEQ ID NO: 604 or 605, or the amino acid sequence of SEQ ID NO: 604 or 605 containing one or more conservative substitutions thereof.

[0239] The above examples are not intended to limit the scope of the present disclosure in any way, and show that linkers containing randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline are suitable for binding proteins. For further description of linker sequences, see, for example, WO2012135345 and PCT / US2017 / 027488.

[0240] The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structure element that needs to be achieved in the linker. For example, glycine, serine, and alanine are best suited for linkers with maximum flexibility. Some combinations of glycine, proline, threonine, and serine are useful when a more rigid and extended linker is required. Any amino acid residue can be considered as a linker, combined with other amino acid residues to construct a longer peptide linker, depending on the desired properties and other needs.

[0241] Fc region and constant domains The present invention also relates to trispecific antibodies comprising an Fc region. In some embodiments, the binding proteins of the present disclosure comprise antibody fragments, including but not limited to, Fab, (Fab')2, Fab'-SH, Fv, or scFv fragments of antibodies. In some embodiments, the binding proteins of the present disclosure comprise antibody fragments, including but not limited to, Fab, (Fab')2, Fab'-SH, Fv, or scFv fragments of antibodies, that comprise an Fc region.

[0242] In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region comprises the hinge, CH1, CH2, CH3, and optionally the CH4 domains of an antibody. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG2 Fc region. In some embodiments, the Fc region is a human IgG3 Fc region, and in some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more of the mutations described herein.

[0243] In certain embodiments, the trispecific antibodies or antibody fragments thereof of the invention comprise a non-naturally occurring Fc domain.

[0244] In some embodiments, the binding proteins of the present disclosure comprise one or two Fc variants. As used herein, the term "Fc variant" refers to a molecule or sequence that has been modified from a native Fc but still contains the binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can encompass molecules or sequences that have been humanized from a non-human native Fc. Additionally, native Fcs contain regions that can be removed because they provide structural features or biological activity not required for the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack or have altered one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0245] As used herein, the term "parent antibody" or "parent immunoglobulin" encompasses unmodified antibodies that are subsequently modified to generate variants. The parent antibody may be a naturally occurring antibody, a non-naturally occurring antibody, or a variant or genetically engineered version of a naturally occurring antibody. The parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or the amino acid sequence encoding it. In preferred embodiments of the present invention, the parent antibody comprises an Fc region. More specifically, the Fc region of the parent antibody according to the present invention is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region of the parent antibody according to the present invention is a modified or unmodified IgG1 Fc region.

[0246] In certain embodiments, the antibodies of the invention are heterodimeric antibodies or antibody fragments thereof with engineered Fc comprising first and second engineered CH3 domains, wherein the first engineered CH3 domain comprises a substitution that promotes heterodimerization. In certain embodiments of the invention, the antibodies are constructed using the aforementioned BEAT® heavy chain (He) heterodimerization technology (Skegro et al., (2017) J Biol Chem 292(23):9745-9759 and Stutz et al., (2020) J Biol Chem 295(28):9392-9408, WO 2012131555), wherein the BEAT(A) chain, also referred to herein as BEAT(A), comprises a first engineered CH3 domain, and the BEAT(B) chain, also referred to herein as BEAT(B), comprises a second engineered CH3 domain.

[0247] In a more particular embodiment, the first engineered CH3 domain comprises one or more substitutions selected from the group comprising: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and said second engineered CH3 domain comprises a substitution from the group comprising: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and in a particular aspect, the heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via said first and second engineered CH3 domains.

[0248] In a more particular embodiment, the present invention discloses a trispecific antibody or antibody fragment thereof constructed based on the BEAT® platform, wherein the BEAT(A) CH3 domain comprises one or more substitutions selected from the group comprising Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and optionally the BEAT(A) CH3 domain comprises one or more substitutions selected from the group comprising D356E, L358M, N384S, V397M, V42 and the BEAT(B) CH3 domain further comprises a substitution selected from the group comprising Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and optionally BEAT(b) further comprises the substitution D401Q (EU numbering).

[0249] In certain aspects, the present invention also relates to a trispecific heterodimeric antibody or antibody fragment thereof comprising a first and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises substitutions from the group comprising Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises substitutions from the group comprising Q347E, Y349A, L351F, S364T , T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), wherein the trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding moieties, each binding to a different antigen, and wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via the first and second engineered CH3 domains.

[0250] In certain embodiments, bispecific antibodies of the invention comprise a variant Fc region that comprises at least one amino acid modification compared to the Fc region of a parent antibody, such that the antibody comprising the variant Fc region exhibits altered effector function compared to the parent antibody. More specifically, the CH2 domain of the Fc region comprises at least one amino acid modification.

[0251] In a specific embodiment of the present invention, BEAT(A) and BEAT(B) are engineered to increase Fc effector function. More specifically, the CH2 domain of the Fc region is engineered to contain at least one amino acid modification. More specifically, BEAT(A) contains one or more substitutions at positions selected from the group consisting of 324, 334, 269, 298, 239, 332, and 333, and BEAT(B) contains one or more substitutions at positions selected from the group consisting of 324, 334, 269, 298, 239, 332, and 333, preferably at positions 324, 334, 269, 289, 298, and 333. Even more specifically, BEAT(A) comprises one or more substitutions selected from the group comprising S324N, K334E, K334A, E269D, S298A, S239D, I332E, and E333A, and BEAT(B) comprises one or more substitutions at positions selected from the group comprising S324N, K334E, K334A, E269D, S289A, K334A, E333A. In certain specific embodiments, BEAT(A) comprises a set of mutations selected from the group comprising: S324N; or S324N and K334E; or E269D, S298A, S324N, and K334A; or S239D, I332E, and S324N; or E269D, S298A, S324N, and E334A; or S298A, S324N, and E333A; or S298A, S324N, and K334A; or S324N, S298A, E269D, and E333A; or S324N, S298A, E269D, and K334A. In other specific embodiments, BEAT(B) comprises a set of mutations selected from the group comprising: S324N; or S324N and K334E; or E269D, S298A, S324N, and K334A; or S239D, I332E, and S324N; or E269D; or E269D, S298A, S324N, and E334A; or S298A, S324N, and E333A; or S298A, S324N, and K334A; or S324N, S298A, E269D, and E333A; or S324N, S298A, E269D, and K334A.In certain preferred embodiments, BEAT(A) and BEAT(B) comprise an S324N mutation; or an S324N and a K334E mutation; or an E269D, S298A, S324N, and a K334A mutation; or an E269D, S298A, S324N, and an E334A mutation; or an S298A, S324N, and an E333A mutation; or an S298A, S324N, and a K334A mutation; or an S324N and a K334E mutation. In a particularly preferred embodiment, BEAT(A) comprises an S239D, an I332E, and an S324N mutation, and BEAT(B) comprises an S324N mutation.

