CD3 / BCMA / CD38 trispecific antibodies

Abstract

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

Description

Technical Field

[0001] The present invention relates to novel trispecific heterodimeric immunoglobulins. More specifically, the present invention relates to trispecific heterodimeric immunoglobulins that target human CD3, human BCMA, and human CD38 epitopes. The present invention also relates to such novel trispecific heterodimeric immunoglobulins for the treatment of proliferative diseases, and in particular cancers, such as hematological cancers. Background Art

[0002] Multispecific antibodies targeting one or more immune-related molecules and one or more tumor antigens to recruit immune cells (such as T cells) to tumor cells have been developed for cancer immunotherapy in the past few decades. For example, antibodies bispecific to CD3 on T cells and surface target antigens on cancer cells can connect T cells to cancer cells without relying on T cell receptor specificity, co-stimulation or peptide antigen presentation (WO2020204708A1). When two proteins target T cells and one targets tumor cells, trispecific T cell engagement antibodies have also been shown to be used to treat various cancers (including breast cancer, lymphoma and multiple myeloma) with promising results (US10882922B2). In addition, it has been proposed to use trispecific binding molecules to crack tumor cells, which, in addition to engaging other components of the TCR complex on CD3 or T cells, also engage two tumor-associated antigens expressed on cancer cells (US20210163620A1).

[0003] Multispecific immune cell redirecting antibodies have been shown in preclinical and clinical studies to mediate T cell redirection (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 for the treatment of complex diseases such as inflammatory diseases, autoimmune disorders and cancers, such as hematological cancers, including multiple myeloma (MM), for which curative treatment is still needed. Multiple myeloma is a neoplastic plasma cell disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow (BM) microenvironment, monoclonal proteins in the blood or urine, and associated organ dysfunction. Multiple myeloma accounts for 1%-2% of all new cancer diagnoses and approximately 20% of all deaths from hematological malignancies. The disease is more common in men and African Americans than in the general population. Multiple myeloma remains an incurable cancer, although recent improvements in the understanding of myeloma pathogenesis have led to the development of new treatments and improved survival rates.

[0004] The diagnosis of multiple myeloma requires the presence of one or more myeloma-defining events (MDEs) and evidence of 10% or more clonal plasma cells on BM examination or biopsy-proven plasmacytoma. MDEs include the so-called CRAB (hypercalcemia, renal failure, anemia, or osteolytic lesions) features as well as the following three specific biomarkers: clonal BM plasma cells >60%, a serum free light chain (sFLC) ratio >100 (provided the affected sFLC level is >100 mg / L), and more than one focal lesion on magnetic resonance imaging. Several genetic abnormalities occurring in neoplastic plasma cells play a major role in the pathogenesis of myeloma and determine disease prognosis.

[0005] The uncontrolled growth of myeloma cells has many consequences, including bone destruction, BM failure, increased plasma volume and viscosity, suppression of normal immunoglobulin production, and renal impairment.

[0006] Symptomatic (active) disease should be treated promptly, whereas asymptomatic (smoldering) myeloma requires only clinical observation, as early treatment with conventional chemotherapy has not shown a clear benefit. Investigational trials are currently evaluating the ability of immunomodulatory drugs to delay progression from asymptomatic to symptomatic myeloma. For active myeloma, current data support the initiation of an induction regimen consisting of thalidomide, lenalidomide, and / or bortezomib, followed by autologous hematopoietic stem cell transplantation (HSCT) after a major disease response in patients who can tolerate an autologous HSCT conditioning regimen. Consideration of physiologic age (which may differ from chronological age) and the presence of comorbidities dictates treatment choice and drug dosing. For example, for patients with severe comorbidities, including cardiopulmonary or hepatic impairment, a less intensive approach may be desirable to limit treatment-related mortality and reduce the risk of treatment interruption.

[0007] The treatment of relapsed / refractory multiple myeloma (RRMM) presents a unique therapeutic challenge due to the heterogeneous nature of the disease at relapse and the lack of clear biologically based recommendations for the selection of salvage therapies at different points in disease progression. With the increasing recognition that the inherent clonal heterogeneity and genomic instability of plasma cells can influence both intrinsic and acquired treatment resistance, determining the optimal treatment selection and sequencing has become crucial. In recent years, the United States (US) Food and Drug Administration (FDA) has approved new agents for relapsed / refractory myeloma, including proteasome inhibitors (carfilzomib and ixazomib), immunomodulatory drugs such as the thalidomide derivatives pomalidomide and lenalidomide, and the histone deacetylase inhibitor panobinostat. Additional molecularly targeted therapies targeting specific cell signaling pathways and those controlling survival and proliferation (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 nearly all patients. Myeloma relapses are often more aggressive with each relapse, which is associated with shorter survival. Therefore, additional treatment options are needed.

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

[0009] CD38 (UniProt P28907) was first identified in 1980 as a surface marker of thymocyte lymphocytes (cluster of differentiation) [Lee, HC ed. (2002). A Natural History of the Human CD38 Gene. Cyclic ADP-Ribose and NAADP. Springer Publishing], and in 1992, it was further described as a surface marker on B cells, monocytes, and natural killer cells (NK cells). Around the same time, CD38 was discovered to be a marker for B cell 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, also known as BCMA, CD269, TNFRSF17 (UniProt Q02223), is a member of the tumor necrosis receptor superfamily that is preferentially expressed in 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 that is involved in B cell maturation, growth, 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 BAFF (THANK, BlyS, B lymphocyte stimulator, TALL-1 and zTNF4), a low-affinity ligand for BCMA. APRIL and BAFF show structural similarities and overlapping but distinct receptor binding specificities. Negative regulator TACI also binds to both BAFF and APRIL. The coordination of APRIL and BAFF with BCMA and / or TACI activates transcription factor NF-KB and increases the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1), and downregulates the expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thereby inhibiting apoptosis and promoting survival. This combined effect promotes B cell differentiation, proliferation, survival and antibody production (as reviewed in Rickert RC et al., Immunol Rev (2011) 244 (1): 115-133). According to this discovery, BCMA also supports the growth and survival of malignant human B cells (including multiple myeloma cells).

[0011] Bispecific antibodies that bind to CD3 and BCMA, such as teclistamab, have been developed for the treatment of MM to induce T cell-mediated cytotoxicity against BCMA-expressing MM cells and / or RRMM cells, alone or in combination with immunotherapeutic drugs such as thalidomide or immunotherapeutic derivatives such as anti-PD-1 antibodies and anti-PD-L1 antibodies ( US11124577B2 ).

[0012] Exceptional overall response rates have been observed using bispecific antibodies or chimeric antigen receptor (CAR) T cell immunotherapy, in which T lymphocytes are engineered with synthetic chimeric antigen receptors. However, durable responses over 2 years remain limited (for treatment with idecabtagene vicleucel (idecabtagene vicleucel) (Munshi, NC et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N. Engl. J. Med. 384, 705-716 (2021)) or teritomab (Moreau, P. et al. Teclistamab in Relapsed or Refractory Multiple Myeloma. N. Engl. J. Med. NEJ Moa 2203478 (2022) doi: 10.1056 / NEJ Moa 2203478), progression-free survival rates are less than 40%. It has been suggested that one of the potential causes of patient relapse may be downregulation of the target or expansion of clones lacking sufficient target expression (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 the target in MM populations has been observed after treatment with CD38-targeting daratumumab (Nijhof, IS 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, AD et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J. Clin. Invest. 129, 2210-2221 (2019)). Therefore, it remains a challenge to develop approaches to prevent tumor escape, which is often associated with lower surface expression of targeted tumor-associated antigens.

[0013] Although promising treatments for hematological cancers such as multiple myeloma are currently available, there remains a need for improved therapies, such as those based on effector cell redirecting antibodies. Indeed, despite the increase in the 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

[0014] 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.

[0015] The present invention also relates to a trispecific antibody or an 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 portions, 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 portion that binds to human CD3 comprises a heavy chain CDR set comprising an amino acid sequence 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.

[0017] The present invention also relates to a trispecific antibody or antibody fragment thereof, comprising at least three binding portions, 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 portion that binds to human BCMA comprises a heavy chain CDR set comprising an amino acid sequence 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.

[0018] The present invention also relates to a trispecific antibody or antibody fragment thereof, comprising at least three binding portions, 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 portion that binds to human CD38 comprises a heavy chain CDR set comprising an amino acid sequence 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 NO: 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 portions, 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 portion 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.

[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 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 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 NO: 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 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 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, wherein the amino acid sequences of SEQ ID NOs: 93, 101, 105 and 108 further comprise 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 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 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 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 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, which further comprises 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 consisting of 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 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 BCMA 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 consisting of SEQ ID NOs: 93, 101, 105 and 108, or to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105 and 108; wherein the amino acid sequences of SEQ ID NOs: 93, 101, 105 and 108 further comprise 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 consisting of 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 portions, 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 portion that binds to human CD3 comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; the at least one binding portion that binds to BCMA comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and the at least one binding portion that binds to human CD38 comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491, and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491. The light chain CDR group of the amino acid sequences of SEQ ID NO:721, SEQ ID NO:722 and SEQ ID NO:723.

[0032] The present invention also relates to a trispecific antibody or antibody fragment thereof, wherein the at least one binding portion that binds to human CD3 comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 181, SEQ ID NO: 307 and SEQ ID NO: 433, and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723; the at least one binding portion that binds to BCMA comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 234, SEQ ID NO: 360 and SEQ ID NO: 486, and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723; and the at least one binding portion that binds to human CD38 comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 236, SEQ ID NO: 362 and SEQ ID NO: 712, and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: The light chain CDR group of the amino acid sequence of NO:723.

[0033] The present invention also relates to a trispecific antibody or an antibody fragment thereof, wherein the at least one binding portion that binds to human CD3 and / or the at least one binding portion that binds to human BCMA and / or the at least one binding portion that binds to 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 portion that binds to human CD3 and the at least one binding portion that binds to human BCMA, or the at least one binding portion that binds to human CD3 and the at least one binding portion that binds to human CD38, or the at least one binding portion that binds to human BCMA and the at least one binding portion that binds to human CD38 are fused to each other.

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

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

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

[0039] The present invention also relates to a trispecific antibody or an antibody fragment thereof, which comprises a set of three amino acid chains comprising an amino acid sequence 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; 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 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 an antibody fragment thereof, comprising three amino acid chains having an amino acid sequence selected from the group consisting of 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 an antibody fragment thereof, wherein the trispecific antibody or the antibody fragment thereof is a heterodimeric immunoglobulin comprising a first engineered immunoglobulin chain comprising a first engineered domain and a second engineered immunoglobulin chain comprising a second engineered domain, wherein the heterodimeric immunoglobulin is heterodimerized through the first engineered domain and the second engineered domain.

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

[0044] More particularly, wherein the first engineered CH3 domain comprises substitutions selected from the group consisting of Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises substitutions selected from the group consisting of 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, it is used to treat 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 syndrome (MDS), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), high-grade serous ovarian cancer, and peritoneal cancer.

[0047] The present invention also relates to an epitope on the extracellular domain of human CD38 bound by the trispecific antibody or antibody fragment 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 epitope has at least Preferably at least Even more preferably at least In the most preferred example, the resolution is about

[0049] More specifically, the present invention also relates to an epitope on the extracellular domain of human CD38, which is bound by the antibody of any one of the preceding claims and comprises 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 epitope has at least Preferably at least Even more preferably at least In the most preferred example, the resolution is about

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

[0051] The present invention also relates to an epitope on the extracellular domain of human CD3 bound by the trispecific antibody or antibody fragment thereof disclosed herein.

[0052] The present invention also relates to antibodies or antibody fragments or antigen-binding fragments thereof that bind 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, wherein the at least one binding portion 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 light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; the at least one binding portion 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 portion 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. The light chain CDR groups of SEQ ID NO:721, SEQ ID NO:722 and SEQ ID NO:723.

[0053] The present invention also relates to antibodies or antigen-binding fragments that bind to the same epitope on CD3 as a reference antibody, wherein the reference antibody is a trispecific antibody, wherein the at least one binding portion that binds to human CD3 and / or BCMA and / or CD38 comprises a heavy chain CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; the at least one binding portion 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 portion 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: The light chain CDR group of ID NO: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 antibodies or antigen-binding fragments that bind to an epitope on human CD38, wherein the epitope on CD3 comprises 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 antibody or antigen-binding fragment has at least Preferably at least Even more preferably at least In the most preferred example, the resolution is about

[0056] The present invention also relates to an isolated nucleic acid encoding the trispecific antibody disclosed herein.

[0057] The present invention also relates to a host cell comprising the isolated nucleic acid.

[0058] The present invention also relates to an antibody or antibody fragment thereof that binds to human CD3, comprising: a heavy chain CDR group comprising an amino acid sequence 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 group comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0059] The present invention also relates to an antibody or antibody fragment thereof 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: 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 light chain CDR set comprising an amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0060] The present invention also relates to a trispecific heterodimeric antibody comprising a first engineered CH3 domain and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises one or more substitutions selected from the group consisting of: 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 consisting of: Q347E , Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment is heterodimerized by the first engineered CH3 domain and the second engineered CH3 domain, characterized in that the trispecific heterodimeric antibody comprises at least three binding portions, each binding portion binding to a different antigen.

[0061] In particular, the present invention relates to a trispecific heterodimeric antibody comprising a first engineered CH3 domain and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises substitutions selected from the group consisting of: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises substitutions selected from the group consisting of: Q347E, Y349A, L 351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment is heterodimerized by the first engineered CH3 domain and the second engineered CH3 domain, characterized in that the trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding portions, each binding portion binding to a different antigen.

[0062] Provided herein are antibodies and antigen-binding fragments thereof that immunospecifically bind to human BCMA, CD38, and CD3. Also described are related polynucleotides capable of encoding the provided BCMA-specific, CD38-specific, 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. In addition, methods for using the provided antibodies and antigen-binding fragments are described. For example, BCMA-specific antibodies and / or CD38-specific antibodies and antigen-binding fragments can be used to diagnose or monitor the progression, regression, or stability of BCMA-expressing cancers and / or CD38-expressing cancers; determine whether a patient should be treated for cancer; or determine whether a subject has a BCMA-expressing cancer and / or a CD38-expressing cancer and is therefore amenable to treatment with a BCMA-specific anti-cancer therapeutic, and / or a CD38-expressing cancer, such as the multispecific antibodies to BCMA, CD38, and CD3 described herein.

[0063] Also provided herein are multispecific antibodies that immunospecifically bind to BCMA, CD38, and CD3, and multispecific antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided BCMAxCD38xCD3 multispecific antibodies, cells expressing the provided antibodies, and related vectors and detectably labeled multispecific antibodies. Furthermore, methods of using the provided multispecific antibodies are described. For example, the BCMAxCD38xCD3 multispecific antibodies can be used to diagnose or monitor the progression, regression, or stability of BCMA-expressing cancers and / or CD38-expressing cancers to determine whether a patient should receive cancer treatment; or to determine whether a subject has a BCMA-expressing cancer and / or a CD38-expressing cancer and is therefore amenable to 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 used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated. Unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

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

[0066] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid polymer having a length of at least 10 nucleotides. In certain embodiments, the nucleotides comprising the polynucleotide can be modified forms of ribonucleotides or deoxyribonucleotides or any type of nucleotide. This modification includes base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3'-dideoxyribose, and ethyl internucleotide bond modifications such as phosphorothioate, phosphorodithioate, selenophosphate, diselenphosphate, aniline phosphorothioate, aniline phosphate, and phosphoramidate. The term "polynucleotide" specifically includes DNA in single-stranded and double-stranded forms.

[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 part of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to 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 a polypeptide that: (1) is free of at least some other polypeptides with which it is normally found, (2) is substantially free of other polypeptides from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with polypeptide moieties with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or non-covalent interactions) with polypeptides with which the "isolated polypeptide" is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA, or other RNA of a synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment 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 protein such as 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 present disclosure.

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

[0072] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and include intact antibodies and any antigen-binding fragments or single chains thereof. Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of 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 kDa-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 to the target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines the constant domains responsible for effector function. Thus, in naturally occurring antibodies, a full-length heavy chain immunoglobulin polypeptide comprises a variable domain (VH) and three constant domains (CH1, CH2, and CH3), wherein the VH domain is located at the amino terminus of the polypeptide and the CH3 domain is located at the carboxyl terminus, and a full-length light chain immunoglobulin polypeptide comprises a variable domain (VL) and a constant domain (CL), wherein the VL domain is located at the amino terminus of the polypeptide and the CL domain is located at the carboxyl terminus.