[0252] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or effector functions of the Fc region (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)).

[0253] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0254] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcyI and / or FcyII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcyI and / or FcyII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to position 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233 to 236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at position 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233 to 236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at position 236; and / or R409K.

[0255] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve purification, for example, by adjusting affinity for purification reagents. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms when one of the two Fc regions of the heterodimeric form contains a mutation(s) that reduces or eliminates binding to Protein A, because the heterodimeric form has intermediate affinity for Protein A-based purification compared to either homodimeric form and can be selectively eluted from Protein A, for example, by using a different pH (see, e.g., Smith, EJ. et al. (2015) Sci. Rep. 5:17943). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region immunoglobulin heavy chain constant domains CH2 and CH3, wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being H435R and Y436F.

[0256] To improve the yield of some binding proteins (e.g., bispecific or trispecific binding proteins), the CH domain can be modified by BEAT technology, which is described in detail with some examples in WO2012131555.

[0257] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to improve serum half-life (see, e.g., Hinton, P R. et al. (2006) J. Immunol. 176(1):346-56). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and / or second Fc region comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being M428L and N434S. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region.

[0258] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to reduce effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and second Fc regions are human IgG1 Fc regions, and each of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, wherein each Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and second Fc regions are human IgG1 Fc regions, and each of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A.In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, wherein each Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, wherein each Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

[0259] In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations to reduce effector function. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region. For further description of Fc mutations at position 329, see, e.g., Shields, R. Let al. (2001) J. Biol. Chem. 276:6591-6604 and WO 1999051642.

[0260] In a specific embodiment, a trispecific antibody or antibody fragment thereof according to the present invention comprises a non-naturally occurring Fc domain comprising L234A / L235A(LALA) / P329A substitutions into the CH2 domain. More specifically, a trispecific antibody or antibody fragment thereof disclosed herein may comprise an Fc, wherein the Fc may comprise first and second BEAT CH3 domains as described above, or a naturally occurring CHE domain, or any other non-naturally occurring CH3 and CH2 domains as described above or comprising L234A / L235A(LALA) / P329A substitutions.

[0261] Binding Protein Applications The antibodies or antibody fragments thereof of the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, for the detection and quantitation of one or more target antigens. The binding proteins bind to one or more target antigens with an affinity that is specific to the assay method being used.

[0262] For diagnostic applications, in certain embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety can be any moiety that can generate a detectable signal, either directly or indirectly. For example, the detectable moiety can be a radioisotope, such as H, C, P, S, Tc, In, or Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, p-galactosidase, or horseradish peroxidase.

[0263] Binding proteins are also useful for in vivo imaging. Binding proteins labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host can be analyzed. Binding proteins can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0264] For clinical or research applications, in certain embodiments, the binding protein may be conjugated to a cytotoxic agent. Various antibodies conjugated to cytotoxic agents (i.e., antibody-drug conjugates) have been used to target cytotoxic payloads to specific tumor cells. Cytotoxic agents and linkers for conjugating drugs to antibodies are known in the art. See, for example, Parslow, A. C. et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

[0265] The present disclosure also relates to kits containing binding proteins and other reagents useful for detecting target antigen levels in biological samples. Such reagents may include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kits include a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kits include 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, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective by itself for treating, preventing, and / or diagnosing a condition, or that is to be combined with another composition that is effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an IV solution bag or vial with a stopper pierceable by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and / or treating the condition of choice. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0266] In some embodiments, the binding proteins of the present disclosure are administered to a patient in need thereof for the treatment or prevention of cancer. In some embodiments, the disclosure relates to methods for preventing and / or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of a heterodimeric antibody described herein or a pharmaceutical composition related thereto. In some embodiments, the disclosure relates to the use of a heterodimeric antibody described herein or a pharmaceutical composition related thereto for preventing and / or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the disclosure relates to a heterodimeric antibody described herein or a pharmaceutical composition related thereto for use in the manufacture of a medicament for preventing and / or treating a proliferative disease or disorder (e.g., cancer) in a patient in need thereof. In some embodiments, the patient is a human. In some embodiments, the binding protein comprises one antigen-binding site that binds to a T-cell surface protein and another antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide. In some embodiments, the binding protein comprises an antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide, an antigen-binding site that binds to a human CD3 polypeptide, and an antigen-binding site that binds to a human BCMA polypeptide.

[0267] In certain embodiments, the cancer is a BCMA-expressing cancer and / or a CD38-expressing cancer. Examples of cancer include, but are not limited to, multiple myeloma, leukemias such as acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia, acute promyelocytic leukemia, acute myeloblastic leukemia, and acute megakaryoblastic leukemia, chronic myelogenous leukemia, multiple myeloma, plasma cell leukemia, lymphoma, breast cancers such as Her2+ breast cancer, prostate cancer, cervical cancer, germinal center B cell Lymphoma or B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, myelodysplastic syndromes (MDS), breast cancer, e.g., Her2+ breast cancer, prostate cancer, cervical breast cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), high-grade serous ovarian cancer, smoldering myeloma, glioma.