[0073] Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined 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 the full-length light and heavy chains, the variable domains and constant domains are generally connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd edition, 1989), which is incorporated herein by reference in its entirety for all purposes. The variable region of each light chain / heavy chain pair generally forms an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conservative framework regions (FRs) connected by three hypervariable regions (also referred to as complementary determining regions or CDRs). The CDRs from each pair of two chains are typically aligned by the framework regions, which can enable binding to specific epitopes. From amino-terminal to carboxyl-terminal, the light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0074] The term "CDR group" refers to a group of three CDRs that appear in a single variable region that is 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 a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that certain sub-portions within the Kabat CDRs adopt almost identical peptide backbone conformations, despite having great diversity at the amino acid sequence level. These subsections are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light chain region and the heavy chain region, respectively. These regions may be referred to as Chothia CDRs, whose boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by: 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 follow one of the systems described herein but will still overlap with the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, will not significantly affect antigen binding. Although certain embodiments use CDRs defined by Kabat or Chothia, the methods used herein can utilize CDRs defined according to any of these systems. The use of amino acid sequences to identify predicted CDRs is well known in the art, such as in Martin, AC "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Vol. 2. Kontermann R., Dubel S. eds. Springer-Verlag, Berlin, pp. 33-51 (2010).The amino acid sequence of the heavy chain and / or light chain variable domain can also be checked by other conventional methods to identify the sequence of the CDR, for example, by comparing with the known amino acid sequence of other heavy chain and light chain variable regions to determine the sequence hypervariable region. The numbered sequences can be compared by eye, or by using one of the alignment programs such as the CLUSTAL program suite, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular modeling is generally used to correctly depict the framework and CDR regions, and thus to correct the allocation based on the sequence. The present invention encompasses all such alternative definitions, and the sequences provided in this specification are not intended to exclude the CDR sequences of alternative definitions, which may only comprise a portion of the CDR sequences provided in the sequence table. In a preferred embodiment of the present 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] As used herein, the term "antibody fragment" includes a portion of a full-length antibody. Non-limiting examples of antibody fragments include: (i) a crystallizable fragment (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, and which are paired by disulfide bonds and non-covalent interactions; (ii) an antigen-binding fragment (Fab), which consists of VL, CL, VH, and CH1 linked by disulfide bonds; (iii) Fab', which consists of VL, CL, VH, and CH1 linked by disulfide bonds and one or more cysteine ​​residues from the hinge region; (iv) Fab'-SH, which is a Fab' fragment in which the cysteine ​​residue contains a free sulfhydryl group; (v) a F(a) fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region. b') 2; (vi) variable fragment (Fv), which consists of VL and VH chains paired together by non-covalent interactions; (vii) single-chain variable fragment (scFv), which consists of VL and VH chains paired together by a linker; (ix) bispecific single-chain Fv dimer, (x) scFv-Fc fragment; (xi) Fd fragment consisting of VH and CHI domains; (xii) single-domain antibodies, dAbs, which consist of either a VH domain or a VL domain; (xiii) diabodies, which consist of two scFv fragments in which the VH and VL domains are linked by a short peptide that prevents them from 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; etc.

[0076] As used herein, the term "Fc" refers to a molecule comprising a sequence of a non-antigen binding fragment, which is produced by digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may include a hinge region. The original immunoglobulin source of native Fc is preferably human-derived and may be any immunoglobulin. Fc molecules are composed of monomeric polypeptides, which can be linked to dimers or multimers by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomer subunits of a native Fc molecule ranges from 1 to 4, depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, lgGA2, and IgG4). An example of Fc is a disulfide-bonded dimer produced by papain digestion of IgG. As used herein, the term "native Fc" is a general term for monomeric, dimeric, and multimeric forms.

[0077] A F(ab) fragment typically comprises a light chain and the VH and CH1 domains of a heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment is incapable of forming a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment may also comprise a light chain comprising two variable domains separated by an amino acid linker and a heavy chain comprising two variable domains separated by an amino acid linker and a CH1 domain.

[0078] A F(ab') fragment typically comprises one light chain and a portion of one heavy chain containing more constant regions (between the CH1 and CH2 domains) such that interchain disulfide bonds can form between the two heavy chains to form the F(ab')2 molecule.

[0079] One embodiment of the present disclosure provides binding proteins having 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 be bound by a binding protein and / or can additionally be used in an animal to produce an antibody that can bind to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody and / or antibody fragment thereof that recognizes the target antigen. An antigen is bound by a binding protein, such as an antibody, through an antigen binding site, also referred to herein as a "binding portion" or "binding domain."

[0081] In certain embodiments of the present invention, one or more binding portions of the antibodies disclosed herein are antibody fragments selected from the non-limiting examples of antibody fragments listed above. In specific embodiments, one or more binding portions of the antibodies disclosed herein are Fab fragments. In a more specific embodiment, all binding portions of the antibodies 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 with one antigen binding site. For example, a monovalent antibody is an antibody with one antigen binding site. The term "multivalent binding protein" refers to a binding protein with more than one antigen binding site. For example, a multivalent antibody is an antibody with more than one antigen binding site. Non-limiting examples of multivalent binding proteins are bivalent and trivalent. The term "bivalent binding protein" refers to a binding protein with two antigen binding sites. For example, a bivalent antibody is an antibody with 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 with three antigen binding sites.

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

[0084] As used herein, the term "multispecific binding protein" refers to any binding protein, including antibodies or antibody fragments thereof, that has more than one binding site and binds to different epitopes of the same antigen or different antigenic 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 antigenic 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, a trispecific binding protein, such as an antibody or antibody fragment thereof, binds to three different antigens. In some embodiments, a trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0087] A trivalent binding protein can bind to one antigenic target (i.e., a monospecific trivalent binding protein). In other embodiments, a trivalent binding protein can bind to two antigenic targets (i.e., a bispecific trivalent binding protein). In other embodiments, a trivalent binding protein can bind to three antigenic targets (i.e., a trispecific trivalent binding protein).

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

[0089] In a preferred embodiment of the present invention, the antibodies disclosed herein are trivalent, preferably trispecific, more preferably trispecific antibodies comprising at least three binding moieties; in a specific embodiment, the antibodies of the present 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 present disclosure bind to the extracellular domain of one or more 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 set forth in 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 is overexpressed in malignant plasma cells. In some embodiments, the binding proteins of the present disclosure bind to 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" is a 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 to 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 binding proteins directed against the host immune system, more specifically against the cytotoxic activity of T cells and 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, human BCMA and human CD38. In a preferred embodiment of the present invention, the antibody is a monoclonal antibody.

[0095] In a specific embodiment, 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 would interfere with the diagnostic or therapeutic use of the binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be: (1) purified to greater than 95% by weight antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated binding protein includes binding protein in situ within recombinant cells, as at least one component of the binding protein's natural environment will not be present.

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

[0098] A nucleic acid is "isolated" or "substantially pure" when it is purified 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 other techniques well known in the art; see, e.g., F. Ausubel et al., eds. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids of the invention can be, for example, DNA or RNA, and may or may not contain intronic sequences.

[0099] The dissociation constant (K) of the binding protein D ) can be determined, for example, by surface plasmon resonance. Typically, surface plasmon resonance analysis uses a BIAcore system (GE) to measure the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR). 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, the term "K D ” refers to the dissociation constant of the interaction between a specific 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" with respect to a binding protein means that the binding protein or antigen-binding fragment thereof binds to at least about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M or lower K D The ability to bind to an antigen containing the epitope and / or to bind to the epitope with an affinity that is at least two times greater than its affinity for a nonspecific antigen.

[0101] As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the light chain domain and the heavy chain domain to fold into a cross-linked dual variable region immunoglobulin. At the sequence level, a linker can be inserted between variable domains or at the transition between a variable domain and a constant domain, respectively. The transition between domains can be identified because the approximate size of the immunoglobulin domain is well known. The precise location of the domain transition can be determined by locating peptide segments that do not form secondary structural elements such as β-sheets or α-helices, as demonstrated 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 a nucleotide sequence encoding a polypeptide chain that forms a binding protein of the present invention. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a binding protein (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein).

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

[0104] Some vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors and episomal mammalian vectors with bacterial origins of replication). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can instruct the expression of genes operably connected to them. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article. Typically, expression vectors used for recombinant DNA technology are typically in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most commonly used vector forms. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which provide equivalent functions.

[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 cells or host cells refer not only to a particular subject cell, but also to the progeny of such a cell. Due to mutations or environmental influences, certain modifications may occur in the progeny, so such progeny may not actually be the same as the parent cell, but such cells are still included within the scope of the term "host cell" as used herein. A 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). In order to recombinantly express the binding protein, the host cells are transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the polypeptide chain of the binding protein, so that the polypeptide chain is expressed in the host cells and preferably secreted into the culture medium in which the host cells are cultured, from which the binding protein can be recovered.

[0106] As used herein, the term "transformation" refers to the change of the genetic characteristics of a cell, and when a cell is modified to contain new DNA, it has been transformed. For example, a cell is transformed at a position where it is genetically modified from its native state. After transformation, the transforming DNA can be physically integrated into the chromosome of the cell and recombined with the DNA of the cell, or can be maintained instantaneously as an additional element without being replicated, or can be replicated independently as a plasmid. When the DNA replicates as the cell divides, the cell is considered to have been stably transformed. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA by a cell, and when exogenous DNA has been introduced into the interior of the cell membrane, the cell has been "transfected." Many transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.

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

[0108] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional 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 the 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 symbols used herein, according to standard usage and convention, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction.

[0109] Naturally occurring residues can be divided into several categories based on common side chain properties:

[0110] (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0111] (2) Polar hydrophilicity: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0112] (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0113] (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro;

[0114] (5) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0115] (6) Acidic: Asp, Glu;

[0116] (7) Basic: His, Lys, Arg;

[0117] (8) Residues that affect chain orientation: Gly, Pro;

[0118] (9) Aromatic: His, Trp, Tyr, Phe; and

[0119] (10) Aromatic hydrophobicity: Phe, Trp, Tyr.

[0120] Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class.

[0121] Those skilled in the art will be able to determine suitable variants of the polypeptide chain of the binding protein using well-known techniques. For example, one skilled in the art can identify suitable regions of the polypeptide chain that can be altered without destroying the activity by targeting regions believed to be less important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. Furthermore, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying the biological activity or adversely affecting the polypeptide structure.

[0122] In the context of two or more nucleic acids or peptide sequences, the term "identity" percentage refers to identical two or more sequences. When on a designated region or when unspecified, when carrying out the comparison and comparison of maximum correspondence over the entire sequence, if two sequences have the same amino acid residues or nucleotides of a specific percentage (for example, at least 60% identity, for example 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), then two sequences are "substantially identical." For sequence comparison, one sequence typically serves as a reference sequence to which test sequences are compared. Methods of sequence alignment 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 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; Clustal Omega algorithm (Sievers F, Higgins DG. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 2018 Jan; 27(1): 135-145.); MUSCULE algorithm (Edgar, RC MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113(2004). https: / / doi.org / 10.1186 / 1471-2105-5-113).

[0123] The terms "patient" or "subject" are used interchangeably herein. As used herein, the term "patient" includes both human and animal subjects. Animal subjects include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens.

[0124] As used herein, the term "treatment" refers to therapeutic treatment and preventive or prophylactic measures. People in need of treatment include people with disorders and people who are prone to disorders or people who need to prevent disorders. In a specific embodiment of the present invention, the disorder is characterized by expression (e.g., overexpression) of BCMA, and / or expression (e.g., overexpression) of CD38, including in cancer and non-cancer diseases such as gastrointestinal, nervous and pulmonary system disorders (Szlasa W et 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 present invention can be used to treat B cell malignancies and autoimmune disorders. In other specific embodiments, the antibodies or antibody fragments thereof of the present invention can be used to treat people with cancer or people who are prone to cancer, or to improve cancer in human subjects. The antibody can also be used to prevent cancer in human patients. In a more specific embodiment, the antibodies or antibody fragments thereof of the present invention are used to treat cancer, such as hematological malignancies. In a preferred embodiment, the cancer is a BCMA-expressing cancer and / or a CD38-expressing cancer. Non-limiting examples of cancers that can be treated with the antibodies or antibody fragments thereof of the invention 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 myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering Multiple myeloma, active multiple myeloma, plasma cell leukemia, lymphoma, germinal center B-cell lymphoma or B-cell acute lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, myelodysplastic syndrome (MDS), breast cancer such as 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, glioma.

[0125] The present invention also relates to a trispecific antibody or antibody fragment thereof for use as a medicament. In particular, for treating the aforementioned disorders; in particular for treating cancer and autoimmune disorders, and more particularly for treating hematological cancers, including but not limited to those mentioned above. In a preferred embodiment, the trispecific antibody of the present invention is used to treat multiple myeloma, including relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, and active multiple myeloma.

[0126] The present invention also relates to the use of the trispecific antibodies or antibody fragments thereof of the present invention for treating the above-mentioned disorders; in particular, for treating cancer and autoimmune disorders, and more particularly for treating blood 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 treating multiple myeloma, including relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, and active multiple myeloma.

[0127] The present invention also relates to methods for treating cancer and autoimmune disorders, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a trispecific antibody of the present invention. In particular, the present invention also relates to methods for treating blood cancers, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a trispecific antibody of the present invention or an antibody fragment thereof. More particularly, the blood cancer is selected from a list including but not limited to those described above. In a preferred embodiment, the present invention relates to a method for treating multiple myeloma, including relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, active multiple myeloma, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a trispecific antibody or an antibody fragment thereof disclosed herein.

[0128] The present invention also relates to pharmaceutical compositions comprising trispecific antibodies and, for example, containing one or more pharmaceutically acceptable excipients or carriers. To prepare pharmaceutical or sterile compositions comprising the trispecific antibodies or antibody fragments thereof of the present disclosure, the MBM preparation can be combined with one or more pharmaceutically acceptable excipients or pharmaceutically acceptable carriers.

[0129] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0130] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing the delivery of a binding protein.

[0131] When used to refer to a pharmaceutical composition comprising one or more binding proteins, the terms "effective amount" and "therapeutically effective amount" refer to an amount or dosage sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated over a period of time. The effective amount can vary depending on the specific binding protein used and also depends on a variety of factors and conditions associated with the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors to be considered include the patient's age, weight, and health status, as well as dose-response curves and toxicity data obtained in preclinical animal studies. Determination of the effective amount or therapeutically effective amount of a given pharmaceutical composition is within the capabilities of those skilled in the art.

[0132] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0133] Anti-CD3 binding protein

[0134] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a CD3 polypeptide (e.g., human and cynomolgus CD3 polypeptide). In some embodiments, the antigen binding domains and / or binding proteins of the present disclosure "cross-react" with human and cynomolgus CD3 polypeptides.

[0135] 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 portion that binds CD3 (e.g., binds human CD3).

[0136] In certain embodiments, an antibody or antibody fragment of the invention comprises a binding portion that binds human CD3, the binding portion comprising a heavy chain CDR set comprising an amino acid sequence 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: 304, and SEQ ID NO: 435. 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; 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 specific embodiment, the binding portion that binds to human CD3 further comprises a common 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.

[0137] Preferably, the binding portion that binds to human CD3 comprises a heavy chain CDR set comprising an amino acid sequence 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 portion 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 specific embodiment, the binding portion that binds to human CD3 further comprises a common 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. More preferably, the binding portion that binds to human CD3 comprises: a heavy chain CDR set comprising the amino acid sequences of SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; 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.

[0138] In other embodiments, the binding portion 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 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 to 67. In more specific embodiments, the binding portion that binds to human CD3 further 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.

[0139] Preferably, the binding portion 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 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 portion that binds to human CD3 further 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.

[0140] In certain embodiments, the binding portion 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.

[0141] In other embodiments, the binding portion that binds human CD3 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: 34 and 50 to 67. In more specific embodiments, the binding portion that binds human CD3 further 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.

[0142] Preferably, the binding portion that binds human CD3 comprises: a heavy chain variable region comprising a heavy chain that 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%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 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.

[0143] 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-mentioned binding portions that bind to human CD3. In any of the above-mentioned bispecific or trispecific binding proteins, the target antigen other than CD3 can be any of the following exemplary target antigens: 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 (also known as MCP-3), CC L8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-ld), 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), CD160, CD272, CD273 (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, ectonucleotide triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, F0LH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D,Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1RAP, ILILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rILI0, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17 Rb (also known as the receptor for IL25), IL18, IL22, 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), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, 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 antigenic targets are human antigenic targets. In a preferred embodiment, the antibody or antibody fragment thereof of the present invention is a trivalent bispecific antibody comprising at least two binding moieties, at least one of which binds to human CD3 and at least one of which binds to BCMA, preferably at least two of which bind to 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 to human CD3, at least one of which binds to BCMA, and at least one of which binds to CD38.

[0144] The present invention also relates to monospecific antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind CD3 (eg, bind human CD3).

[0145] In a specific aspect, the invention relates to an antibody or antibody fragment thereof 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: 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: 440. 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; 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; and a light chain CDR set of a common light chain comprising the amino acid sequence of SEQ ID NO:1; in particular, the light chain CDR set comprises the amino acid sequences of SEQ ID NO:721, SEQ ID NO:722 and SEQ ID NO:723.

[0146] In a preferred aspect, the antibody or antibody fragment that binds to human CD3 comprises: a heavy chain CDR set comprising an amino acid sequence 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0147] In preferred embodiments, the antibodies disclosed herein that comprise a binding portion that binds human CD3 as described above are monoclonal.

[0148] Anti-BCMA binding protein

[0149] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a BCMA polypeptide (e.g., human and cynomolgus monkey BCMA polypeptide). In some embodiments, the antigen binding domains and / or binding proteins of the present disclosure "cross-react" with human and cynomolgus monkey BCMA polypeptides.

[0150] 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 BCMA (e.g., binds human BCMA).

[0151] In certain embodiments, an antibody or antibody fragment of the invention comprises a binding portion that binds human BCMA, the binding portion comprising a heavy chain CDR set comprising an amino acid sequence 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; 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; 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 specific embodiment, the binding portion that binds to human BCMA further comprises a common 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.

[0152] In a more specific embodiment, the binding portion that binds to human BCMA comprises a heavy chain CDR set comprising an amino acid sequence 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. Preferably, the binding portion that binds to human BCMA comprises a CDR set comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486. In a more specific embodiment, the binding portion that binds to human CD3 further comprises a common 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 sequence of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723. Preferably, the binding portion that binds to human BCMA comprises: a heavy chain CDR set comprising the amino acid sequences of SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486; 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.

[0153] In other embodiments, the binding portion that binds human BCMA comprises a heavy chain variable region 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%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 to 109. In a more specific embodiment, the binding portion that binds human BCMA further comprises a light chain variable region comprising 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.

[0154] In other embodiments, the binding portion 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: 83 to 109, wherein the amino acid sequence of SEQ ID NOs: 83-109 further comprises the mutation N82aS. In a more specific embodiment, the binding moiety that binds human BCMA further 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.