[0268] Therapeutic compositions for binding proteins and their administration Therapeutic or pharmaceutical compositions comprising the binding protein are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of the binding protein or binding protein-drug conjugate mixed with a pharmaceutically or physiologically acceptable compounding agent selected for compatibility with the mode of administration.

[0269] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.

[0270] Pharmaceutical compositions may contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition.Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvent (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohol (e.g., mannitol or sorbitol), suspending agent, surfactant or wetting agent (e.g., pluronic; PEG; sorbitan ester; polysorbate, e.g., polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol, or tyloxapol), stability enhancer (e.g., sucrose or sorbitol), tonicity enhancer (e.g., alkali metal halide, preferably sodium chloride or potassium chloride, or mannitol sorbitol), delivery vehicle, diluent, excipient, and / or pharmaceutical adjuvant (see, e.g., REMINGTON'S See PHARMACEUTICAL SCIENCES (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990) and subsequent editions).

[0271] The optimal pharmaceutical composition can be determined by one of skill in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. Such composition can influence the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of the binding protein.

[0272] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a vehicle or carrier suitable for injection can be water, saline, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in parenteral compositions. Further exemplary vehicles are neutral buffered saline or saline mixed with serum albumin. Furthermore, the binding protein can be formulated as a liquid preparation or as a lyophilizate using appropriate excipients.

[0273] The pharmaceutical compositions of the present disclosure can be selected for intravenous or subcutaneous administration. Alternatively, the compositions can be selected for inhalation or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0274] When parenteral administration is intended, the therapeutic composition used may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable parenteral injection vehicle is sterile distilled water, in which the binding protein is formulated as a sterile, isotonic solution and appropriately maintained. Yet another preparation may involve formulating the molecule of interest with an agent that provides controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, and then the product may be delivered by depot injection. Hyaluronic acid may also be used, which may have the effect of enhancing the duration of circulation. Other suitable means for introducing the molecule of interest include implantable drug delivery devices.

[0275] In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized.

[0276] It is also contemplated that certain formulations may be administered orally. In one embodiment of the present disclosure, binding proteins administered in this manner may be formulated with or without carriers customarily used in formulating solid dosage forms, such as tablets and capsules. For example, capsules may be designed to release the active portion of the formulation at a location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to promote absorption of the binding protein. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[0277] Another pharmaceutical composition may contain an effective amount of the binding protein in a mixture with non-toxic excipients suitable for tablet manufacture. A unit-dose solution may be prepared by dissolving the tablet in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0278] Additional pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including formulations comprising the binding protein in sustained- or controlled-delivery formulations. Techniques for constructing various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0279] Pharmaceutical compositions used for in vivo administration must usually be sterile. Sterility can be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method can be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are usually placed into a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0280] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0281] The present disclosure also encompasses kits for producing single-dose administration units. Each kit may include both a first container with a dried protein and a second container with an aqueous formulation. Kits containing single- or multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also within the scope of the present disclosure.

[0282] The effective amount of a pharmaceutical composition of a binding protein used for treatment will depend, for example, on the therapeutic situation and purpose. Thus, one of skill in the art will understand that appropriate dosage levels for treatment will vary depending, in part, on the molecule being delivered, the indication for which the binding protein is being used, the route of administration, and the size (weight, body surface area, or organ size) and / or condition (age and health) of the patient. Thus, a clinician can titer the dosage and modify the route of administration to obtain optimal therapeutic effect.

[0283] The frequency of administration depends on the pharmacokinetic parameters of the binding protein in the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition can be administered as a single dose, as two or more doses over time (which may or may not contain the same amount of the molecule of interest), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of tasks routinely performed by those skilled in the art. The appropriate dosage can be ascertained using appropriate dose-response data.

[0284] The route of administration of the pharmaceutical composition is in accordance with known methods, for example, by injection via oral, subcutaneous, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes, by sustained release system, or by implantation device. If desired, the composition can be administered by bolus injection or continuously by infusion, or by implantation device.

[0285] The compositions may be administered locally by implantation of a membrane, sponge, or another suitable material into which the molecule of interest has been absorbed or encapsulated. When an implantable device is used, the device may be implanted into any suitable tissue or organ, and delivery of the molecule of interest may be by diffusion, slow release bolus, or continuous administration.

[0286] Treatment regimen The present disclosure also provides methods of treating multiple myeloma in a subject in need thereof. The methods use heterodimeric antibodies that engage CD3, BCMA, and CD38 in a manner that transiently connects malignant cells with committed T cells, thereby inducing T cell-mediated killing of the bound malignant cells. The methods described herein utilize heterodimeric antibodies that bind CD3, BCMA, and CD38 in a manner that maximizes destruction of target cells while reducing undesirable side effects (e.g., uncontrolled cytokine release).

[0287] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. The antibodies of the present invention are administered to a subject in need thereof, including patients with a disorder and patients susceptible to a disorder, or patients in whom a disorder is to be prevented. The antibodies of the present invention can be used to treat humans with or susceptible to cancer, or to ameliorate cancer in human subjects, as well as to prevent cancer in human patients. The antibodies of the present invention, or antibody fragments thereof, are administered to a subject in need thereof, for example, a human subject suffering from a BCMA-expressing cancer and / or a CD38-expressing cancer, such as multiple myeloma, e.g., relapsed / refractory multiple myeloma. Relapsed myeloma is characterized by the recurrence of disease after successful prior treatment. Examples of laboratory and radiological criteria indicative of disease include, but are not limited to, a greater than 25% increase in serum or urinary monoclonal protein (M protein) from nadir, or a greater than 25% difference between tumor-derived and non-tumor-derived serum free light chains, or the development of new plasmacytoma or hypercalcemia. Sonneveld et al., Haematologica. 2016 Apr;101(4):396-406.