[0155] Preferably, the binding portion that binds human BCMA comprises a heavy chain variable region 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%, 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 specific embodiment, the binding portion that binds human BCMA further comprises a light chain variable region comprising 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.

[0156] Preferably, the binding portion 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, wherein the amino acid sequences of SEQ ID NOs: 93, 101, 105 and 108 further comprise the substitution N82aS. In a more specific embodiment, the binding moiety that binds human BCMA further 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.

[0157] In a more specific embodiment, the binding portion 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.

[0158] In other embodiments, the binding portion 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 to 109. In a more specific embodiment, the binding portion that binds human BCMA further 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.

[0159] In other embodiments, the binding portion that binds to 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 to 109, wherein the amino acid sequence of SEQ ID NOs: 83-109 further comprises the mutation N82aS. In a more specific embodiment, the binding portion that binds to human BCMA further 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 an amino acid sequence of SEQ ID NO: 1.

[0160] Preferably, the binding portion that binds human BCMA comprises: a heavy chain variable region comprising a heavy chain that 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%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 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.

[0161] Preferably, the binding portion 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, 108, wherein the amino acid sequence of SEQ ID NOs: 93, 101, 105 and 108 further comprises the amino acid substitution N82aS; 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 NO:1.

[0162] 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-mentioned binding moieties that bind to human BCMA. In any of the above-mentioned bispecific or trispecific binding proteins, the target antigen other than BCMA can be any of the following exemplary target antigens: 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 (also known as MCP-3 ), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-ld), 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,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), CD27 5 (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, CXCR 3. DNGR-1, ectonucleotide triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, F0LH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1RAP, ILILIA, IL1B, IL1F10, IL2, IL4, IL4Ra , IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rILI0, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, 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, PROMI, 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, TNFa, 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 antigenic targets are human antigenic targets. In a preferred embodiment, the antibodies of the present invention or their antibody fragments are trivalent bispecific antibodies comprising at least two binding moieties, at least one of which binds to human CD3 and at least one of which binds to BCMA, preferably at least two of which bind to BCMA. In a more preferred embodiment, the antibodies of the present invention are trivalent trispecific antibodies 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.

[0163] The present invention also relates to monospecific antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind BCMA (e.g., bind human BCMA).

[0164] In a specific aspect, the invention relates to an antibody or antibody fragment thereof 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: 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. 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 SEQ ID NO:222, SEQ ID NO:348, and SEQ ID NO:474; 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; and a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0165] In a more specific aspect, the present invention relates to an antibody or antibody fragment thereof 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: 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 light chain CDR set comprising an amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0166] In preferred embodiments, the antibodies disclosed herein that comprise a binding portion that binds human BCMA as described above are monoclonal.

[0167] Anti-CD38 binding protein

[0168] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a CD38 polypeptide (e.g., human and cynomolgus CD38 polypeptide). In some embodiments, the antigen binding domains and / or binding proteins of the present disclosure "cross-react" with human and cynomolgus CD38 polypeptides.

[0169] 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 portion that binds CD38 (e.g., binds human CD38).

[0170] In certain embodiments, an antibody or antibody fragment of the invention comprises a binding portion that binds human CD38, the binding portion comprising a heavy chain CDR set comprising an amino acid sequence 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: 377, and SEQ ID NO: 498; 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: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:236, SEQ ID NO:362, and SEQ ID NO:712; ID NO:237, SEQ ID NO:363 and SEQ ID NO:700. In a more specific embodiment, the binding portion that binds human CD38 further comprises the CDR set of the common light chain 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.

[0171] In a more specific embodiment, the binding portion that binds to human CD38 comprises a heavy chain CDR set comprising an amino acid sequence 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. Preferably, the binding portion that binds to human CD38 comprises a CDR set 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 more specific embodiment, the binding portion that binds to human CD38 further comprises a common 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.

[0172] In other embodiments, the binding portion 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 to 127. In more specific embodiments, the binding portion that binds human CD38 further 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.

[0173] Preferably, the binding portion 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 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 portion that binds to human CD38 further 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 portions, 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 portion that binds to 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.

[0174] In other embodiments, the binding portion that binds human CD38 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: 110 to 127. In a more specific embodiment, the binding portion that binds human BCMA further 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.

[0175] Preferably, the binding portion that binds human CD38 comprises: a heavy chain variable region comprising a heavy chain that 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%, 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 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.

[0176] The present invention relates to multispecific (eg, bispecific or trispecific) and multivalent (eg, bivalent or trivalent) antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind human CD38.

[0177] In any of the above trispecific binding proteins, the target antigen other than CD38 can be any of the following exemplary target antigens: 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 (also known as MCP-3), C CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-ld), 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,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), CD27 5 (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, CXCR 3. DNGR-1, ectonucleotide triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, F0LH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1RAP, ILILIA, ILIB, IL1F10, IL2, IL4, IL4Ra , IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rlLI0, IL12, 1L13, 1L13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, 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, PROMI, 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, TNFa, 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 antigenic targets are human antigenic targets. In a preferred embodiment, the antibodies of the present invention are trivalent trispecific antibodies 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.

[0178] The present invention also relates to monospecific antibodies or antibody fragments thereof comprising any of the above-described binding moieties that bind CD38 (eg, bind human CD38).

[0179] In a specific aspect, the invention relates to an antibody or antibody fragment thereof 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: 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: 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: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 group comprising the amino acid sequence of SEQ ID NO:721, SEQ ID NO: ID NO:722 and SEQ ID NO:723.

[0180] In a more specific aspect, the present invention relates to an antibody or antibody fragment thereof that binds to human CD38, comprising: a heavy chain CDR set comprising an amino acid sequence 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.

[0181] In preferred embodiments, the antibodies disclosed herein comprising a binding portion that binds human CD38 as described above are monoclonal.

[0182] Trivalent bispecific anti-CD3 and anti-BCMA antibody

[0183] In certain aspects, the invention relates to trivalent bispecific antibodies or antibody fragments 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.

[0184] In a specific embodiment of the invention, the trivalent bispecific anti-CD3 and anti-BCMA antibody is a heterodimeric immunoglobulin, in particular using the previously described The heavy chain (He) heterodimerization technology was constructed (Skegro et al., (2017) J Biol Chem 292(23):9745-9759 and Stutz et al., (2020) J Biol Chem 295(28):9392-9408, WO2012131555), including BEAT (A) chain, also referred to herein as BTA, and BEAT (B) chain, also referred to herein as BTB. More specifically, a trivalent bispecific anti-CD3 and anti-BCMA antibody was designed by combining a CD3 binder as a Fab and a BCMA bi-dAb from the prior art on a silent BEAT Fc backbone (LALA mutation, Hezareh et al., 2001, J Virol, 75(24):12161-12168).

[0185] 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 a trivalent bispecific anti-CD3 and anti-BCMA antibody comprising a BTA chain and / or a BTB chain and / or a common 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 above amino acid sequence.

[0186] Trispecific anti-CD3, anti-BCMA, and anti-CD38 protein

[0187] The present invention relates to 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, also referred to herein as CD3 / BCMA / CD38 antibodies. In preferred embodiments, the present invention provides trispecific antibodies or antibody fragments thereof that bind to human CD3, human BCMA, and human CD38. In preferred embodiments of the present invention, the antibodies are monoclonal antibodies. The binding moieties that bind to CD3, BCMA, or CD38 according to the present invention are described above.

[0188] Trispecific antibodies are a group of engineered antibody derivatives that recognize three different target antigens. In a specific embodiment of the present invention, the trispecific anti-CD3, anti-BCMA, and anti-CD38 antibodies are heterodimeric immunoglobulins, particularly those based on Platform Construction of trispecific antibody technology. The platform is based on replacing the protein-protein interface of the CH3 domain pair of the antibody Fc region with the protein-protein interface of the T cell receptor (TCR) α-β constant region. Interface drives heterodimer bispecific or trispecific antibodies (bsAb or TriAb) relative to the preferential formation of homodimer pollutants.In addition, CD3 / BCMA / CD38 antibodies utilize Fab CD38 binding arms as Fab units, utilizing FabBCMA binding arms and Fab CD3 binding arms, which all utilize common light chains to prevent light chain mispairing, and light chain mispairing is a common challenge related to BSab and even more TriAb platforms. Additional engineering has also been carried out to allow for easier and faster purification processes, maintaining the natural conformation, stability and combination with neonatal Fc receptors (FcRn) of antibody structure, so as to maximize 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).

[0189] The binding moiety that binds CD3, BCMA or CD38 according to the present invention may be any of the binding moieties that bind CD3, BCMA or CD38 described above.

[0190] In certain specific embodiments, at least one binding portion that binds human CD3 comprises a heavy chain CDR set 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; at least one binding portion that binds BCMA comprises a heavy chain CDR set 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: 354, SEQ ID NO: 357; SEQ ID NO: 358, SEQ ID NO: 369, and SEQ ID NO: 470; SEQ ID NO: 361, SEQ ID NO: 362, and SEQ ID NO: 471; SEQ ID NO: 363, SEQ ID NO: 364, and SEQ ID NO: 472; SEQ ID NO: 365, SEQ ID NO: 366, and SEQ ID NO: 473; SEQ ID NO: 367, SEQ ID NO: 368, and SEQ ID NO: 474; SEQ ID NO: 369, SEQ ID NO: 370, and SEQ ID NO: 475. NO:231, SEQ ID NO:357 and SEQ ID NO:483; and at least one binding portion that binds human CD38 comprises a heavy chain CDR set 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.

[0191] More specifically, the trispecific antibodies of the invention comprise: at least one binding portion that binds to human CD3, the at least one binding portion comprising a heavy chain CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; at least one binding portion that binds to BCMA, the at least one binding portion comprising a heavy chain CDR set comprising SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486; and at least one binding portion that binds to human CD38, the at least one binding portion comprising a heavy chain CDR set 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 portion that binds to human CD38 comprises a heavy chain CDR set comprising the amino acid sequence of SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491.

[0192] 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.

[0193] 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 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 light chain CDR set 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 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: NO:722 and the light chain CDR group of SEQ ID NO:723.

[0194] 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 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 light chain CDR set 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 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: NO:722 and the light chain CDR group of SEQ ID NO:723.

[0195] The present invention also relates to antibodies or antibody fragments or antigen-binding fragments thereof that bind 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 portions, 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 portion that binds human CD3 comprises a heavy chain CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307 and SEQ ID NO: 433 and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723; the at least one binding portion that binds 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 portion that binds human CD38 comprises a heavy chain CDR set comprising SEQ ID NO: 239, SEQ ID NO: 365 and SEQ ID NO: 370. The heavy chain CDR group comprises SEQ ID NO: 491 and the light chain CDR group comprises SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0196] The present invention also relates to antibodies or antibody fragments or antigen-binding fragments thereof that bind 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 portions, 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 portion that binds human CD3 comprises a heavy chain CDR set comprising SEQ ID NO: 181, SEQ ID NO: 307 and SEQ ID NO: 433 and a light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723; the at least one binding portion that binds 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 portion that binds human CD38 comprises a heavy chain CDR set comprising SEQ ID NO: 236, SEQ ID NO: 362 and SEQ ID NO: The heavy chain CDR group comprises SEQ ID NO: 712 and the light chain CDR group comprises SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0197] In a specific aspect of the invention, the trispecific antibody comprises: at least one binding portion that binds to human CD3, the at least one binding portion 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 NO: 55, 58, 60, 62 and 66; at least one binding portion that binds to human BCMA, the at least one binding portion 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: NO:93, 101, 105 and 108 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 NO:93, 101, 105 and 108; and at least one binding portion that binds to human CD38, the at least one binding portion comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:93, 101, 105 and 108; NO: 110, 113, 122 and 111. In one 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.

[0198] In a specific aspect of the invention, the trispecific antibody comprises: at least one binding portion that binds to human CD3, the at least one binding portion 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 NO: 55, 58, 60, 62 and 66; at least one binding portion that binds to human BCMA, the at least one binding portion 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: NO: 93, 101, 105 and 108, wherein the amino acid sequence of SEQ ID NO: 93, 101, 105 and 108 further comprises the substitution N82aS; and at least one binding portion that binds to human CD38, the at least one binding portion comprising a polypeptide comprising a polypeptide selected from the group consisting of SEQ ID NO: 93, 101, 105 and 108. In one 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.

[0199] In a specific aspect of the invention, the trispecific antibody comprises: at least one binding portion that binds to human CD3, the at least one binding portion 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, 58, 60, 62 and 66; at least one binding portion that binds to human BCMA, the at least one binding portion 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: 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 specific aspect, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0200] In a specific aspect of the invention, the trispecific antibody comprises: at least one binding portion that binds to human CD3, the at least one binding portion 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, 58, 60, 62 and 66; at least one binding portion that binds to human BCMA, the at least one binding portion 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: NO:93, 101, 105 and 108, wherein the amino acid sequence of SEQ ID NO:93, 101, 105 and 108 further comprises the substitution N82aS; and at least one binding portion that binds to human CD38, the at least one binding portion 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:93, 101, 105 and 108, wherein the amino acid sequence of SEQ ID NO:93, 101, 105 and 108 further comprises the substitution N82aS; In one embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising an amino acid sequence of SEQ ID NO: 1.

[0201] More specifically, the trispecific antibodies or antibody fragments thereof of the present invention comprise: at least one binding portion that binds to human CD3, the at least one binding portion 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 NO: 55; at least one binding portion that binds to human BCMA, the at least one binding portion 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: 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 108; and at least one binding portion that binds to human CD38, the at least one binding portion comprising 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%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 110 and 113. In a more specific aspect, 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.

[0202] More specifically, the trispecific antibodies or antibody fragments thereof of the present invention comprise: at least one binding portion that binds to human CD3, the at least one binding portion 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 NO: 55; at least one binding portion that binds to human BCMA, the at least one binding portion 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: NO:108, wherein the amino acid sequence of SEQ ID NO:108 further comprises the mutation N82aS; and at least one binding portion that binds to human CD38, the at least one binding portion comprising a polypeptide comprising a polypeptide selected from the group consisting of SEQ ID NO:108. In one 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.

[0203] Even more specifically, the trispecific antibodies or antibody fragments thereof of the invention comprise: at least one binding portion that binds to human CD3, the at least one binding portion 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; at least one binding portion that binds to human BCMA, the at least one binding portion 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: : The invention also provides 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, preferably SEQ ID NO: 113. In a more specific aspect, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0204] Even more specifically, the trispecific antibodies or antibody fragments thereof of the invention comprise: at least one binding portion that binds to human CD3, the at least one binding portion 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; at least one binding portion that binds to human BCMA, the at least one binding portion 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: NO: 108, wherein the amino acid sequence of SEQ ID NO: 108 further comprises the mutation N82aS; and at least one binding portion that binds to human CD38, the at least one binding portion 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: 108, wherein the amino acid sequence of SEQ ID NO: 108 further comprises the mutation N82aS; and In one embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0205] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the present invention comprises: at least one binding portion that binds to human CD3, the at least one binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, the at least one binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 108; and at least one binding portion that binds to human CD38, the at least one binding portion 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.

[0206] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the present invention comprises: at least one binding portion that binds to human CD3, the at least one binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, the at least one binding portion 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 portion that binds to human CD38, the at least one binding portion 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.

[0207] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the present invention comprises: at least one binding portion that binds to human CD3, the at least one binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, the at least one binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 108; and at least one binding portion that binds to human CD38, the at least one binding portion 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.

[0208] In a preferred embodiment, the trispecific antibody or antibody fragment thereof of the present invention comprises: at least one binding portion that binds to human CD3, the at least one binding portion comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, the at least one binding portion 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 portion that binds to human CD38, the at least one binding portion 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.

[0209] In a more specific aspect of the invention, the trispecific antibody comprises: at least one binding portion that binds to human CD3, the at least one binding portion 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; and at least one binding portion that binds to human BCMA, the at least one binding portion 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. 591; and at least one binding portion that binds to human CD38, the at least one binding portion 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 consisting of SEQ ID NOs: 593 and 594, preferably SEQ ID NO: 594. In a more specific aspect, 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.

[0210] The present invention also relates to antibodies or antigen-binding fragments that bind 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.

[0211] In particular, the present invention also relates to antibodies or antibody fragments thereof or antigen-binding fragments that bind 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 human BCMA, and one binding portion that binds to CD38, wherein the antibody binds to human CD3, human BCMA, and human CD38, wherein the reference antibody is a trispecific antibody or antibody fragment thereof comprising at least three binding portions, 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 portion 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 light chain CDR set comprising SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and the at least one binding portion 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: and the at least one binding portion 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.

[0212] 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.

[0213] The present invention also relates to antibodies or antigen-binding fragments that bind to an epitope on human CD38, wherein the epitope on CD38 comprises 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 antibody or antigen-binding fragment has at least Preferably at least Even more preferably at least In the most preferred example, the resolution is about

[0214] 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, an epitope having at least Preferably at least Even more preferably at least In the most preferred example, the resolution is about

[0215] The present invention also provides full-length antibodies that bind to human CD3 and / or human BCMA and / or human CD38, as well as antibody fragments and / or binding portions thereof. Antibody fragments include, but are not limited to: (i) Fab fragments consisting of the VL, VH, CL, and CHI domains, including Fab' and Fab'-SH; (ii) Fd fragments consisting of the VH and CHI domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments (Ward ES et al., (1989) Nature, 341:544-546), which consist of a single variable domain; (v) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vi) single-chain Fv molecules (scFv), in which the VH and VL domains are linked by a peptide linker, which allows the two domains to associate to form an antigen binding site (Bird RE et al., (1988) Science 242:423-426; Huston et al., (1990) Science 243:424-426). JS et al., (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83), (vii) bispecific single chain Fv dimers (PCT / US92 / 09965); (viii) "diabodies" or "tribodies", multivalent or multispecific fragments constructed by genetic fusion (Tomlinson I & Hollinger P (2000) Methods Enzymol. 326: 461-79; WO94 / 13804; Holliger P et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-48), and (ix) scFv genetically fused to the same or different antibodies (Coloma MJ & Morrison SL (1997) Nature Biotechnology, 15(2): 159-163).