[0288] In patients with non-secretory disease, relapse is characterized by an increase in bone marrow plasma cells. Signs of recurrent disease are also characterized by the appearance or reappearance of one or more CRAB criteria or a rapid and consistent biochemical relapse. Refractory myeloma is myeloma for which treatment is ineffective. Relapsed / refractory multiple myeloma refers to disease that becomes ineffective or progressive during treatment or within 60 days of the last treatment in patients who previously achieved at least a minimal response to prior therapy. Sonneveld (supra); Anderson et al., Leukemia. 2008;22(2):231-239.

[0289] The methods of the present disclosure may involve administering a dose of about 0.0001 mg / kg to about 200 mg / kg, for example, about 0.0005 mg / kg to about 200 mg / kg, or about 0.001 mg / kg to about 200 mg / kg, or about 0.0015 mg / kg to about 200 mg / kg, or about 0.005 mg / kg to about 200 mg / kg, or about 0.01 mg / kg to about 200 mg / kg, or about 0.015 mg / kg to about 200 mg / kg, or about 0.03 mg / kg to about 200 mg / kg, or about 0.05 mg / kg to about 200 mg / kg, or about 0.06 mg / kg to about 200 mg / kg, or about 0.09 mg / kg to about 200 mg / kg, or about 0.1 mg / kg to about 200 mg / kg. g / kg, or about 0.12 mg / kg to about 200 mg / kg, or about 0.15 mg / kg to about 200 mg / kg, or about 0.18 mg / kg to about 200 mg / kg, or about 0.2 mg / kg to about 200 mg / kg, or about 0.3 mg / kg to about 200 mg / kg, or about 0.36 mg / kg to about 200 mg / kg, or about 0.5 mg / kg to about 200 mg / kg, or about 0.6 mg / kg to about 200 mg / kg, or about 0.7 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 200 mg / kg, or about 1.2 mg / kg to about 200 mg / kg, or about 1.5 mg / kg to about 200 mg / kg. For example, about 0.0001 mg / kg to about 20 mg / kg, or about 0.0005 mg / kg to about 10 mg / kg, or about 0.0005 mg / kg to about 5 mg / kg, or about 0.0005 mg / kg to about 1.5 mg / kg, or about 0.0005 mg / kg to about 1.2 mg / kg. The present disclosure also includes dosage ranges of any value included between the intervals disclosed above.For example, trispecific antibodies of the invention may have a concentration of at least about 0.0001 mg / kg, at least about 0.0005 mg / kg, at least about 0.001 mg / kg, at least about 0.0015 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.015 mg / kg, at least about 0.03 mg / kg, at least about 0.05 mg / kg, at least about 0.06 mg / kg, at least about 0.09 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.15 mg / kg, at least about 0.18 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, and / or at least about 100 mg / kg, at least about 150 mg / kg, or at least about 200 mg / kg.

[0290] In various embodiments of this method, dosage is adjusted during treatment.For example, a subject is administered an initial dose in one or more administrations, and a higher dose is used in one or more subsequent administrations.In other words, the present disclosure contemplates increasing the dosage of the trispecific antibody at least once over the course of treatment.Alternatively, dosage can be reduced during treatment so that the amount of heterodimeric antibody is reduced as treatment progresses.

[0291] The present disclosure contemplates methods in which repeated (i.e., two or more) doses of a trispecific antibody are administered during the treatment period. Individual doses can be administered at any interval, for example, once a week, twice a week, three times a week, four times a week, or five times a week. Individual doses can be administered once every two weeks, once every three weeks, or once every four weeks. In other words, in some embodiments, a two-week waiting period elapses between administrations of the heterodimeric antibody to a subject. The waiting period between administrations of doses need not be constant throughout the treatment period. In other words, the interval between administrations can be adjusted during the treatment period. In some embodiments, the method comprises administering the heterodimeric antibody to the subject twice weekly during the first and second weeks of treatment (i.e., twice weekly during weeks 1 and 2), administering the heterodimeric antibody to the subject once weekly during the third and fourth weeks of treatment (i.e., once weekly during weeks 3 and 4), and administering the heterodimeric antibody once every two weeks starting at week 5 until the end of treatment (i.e., there is a two-week waiting period between administrations starting at week 5 until the end of treatment).

[0292] Alternatively, in various embodiments, the method comprises administering the trispecific antibody once a week for weeks 1-4 of treatment, and optionally administering the antibody once every two weeks starting at week 5 until the end of treatment.

[0293] Repeated administrations of the heterodimeric antibody are administered over a treatment period of, for example, 3 months to about 18 months, or about 3 months to about 12 months, or about 3 months to about 9 months, or about 3 months to about 6 months, or about 3 months to about 8 months, or about 6 months to about 18 months, or about 6 months to about 12 months, or about 8 months to about 12 months, or about 6 months to about 8 months, or about 8 months to about 12 months (e.g., about 8 months). Optionally, multiple (i.e., two or more) doses of the heterodimeric antibody are administered over a treatment period of about 12 weeks to about 52 weeks, or about 12 weeks to about 36 weeks, or about 24 weeks to about 32 weeks, with doses administered twice a week, once a week, once every two weeks, or once every four weeks.

[0294] "Treating" multiple myeloma refers to achieving any effective response to treatment of the disease. For example, an effective response to treatment includes one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in tumor cell death; (3) an inhibition of neoplastic cell survival; (4) a reduction in paraprotein production by tumor cells; (5) an inhibition of tumor growth (i.e., some delay, preferably cessation, of tumor growth); (6) an increase in patient survival; and (7) some alleviation of one or more symptoms associated with the disease or condition. Anti-tumor effects can be assessed by changes in tumor morphology (i.e., total tumor burden, tumor size, etc.) using screening techniques, such as magnetic resonance imaging (MRI) scans, X-ray imaging, computed tomography (CT) scans, bone scan imaging, endoscopy, and tumor biopsy sampling, including bone marrow aspiration (BMA) and enumeration of circulating tumor cells. A complete response to treatment (i.e., absence of clinically detectable disease with normalization of any previously abnormal radiographic results, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal proteins) is not required; any degree of improvement is contemplated.