[0216] In certain embodiments of the present invention, the trispecific antibody is a full-length antibody or an antibody fragment thereof, wherein at least one binding portion that binds to human CD3 and / or at least one binding portion that binds to human BCMA and / or at least one binding portion that binds to human CD38 is an antibody fragment, such as a Fab region. As used herein, the term "Fab" or "Fab region" or "Fab domain" includes a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to this region in isolation or, in the case of a full-length antibody or antibody fragment, to this region.

[0217] In certain embodiments of the invention, the trispecific antibody comprises at least one binding moiety that binds to human CD3 and at least one binding moiety that binds to human BCMA fused to each other, or at least one binding moiety that binds to human CD3 and at least one binding moiety that binds to human CD38 fused to each other, or at least one binding moiety that binds to human BCMA and at least one binding moiety that binds to human CD38 fused to each other.

[0218] In one embodiment, the trispecific antibody has at least one binding moiety that binds human BCMA fused at the N-terminus 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 at the N-terminus to the C-terminus of at least one binding moiety that binds human CD38.

[0219] In another embodiment, the trispecific antibody has at least one binding moiety that binds human BCMA fused at the C-terminus to the N-terminus of at least one binding moiety that binds human CD3. In an additional embodiment, the trispecific antibody of the invention has at least one binding moiety that binds human BCMA fused at the C-terminus to the N-terminus of said at least one binding moiety that binds human CD38.

[0220] In another embodiment, the trispecific antibody has at least one binding moiety that binds human CD3 fused at the C-terminus to the N-terminus of at least one binding moiety that binds human CD38. In additional embodiments, the trispecific antibody of the invention has at least one binding moiety that binds human CD3 fused at the N-terminus to the C-terminus of said at least one binding moiety that binds human CD38.

[0221] In a specific aspect, the above binding moieties are fused to each other via a linker (eg, a peptide linker).

[0222] In a specific aspect, the binding portion of the trispecific antibody of the invention is a Fab fragment.

[0223] In an even more specific embodiment, an antibody of the invention has three binding moieties that are Fab fragments, wherein the binding moiety that binds human BCMA is fused at the C-terminus to the N-terminus of the Fab heavy or light chain of the binding moiety that binds human CD3, or wherein the binding moiety that binds human BCMA is fused at the C-terminus to the N-terminus of the Fab heavy or light chain of the binding moiety that binds human CD38.

[0224] In an even more specific embodiment, an antibody of the invention has three binding moieties that are Fab fragments, wherein the binding moiety that binds human BCMA is fused at the N-terminus to the C-terminus of the Fab heavy or light chain of the binding moiety that binds human CD3, or wherein the binding moiety that binds human BCMA is fused at the N-terminus to the C-terminus of the Fab heavy or light chain of the binding moiety that binds human CD38.

[0225] In a specific aspect of the invention, the binding portion of the antibody fused as described above is fused via a peptide linker. Preferably, the linker has a sequence selected from SEQ ID NO: 604 and 605. Other preferred peptide linkers are listed in the "Linkers" section below.

[0226] Preferably, the trispecific antibody according to the invention comprises a set of three amino acid chains having a sequence that is 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 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; 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; SEQ ID NO: 548, SEQ ID NO: 549 and SEQ ID NO: 1; 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.

[0227] 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 consisting of: 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.

[0228] In a more preferred embodiment, the trispecific antibody or antibody fragment thereof according to the present invention comprises a set of three amino acid chains of amino acid sequences SEQ ID NO: 546, SEQ ID NO: 547 and SEQ ID NO: 1.

[0229] Nucleic Acids

[0230] The polynucleotides of coding for forming protein-binding polypeptides are incorporated into recombinant expression vectors by standard recombinant DNA methods, and such vectors are introduced into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING:A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd edition). Enzymatic reaction and purification techniques can be carried out according to the manufacturer's instructions, as generally completed in this area or as described herein. Unless specifically defined, the nomenclature and laboratory procedures and techniques related to analytical chemistry as described herein, synthetic organic chemistry, and medical science and pharmaceutical chemistry are well known in the art and commonly used those. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, preparation, delivery and patient treatment.

[0231] Other aspects of the present disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid molecules comprise 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.

[0232] Some aspects of the present disclosure relate to kits for polynucleotides. In some embodiments, one or more polynucleotides are vectors (e.g., expression vectors). The kit is particularly useful for producing one or more binding proteins as described herein, such as bispecific or trispecific binding proteins of the present disclosure. In some embodiments, the kit comprises one, two, three, or four polynucleotides.

[0233] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of a binding protein encoding nucleic acid sequence. A promoter can refer to a nucleic acid control sequence that directs 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 in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a binding protein encoding sequence if the promoter affects the transcription or expression of the encoding sequence. Examples of promoters may include, but are not limited to, promoters obtained from viral genomes (e.g., polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, polyomavirus, such as simian virus 40 (SV40), etc.), heterologous eukaryotic promoters (e.g., actin promoter, immunoglobulin promoter, heat shock promoter, etc.), 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), lac system, tip system, tac system, trc system, major operator and promoter region of bacteriophage λ, promoter of 3-phosphoglycerate kinase, promoter of yeast acid phosphatase, and promoter of yeast α-mating factor. The polynucleotide encoding the binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or readily identified by one skilled in the art.

[0234] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably connected to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, alpha-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic cell viruses (such as the SV40 enhancer (bp 100-270) on the late side of the replication origin, cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, adenovirus enhancer, etc.). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, sindbis virus, measles virus, herpes virus, lentivirus, retrovirus, adeno-associated virus vector, etc.). Expression vectors may be used to transfect host cells, such as 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 can be used to transfect any cell of interest.

[0235] Other aspects of the present disclosure relate to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any binding protein 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 vector described herein. In some embodiments, the one or more vectors are expression vectors.

[0236] Isolated host cells

[0237] Other aspects of the present disclosure relate to isolated host cells 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 Saccharomyces 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 TM 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 may include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture, and their variants 293T), Expi293 TM 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 cancer cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma cell lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0238] Other aspects of the present disclosure relate to a method for producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any host cell described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any isolated nucleic acid, vector, and / or vector system described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell.

[0239] Methods for culturing host cells under conditions that express proteins are well known to those of ordinary skill in the art. Cell culture is the process of growing cells under controlled conditions in an artificial environment. The terms "cell culture," "cultivation," or "host cell culture" refer to the growth, propagation, and / or maintenance of cells under controlled, artificial conditions. Optimal culture conditions are achieved by controlling and adjusting several parameters, including the formulation of the cell culture medium, the operating parameters of the bioreactor, the nutrient supply pattern, and the duration of the culture. The culture medium formulation is optimized to promote cell viability and growth; 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 include 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 the initial basal medium and are used until depleted, while waste products accumulate. In fed-batch culture, additional feed medium is supplied to prevent nutrient depletion and prolong the culture. Separately, in perfusion mode, cells in culture are continuously replenished with fresh medium containing nutrients, which flows through the bioreactor to remove cellular waste products. The skilled artisan understands that the culture period is important in that it needs to be long enough for the cells to produce consistent amounts of product, but it cannot be so long as to compromise cell viability.

[0240] Methods for isolating proteins from cultured host cells are well known to those of ordinary skill in the art and include, for example, chromatographic techniques. The term "chromatography" refers to the operation of separating compounds in a mixture based on their ability to interact with the stationary phase of a chromatographic resin, from which these compounds can be retained or eluted. Chromatographic techniques are known in the art, such as ion exchange chromatography, which separates ions and polar molecules based on their charge differences. Examples of ion exchange chromatography techniques are cation exchange chromatography and anion exchange chromatography. Affinity chromatography relies on the specific interaction of proteins with fixed ligands. Non-limiting examples of ligands that can be used to purify antibodies by affinity chromatography are protein A and protein G. Protein A is a cell wall protein isolated from Staphylococcus aureus that has the property of binding to immunoglobulin Fc (but not to the antigen binding site). In this article, the term "protein A chromatography" refers to affinity chromatography in which the ligand that binds to the antibody is protein A, wherein the term "protein A" includes native protein A, recombinant protein A, and analogs or derivatives thereof. Protein analogs and derivatives all retain their binding activity to antibodies (e.g., IgG Fc). Protein G is a cell wall protein isolated from group G Streptococcus, and its N-terminal portion is an albumin binding domain, and its C-terminal portion is an IgG binding domain and a cell wall binding domain. In this article, the term "protein G chromatography" refers to affinity chromatography, wherein the ligand that binds to the antibody is protein G, wherein the term "protein G" includes native protein G, recombinant protein G and its analogs or derivatives. Methods for isolating proteins from cultured host cells may include an affinity chromatography step (such as protein A affinity chromatography) followed by one or more steps of ion exchange chromatography and / or size exclusion chromatography.