[0295] The antibody or antibody fragment thereof can be administered to a subject via any suitable means, for example, intravenously, subcutaneously, intraarterially, intralymphatically, intrathecally, intracerebrally, intraperitoneally, intracerebrospinally, intradermally, intra-articularly, intrasynovially, orally, topically, or via inhalation routes, osmotic pumps, drug encapsulation, e.g., nanoparticle encapsulation, or surface coating-mediated drug delivery. For example, the antibody can be administered as a bolus via intravenous administration or by continuous infusion over a period of time. In certain embodiments, the antibody is administered via intravenous infusion, e.g., over a period of about 30 minutes to about 4 hours. Optionally, the infusion time is decreased for subsequent administrations. For example, in one embodiment, the first administration of the antibody is administered over about 4 hours, and subsequent administrations are administered over a period of 2 hours or less. In this regard, the first administration of the antibody is optionally administered over about 4 hours, the second administration of the antibody is optionally administered over about 2 hours, and subsequent administrations are optionally administered over about 30 minutes. In another specific embodiment, the trispecific antibody of the invention is administered subcutaneously.

[0296] In some cases, the subject has been previously treated for cancer, such as multiple myeloma. For example, the subject may have previously received an immunomodulatory agent (thalidomide, lenalidomide, pomalidomide), a proteasome inhibitor (e.g., pomalidomide, bortezomib, or carfilzomib), dexamethasone, doxorubicin, or a combination thereof.

[0297] Optionally, the subject has been previously treated with an anti-CD38 monospecific antibody, e.g., daratumumab (DARZALEX®). In various embodiments, the subject is relapsed or refractory to prior treatment with an anti-CD38 monospecific antibody. The method may include a waiting period between prior administration of the anti-CD38 monospecific antibody and administration of the heterodimeric antibody.

[0298] Combination therapy Optionally, the antibody of the invention or antibody fragment thereof is part of a treatment regimen that includes administration of one or more other therapeutic agents, radiation therapy, stem cell transplantation, or the like.

[0299] The methods of the present disclosure optionally further comprise administering dexamethasone to the subject. Dexamethasone can be administered by any route, such as those described herein. Preferably, dexamethasone is administered intravenously or orally. When dexamethasone is administered intravenously, it is optionally administered to the subject within 1 hour before the administration of the antibody. Dexamethasone is optionally administered in an amount of about 8 mg or about 4 mg.

[0300] In various embodiments, the methods of the present disclosure further comprise administering a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents that damage DNA include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and its analogs or metabolites, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, etc.). DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[0301] Chemotherapeutic agents that interfere with cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib, CEP-18770, MG132, peptide vinyl sulfones, peptide epoxyketones (e.g., epoxomicin and carfilzomib), β-lactone inhibitors (e.g., lactacystin, MLN 519, NPI-0052, Salinosporamide A), compounds that form dithiocarbamate complexes with metals (e.g., disulfiram), and certain antioxidants (e.g., epigallocatechin-3-gallate, catechin-3-gallate, and Salinosporamide A); NF-kB inhibitors, including inhibitors of IKB kinase; antibodies that bind to proteins overexpressed in cancer, thereby downregulating cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and other inhibitors of proteins or enzymes known to be upregulated, overexpressed, or activated in cancer, whose inhibition downregulates cell replication.

[0302] Treatment regimens may include administration of other antibody therapeutics, such as elotuzumab (humanized monoclonal against SLAMF7; Tai et al., Blood, 2008;112:1329-37); daratumumab, MOR202, and isatuximab, which target CD38; nBT062-SMCC-DMI, nBT062-SPDB-DM4, and nBT062-SPP-DMI, which target CD138; lucatumumab (also known as HCD122) and dacetuzumab (also known as SGN-40), which target CD40; and lorvotuzumab, which targets CD56. For a review of antibody therapeutics for the treatment of multiple myeloma, see, e.g., Tandon et al., Oncology & Hematology Review, 2015;11(2):115-21, and Sondergeld et al., Clinical Advances in Hematology & Oncology, 2015;13(9),599 (both incorporated by reference).

[0303] In some embodiments, the heterodimeric antibody is administered before, concurrently with, or after treatment with proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), immunomodulatory drugs (IMiDs) such as the thalidomide derivatives pomalidomide and lenalidomide, antibodies such as the histone deacetylase inhibitors panobinostat, daratumumab, teclistamab, isatuximab, elotuzumab talquetamab, alnuctamab, belantamab, erlanatamab, or any other of the above compounds and / or drugs and / or biotherapeutics, including venetoclax, mafodotin, selinixol, dexamethasone, doxorubicin, melflufen, CAR-T cell therapy, and combinations thereof.

[0304] All cited references are expressly incorporated herein by reference in their entirety. Although specific embodiments of the present invention have been described above for purposes of illustration, those skilled in the art will appreciate that many changes in detail may be made therein without departing from the invention as set forth in the appended claims. [Brief explanation of the drawings]

[0305] [Figure 1A] Anti-human CD3 C1 candidates bind to human CD3 and induce T cell activation. Binding of anti-CD3 candidates to human PBMCs was assessed by flow cytometry (A). Concurrently, the ability of anti-CD3 candidates to induce activation of human purified T cells for 48 hours via upregulation of CD69 on CD4+ T cells was assessed by flow cytometry (B). Each point represents the mean + / - standard deviation of three independent measurements from one representative experiment. The experimental setup and analysis are described in Example 1. [Figure 1B] Anti-human CD3 C1 candidates bind to human CD3 and induce T cell activation. Binding of anti-CD3 candidates to human PBMCs was assessed by flow cytometry (A). Concurrently, the ability of anti-CD3 candidates to induce activation of human purified T cells for 48 hours via upregulation of CD69 on CD4+ T cells was assessed by flow cytometry (B). Each point represents the mean + / - standard deviation of three independent measurements from one representative experiment. The experimental setup and analysis are described in Example 1. [Figure 2] Anti-CD3-C1 candidates and anti-CD3-C1 optimized candidates specifically bind to human CD3. Binding of anti-CD3 candidates to HPB-ALL wild-type and knockout CD3 cell lines was assessed by flow cytometry. Each bar represents the geometric mean of fluorescence of one replicate tested at 8 nM from one representative experiment. Experimental setup and analysis are described in Example 2. [Figure 3] Format of a tool molecule used to evaluate the T cell activation propensity of CD3 binders. [Figure 4A]The 2+1 C1-D6 / BCMA candidate induces the most potent tumor cell killing and T cell activation in a redirected lysis assay. The ability of the 2+1 CD3xBCMA-dAbx2 candidate to induce T cell-mediated killing of NCI-H929 tumor cells (A) was evaluated in a redirected lysis assay using a 5:1 effector-to-target ratio and measured by flow cytometry after 48 hours of incubation. In the same assay, human T cell activation was assessed by flow cytometry as measured by the percentage of CD69 upregulation on CD4 T cells (B). Each point represents the mean + / - standard deviation of two technical replicates from one representative donor. The experimental setup and analysis are described in Example 3. [Figure 4B] The 2+1 C1-D6 / BCMA candidate induces the most potent tumor cell killing and T cell activation in a redirected lysis assay. The ability of the 2+1 CD3xBCMA-dAbx2 candidate to induce T cell-mediated killing of NCI-H929 tumor cells (A) was evaluated in a redirected lysis assay using a 5:1 effector-to-target ratio and measured by flow cytometry after 48 hours of incubation. In the same assay, human T cell activation was assessed by flow cytometry as measured by the percentage of CD69 upregulation on CD4 T cells (B). Each point represents the mean + / - standard deviation of two technical replicates from one representative donor. The experimental setup and analysis are described in Example 3. [Figure 5-1] HDX epitope mapping of C1-D6 binders to hCD3yε. hCD3yε showed a significant decrease in deuterium uptake (-10) upon binding to IgG with AA182-197, YVCYPRGSKPEDANFY (SEQ ID NO: 726), indicating that this is the predominant epitope. Figure 5 shows a differential heat map comparing the hydrogen / deuterium exchange of hsCD3y(23-103)-hsCD3ε(23-118) alone with the hydrogen / deuterium exchange of hsCD3y(23-103)-hsCD3ε(23-118) and anti-CD3-C1-D6 IgG1 LALA. [Figure 5-2]HDX epitope mapping of C1-D6 binders to hCD3yε. hCD3yε showed a significant decrease in deuterium uptake (-10) upon binding to IgG with AA182-197, YVCYPRGSKPEDANFY (SEQ ID NO: 726), indicating that this is the predominant epitope. Figure 5 shows a differential heat map comparing the hydrogen / deuterium exchange of hsCD3y(23-103)-hsCD3ε(23-118) alone with the hydrogen / deuterium exchange of hsCD3y(23-103)-hsCD3ε(23-118) and anti-CD3-C1-D6 IgG1 LALA. [Figure 6A] Blockade of human BCMA / human APRIL and human BCMA / human BAFF interactions upon binding of anti-BCMA-D3 Fab and anti-BCMA-E6 Fab to human BCMA, as assessed by surface plasmon resonance. Recombinant human APRIL-his protein or recombinant human BAFF-his protein was loaded onto a CM5 sensor chip pre-coated with an anti-histidine antibody. A solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations of anti-BCMA-D3 Fab (0 nM, 25 nM, 50 nM, or 200 nM) was injected over A) immobilized human APRIL or B) immobilized human BAFF. Similarly, a solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-E6 Fab was injected over C) immobilized human APRIL or D) immobilized human BAFF. Plots show data expressed as number of resonance units (abbreviated RU; Y-axis) versus time (X-axis). [Figure 6B]Blockade of human BCMA / human APRIL and human BCMA / human BAFF interactions upon binding of anti-BCMA-D3 Fab and anti-BCMA-E6 Fab to human BCMA, as assessed by surface plasmon resonance. Recombinant human APRIL-his protein or recombinant human BAFF-his protein was loaded onto a CM5 sensor chip pre-coated with an anti-histidine antibody. A solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations of anti-BCMA-D3 Fab (0 nM, 25 nM, 50 nM, or 200 nM) was injected over A) immobilized human APRIL or B) immobilized human BAFF. Similarly, a solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-E6 Fab was injected over C) immobilized human APRIL or D) immobilized human BAFF. Plots show data expressed as number of resonance units (abbreviated RU; Y-axis) versus time (X-axis). [Figure 6C] Blockade of human BCMA / human APRIL and human BCMA / human BAFF interactions upon binding of anti-BCMA-D3 Fab and anti-BCMA-E6 Fab to human BCMA, as assessed by surface plasmon resonance. Recombinant human APRIL-his protein or recombinant human BAFF-his protein was loaded onto a CM5 sensor chip pre-coated with an anti-histidine antibody. A solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations of anti-BCMA-D3 Fab (0 nM, 25 nM, 50 nM, or 200 nM) was injected over A) immobilized human APRIL or B) immobilized human BAFF. Similarly, a solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-E6 Fab was injected over C) immobilized human APRIL or D) immobilized human BAFF. Plots show data expressed as number of resonance units (abbreviated RU; Y-axis) versus time (X-axis). [Figure 6D]Blockade of human BCMA / human APRIL and human BCMA / human BAFF interactions upon binding of anti-BCMA-D3 Fab and anti-BCMA-E6 Fab to human BCMA, as assessed by surface plasmon resonance. Recombinant human APRIL-his protein or recombinant human BAFF-his protein was loaded onto a CM5 sensor chip pre-coated with an anti-histidine antibody. A solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations of anti-BCMA-D3 Fab (0 nM, 25 nM, 50 nM, or 200 nM) was injected over A) immobilized human APRIL or B) immobilized human BAFF. Similarly, a solution of 50 nM recombinant BCMA-Fc protein in running buffer pre-mixed with various concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-E6 Fab was injected over C) immobilized human APRIL or D) immobilized human BAFF. Plots show data expressed as number of resonance units (abbreviated RU; Y-axis) versus time (X-axis). [Figure 7] Anti-BCMA-D3 and anti-BCMA-E6 affinity matured candidates specifically bind to BCMA. Binding of anti-BCMA candidates to NCI-H929 wild-type and knockout BCMA cell lines was assessed by flow cytometry. Each bar represents the geometric mean of fluorescence of one replicate tested at 10 nM from one representative experiment. Experimental setup and analysis are described in Example 6. [Figure 8A]Epitope binning of anti-CD38-E2 Fab, anti-CD38-B3 Fab, and daratumumab Fab against human CD38 using Octet Bio-Layer Interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin (SA) biosensor. 200 nM of Fab antibody 1 (Ab1) in kinetic buffer was injected over immobilized human CD38 until surface saturation was reached. Then, a premixed solution of Ab1 and Fab antibody 2 (Ab2) at a final concentration of 200 nM was injected over the saturated surface (competition phase). As a control for surface saturation, the same experimental procedure was performed using only 400 nM of Ab1 in the competition phase. Plots show binding to the sensor tip as wavelength shift (response in nm; Y-axis) versus time (X-axis) for A) anti-CD38-E2 Fab as Ab1, B) anti-CD38-B3 Fab as Ab1, and C) daratumumab Fab as Ab1. Curves are labeled with Ab2 clone names. [Figure 8B] Epitope binning of anti-CD38-E2 Fab, anti-CD38-B3 Fab, and daratumumab Fab against human CD38 using Octet Bio-Layer Interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin (SA) biosensor. 200 nM of Fab antibody 1 (Ab1) in kinetic buffer was injected over immobilized human CD38 until surface saturation was reached. Then, a premixed solution of Ab1 and Fab antibody 2 (Ab2) at a final concentration of 200 nM was injected over the saturated surface (competition phase). As a control for surface saturation, the same experimental procedure was performed using only 400 nM of Ab1 in the competition phase. Plots show binding to the sensor tip as wavelength shift (response in nm; Y-axis) versus time (X-axis) for A) anti-CD38-E2 Fab as Ab1, B) anti-CD38-B3 Fab as Ab1, and C) daratumumab Fab as Ab1. Curves are labeled with Ab2 clone names. [Figure 8C]Epitope binning of anti-CD38-E2 Fab, anti-CD38-B3 Fab, and daratumumab Fab against human CD38 using Octet Bio-Layer Interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin (SA) biosensor. 200 nM of Fab antibody 1 (Ab1) in kinetic buffer was injected over immobilized human CD38 until surface saturation was reached. Then, a premixed solution of Ab1 and Fab antibody 2 (Ab2) at a final concentration of 200 nM was injected over the saturated surface (competition phase). As a control for surface saturation, the same experimental procedure was performed using only 400 nM of Ab1 in the competition phase. Plots show binding to the sensor tip as wavelength shift (response in nm; Y-axis) versus time (X-axis) for A) anti-CD38-E2 Fab as Ab1, B) anti-CD38-B3 Fab as Ab1, and C) daratumumab Fab as Ab1. Curves are labeled with Ab2 clone names. [Figure 9A] Schematic diagram of CD3 / BCMA / CD38 trispecific antibodies. The CD3 / BCMA / CD38 antibody was constructed with three Fabs targeting three distinct antigens, whereby the "Fc-distal" Fab domain was fused to the N-terminus of the VH domain of the "Fc-proximal" Fab. A) Construct BO (BTB Outer) CD3 / BCMA / CD38 had a CD38-binding agent on arm A, a CD3-binding arm in the Fc-distal position of the outer B arm, and BCMA in the "Fc-proximal" position of the B arm, while B) for construct C, CD3 / BCMA / CD38 had a CD3-binding arm on arm A, a BCMA-binding agent in the Fc-distal position of arm B, and a CD38-binding agent in the Fc-proximal position of the B arm. [Figure 9B]Schematic diagram of CD3 / BCMA / CD38 trispecific antibodies. The CD3 / BCMA / CD38 antibody was constructed with three Fabs targeting three distinct antigens, whereby the "Fc-distal" Fab domain was fused to the N-terminus of the VH domain of the "Fc-proximal" Fab. A) Construct BO (BTB Outer) CD3 / BCMA / CD38 had a CD38-binding agent on arm A, a CD3-binding arm in the Fc-distal position of the outer B arm, and BCMA in the "Fc-proximal" position of the B arm, while B) for construct C, CD3 / BCMA / CD38 had a CD3-binding arm on arm A, a BCMA-binding agent in the Fc-distal position of arm B, and a CD38-binding agent in the Fc-proximal position of the B arm. [Figure 10A] Differential scanning calorimetry (DSC) (thermal transition) for CD3 / BCMA / CD38 antibodies

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Claims

1. A trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38.

2. The trispecific antibody or antibody fragment thereof of claim 1 , further comprising a common light chain.

3. 3. The trispecific antibody or antibody fragment thereof of claim 1 or 2, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain CDR set comprising amino acid sequences selected from the group comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; SEQ ID NO: 184, SEQ ID NO: 310, and SEQ ID NO: 436; SEQ ID NO: 186, SEQ ID NO: 312, and SEQ ID NO: 438; SEQ ID NO: 188, SEQ ID NO: 314, and SEQ ID NO: 440; SEQ ID NO: 192, SEQ ID NO: 318, and SEQ ID NO:

444.

4. The trispecific antibody or antibody fragment thereof of any one of claims 1 to 3, wherein the at least one binding moiety that binds to human BCMA comprises a heavy chain CDR set comprising amino acid sequences selected from the group comprising: SEQ ID NO:234, SEQ ID NO:360, and SEQ ID NO:486; SEQ ID NO:219, SEQ ID NO:345, and SEQ ID NO:471; SEQ ID NO:227, SEQ ID NO:353, and SEQ ID NO:479; SEQ ID NO:231, SEQ ID NO:357, and SEQ ID NO:

483.

5. 5. The trispecific antibody or antibody fragment thereof of any one of claims 1 to 4, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain CDR set comprising amino acid sequences comprising SEQ ID NO:236, SEQ ID NO:362, and SEQ ID NO:712; SEQ ID NO:239, SEQ ID NO:365, and SEQ ID NO:491; SEQ ID NO:237, SEQ ID NO:363, and SEQ ID NO:700; SEQ ID NO:248, SEQ ID NO:374, and SEQ ID NO:

500.

6. The trispecific antibody or antibody fragment thereof according to any one of claims 1 to 5, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NOs: 55; 58; 60; 62 and 66.

7. 7. The trispecific multispecific antibody or antibody fragment thereof according to any one of claims 1 to 6, wherein said at least one binding moiety that binds to human BCMA comprises a heavy chain comprising an amino acid sequence selected from the group comprising SEQ ID NOs: 93, 101, 105 and 108, or an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NOs: 93, 101, 105 and 108, wherein said amino acid sequence further comprises the amino acid substitution N82aS.

8. The trispecific antibody or antibody fragment thereof according to any one of claims 1 to 7, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain comprising an amino acid sequence that is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group comprising SEQ ID NOs: 110; 113, 122 and 111.

9. 9. The trispecific antibody or antibody fragment thereof according to any one of claims 1 to 8, wherein said at least one binding moiety binding to human CD3 comprises a heavy chain CDR set comprising the amino acid sequences of SEQ ID NOs: 181, 307, and 433, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723; said at least one binding moiety binding to human BCMA comprises a heavy chain CDR set comprising the amino acid sequences of SEQ ID NOs: 234, 360, and 486, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723; and said at least one binding moiety binding to human CD38 comprises a heavy chain CDR set comprising the amino acid sequences of SEQ ID NOs: 239, 365, and 491, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

10. 10. The trispecific antibody or antibody fragment thereof of claim 9, comprising three amino acid chains having the amino acid sequences of SEQ ID NO:546, SEQ ID NO:547 and SEQ ID NO:

1.

11. The trispecific antibody or antibody fragment thereof according to any one of claims 1 to 10, wherein the at least one binding moiety that binds to human BCMA is fused at its N-terminus to the C-terminus of the at least one binding moiety that binds to human CD3 via a peptide linker.

12. The trispecific antibody or antibody fragment thereof of claim 1, wherein the at least one binding moiety that binds to human BCMA is fused at its C-terminus to the N-terminus of the at least one binding moiety that binds to human CD3 via a peptide linker, or the at least one binding moiety that binds to human BCMA is fused at its C-terminus to the N-terminus of the at least one binding moiety that binds to human CD38 via a peptide linker, or the at least one binding moiety that binds to human BCMA is fused at its N-terminus to the C-terminus of the at least one binding moiety that binds to human CD38 via a peptide linker.

13. The trispecific antibody or antibody fragment thereof according to any one of claims 1 to 12, wherein the trispecific antibody or antibody fragment thereof comprises a non-naturally occurring Fc domain.

14. A trispecific antibody or an antibody fragment thereof according to any one of claims 1 to 13 for use as a medicament.

15. The trispecific antibody or antibody fragment thereof according to claim 14 for use in the treatment of BCMA and / or CD38 expressing cancer.

16. 16. The trispecific antibody or antibody fragment thereof according to claim 14 or 15 for use in the treatment of multiple myeloma, relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, active multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, prostate cancer, cervical cancer, germinal center B-cell lymphoma or B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), high-grade serous ovarian cancer, peritoneal cancer.

17. An antibody or antibody fragment thereof or antigen-binding fragment thereof that binds to the same epitope on human CD3 as a reference antibody or antibody fragment thereof, comprising at least one binding portion that binds to human CD3, one binding portion that binds to BCMA, and one binding portion that binds to CD38, as described in claim 9.

18. 18. The epitope on the human CD38 extracellular domain bound by the antibody or antibody fragment thereof of any one of claims 1 to 17, comprising residues Glu103, Gln107, Thr114, Thr116, Arg194, Arg195, Glu198, Ala199, Asp202, Ser224, His228, Asn229, Gln231, Pro232, Glu233, Lys234, Val235, Gln236, Ile265, Ser267, Lys268, Arg269 and Asn270 as detected by X-ray crystallography.

19. An antibody or antibody fragment thereof that binds to human CD3, comprising a heavy chain CDR set comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 181, 307, and 433; SEQ ID NOs: 184, 310, and 436; SEQ ID NOs: 186, 312, and 438; SEQ ID NOs: 188, 314, and 440; SEQ ID NOs: 192, 318, and 444, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs: 721, 722, and 723.

20. An antibody or antibody fragment thereof that binds to human BCMA, comprising a heavy chain CDR set comprising amino acid sequences selected from the group consisting of SEQ ID NOs:234, 360, and 486; SEQ ID NOs:219, 345, and 471; SEQ ID NOs:227, 353, and 479; SEQ ID NOs:231, 357, and 483, and a light chain CDR set comprising the amino acid sequences of SEQ ID NOs:721, 722, and 723.

21. A trispecific heterodimeric antibody or antibody fragment thereof comprising a first and a second engineered CH3 domain, wherein said first engineered CH3 domain comprises substitutions of the group comprising: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and said second engineered CH3 domain comprises substitutions of the group comprising: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and 1. A trispecific heterodimeric antibody or antibody fragment thereof comprising a substitution of the group comprising: L, D399E, F405A, Y407S, K409R, T411R (EU numbering), wherein said trispecific heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via said first and second engineered CH3 domains, and wherein said trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding moieties, each binding moiety binding to a different antigen.

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