[0241] Antibody framework

[0242] In certain embodiments of the present invention, the binding portions of the trispecific antibodies or antibody fragments thereof are fused to each other (i.e., linked together). In specific embodiments, at least one binding portion that binds to human CD3 and at least one binding portion that binds to human BCMA, or at least one binding portion that binds to human CD3 and at least one binding portion that binds to human CD38, or at least one binding portion that binds to human BCMA and at least one binding portion that binds to human CD38 are fused to each other. In more specific embodiments, at least one binding portion that binds to human CD3 and at least one binding portion that binds to human BCMA, or at least one binding portion that binds to human CD3 and at least one binding portion that binds to human CD38, or at least one binding portion that binds to human BCMA and at least one binding portion that binds to human CD38 are fused to each other via a linker (e.g., a peptide linker).

[0243] In certain embodiments, the binding moiety that binds to human BCMA is fused at the N-terminus to the C-terminus of the binding moiety that binds to human CD3. In other embodiments, the binding moiety that binds to human BCMA is fused at the N-terminus to the C-terminus of the binding moiety that binds to human CD38. In other embodiments, the binding moiety that binds to human BCMA is fused at the C-terminus to the N-terminus of the binding moiety that binds to human CD3. In other embodiments, the binding moiety that binds to human BCMA is fused at the C-terminus to the N-terminus of the binding moiety that binds to human CD38.

[0244] In certain embodiments, the binding moiety that binds to human CD3 is fused at the N-terminus to the C-terminus of the binding moiety that binds to human BCMA. In other embodiments, the binding moiety that binds to human CD3 is fused at the N-terminus to the C-terminus of the binding moiety that binds to human CD38. In other embodiments, the binding moiety that binds to human CD3 is fused at the C-terminus to the N-terminus of the binding moiety that binds to human BCMA. In other embodiments, the binding moiety that binds to human CD3 is fused at the C-terminus to the N-terminus of the binding moiety that binds to human CD38.

[0245] In certain embodiments, the binding moiety that binds to human CD38 is fused at the N-terminus to the C-terminus of the binding moiety that binds to human BCMA. In other embodiments, the binding moiety that binds to human CD38 is fused at the N-terminus to the C-terminus of the binding moiety that binds to human CD3. In other embodiments, the binding moiety that binds to human CD38 is fused at the C-terminus to the N-terminus of the binding moiety that binds to human BCMA. In other embodiments, the binding moiety that binds to human CD38 is fused at the C-terminus to the N-terminus of the binding moiety that binds to human CD3.

[0246] In preferred embodiments, the binding moiety that binds human BCMA is fused at the N-terminus to the C-terminus of the binding moiety that binds human CD3, or the binding moiety that binds human BCMA is fused at the C-terminus to the N-terminus of the binding moiety that binds human CD38.

[0247] In a preferred embodiment, the binding moieties linked as described above are fused to each other via a linker (eg, a peptide linker).

[0248] In a preferred embodiment, the binding moiety that binds human BCMA is fused at the 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 the C-terminus to the N-terminus of the binding moiety that binds human CD38 via a peptide linker.

[0249] In an even more preferred embodiment, the binding moiety that binds human BCMA is fused at the N-terminus to the C-terminus of the binding moiety that binds human CD3 via a peptide linker of SEQ ID NO: 605, or the binding moiety that binds human BCMA is fused at the C-terminus to the N-terminus of the binding moiety that binds human CD38 via a peptide linker of SEQ ID NO: 604.

[0250] In a specific embodiment of the invention, the binding portion of the antibodies disclosed herein is an antibody fragment selected from the previously mentioned non-limiting list of "antibody fragments". In a more specific embodiment, 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.

[0251] In certain embodiments, the antibodies of the present invention comprise an Fc domain and at least three binding moieties. In more specific embodiments, at least the first binding moiety is fused at the N-terminus to the C-terminus of the second binding moiety. In certain aspects, the first binding moiety (e.g., Fab domain) located between the Fc domain and the second binding moiety (e.g., the second Fab domain) of the antibody is referred to as the "Fc proximal end," while the second binding moiety is referred to as the "Fc distal end."

[0252] In specific embodiments, the second binding moiety (eg, Fc-distal binding moiety) is fused to the N-terminus of the VH (variable heavy chain) domain of the first binding moiety (eg, Fc-proximal binding moiety).

[0253] In a preferred embodiment, the binding moiety that binds CD38 is Fc proximal and the binding moiety that binds CD3 or the binding moiety that binds BCMA is Fc distal; preferably, the binding moiety that binds CD38 is proximal and the binding moiety that binds BCMA is Fc distal. In a preferred embodiment, 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.

[0254] In a specific embodiment of the present invention, the trispecific antibody or antibody fragment thereof of the present invention comprises three binding moieties, one binding to human CD3, one binding to human BCMA and one binding to human CD38, said three binding moieties being Fab fragments, and the antibody is prepared using a method based on the method described above. Platform The invention relates to a trispecific antibody constructed using trispecific antibody technology. In one embodiment, the binding portion that binds human BCMA and the binding portion that binds human CD38 are located on the same arm of the BEAT antibody. More specifically, the binding portion of the binding arm that binds human BCMA is distal to the Fc, and the binding portion that binds human CD38 is proximal to the Fc. In a more specific embodiment, the binding portion that binds human BCMA and the binding portion that binds human CD38 are fused via a flexible linker; the flexible linker preferably comprises the amino acid sequence of SEQ ID NO: 604. In a preferred embodiment, the binding portion that binds human CD3 and the binding portion that binds human BCMA are located on the same arm of the BEAT antibody. More specifically, the binding portion of the binding arm that binds human CD3 is distal to the Fc, and the binding portion that binds human BCMA is proximal to the Fc. In a more specific embodiment, the binding portion that binds human CD3 and the binding portion that binds human BCMA are fused via a flexible linker; the flexible linker preferably comprises the amino acid sequence of SEQ ID NO: 605.

[0255] connector

[0256] In some embodiments, linkers of the invention, such as L1, L2, L3, and L4, range from no amino acids (length = 0) to about 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. 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 binding protein can all have the same amino acid sequence, or can all have different amino acid sequences.

[0257] Examples of suitable linkers include single glycine (Gly) residues; diglycine peptides (Gly-Gly); tripeptides (Gly-Gly-Gly); peptides with four glycine residues; peptides with five glycine residues; peptides with six glycine residues; peptides with seven glycine residues; and peptides with eight glycine residues. Other combinations of amino acid residues may be used, such as the peptides GGGT or GGGGS, or repeats of the peptides, such as the peptides GGGGS GGGGS GGGGS. In a preferred embodiment, the linker of the invention connecting the 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 comprising one or more conservative substitutions thereof.

[0258] The examples listed above are not intended to limit the scope of the present disclosure in any way, and linkers comprising 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 have been shown to be suitable for use in conjunction with proteins. For other descriptions of linker sequences, see, for example, WO2012135345 and International Application No. PCT / US2017 / 027488.

[0259] The identity and sequence of amino acid residues in the joint can change according to the type of secondary structure element required for realizing in the joint.For example, glycine, serine and alanine are best for the joint with maximum flexibility.If more rigid and extended joints are needed, some combinations of glycine, proline, threonine and serine are useful.Any amino acid residue can be considered as a joint in combination with other amino acid residues, to build a larger peptide joint according to the desired property as required.

[0260] Fc region and constant domains

[0261] The present invention also relates to a trispecific antibody comprising an Fc region. In some embodiments, the binding protein of the present disclosure comprises an antibody fragment, including but not limited to an antibody Fab, (Fab')2, Fab'-SH, Fv, or scFv fragment. In some embodiments, the binding protein of the present disclosure comprises an antibody fragment containing an Fc region, including but not limited to an antibody Fab, (Fab')2, Fab'-SH, Fv, or scFv fragment.

[0262] In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or a polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region includes the antibody hinge, CH1, CH2, CH3, and optionally, the CH4 domain. 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 includes one or more mutations described herein.

[0263] In certain embodiments, the trispecific antibodies or antibody fragments thereof of the invention comprise a non-naturally occurring Fc domain.

[0264] In some embodiments, the binding proteins of the present disclosure include one or two Fc variants. As used herein, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" can include molecules or sequences that are humanized from non-human native Fc. In addition, native Fc contains regions that can be removed because they provide structural features or biological activities that are not required by the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues or in which one or more Fc sites or residues have been modified, which affects or is involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed 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).

[0265] As used herein, the term "parent antibody" or "parent immunoglobulin" includes unmodified antibodies that are subsequently modified to produce variants. The parent antibody may be a naturally occurring antibody, a non-naturally occurring antibody, or a variant or engineered form of a naturally occurring antibody. The parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or an amino acid sequence encoding it. In a preferred embodiment 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.

[0266] In a specific embodiment, the present invention is a heterodimeric antibody or antibody fragment thereof having an engineered Fc comprising a first engineered CH3 domain and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises a substitution that favors heterodimerization. In a specific embodiment of the present invention, the antibody is prepared using the previously described The heavy chain (He) heterodimerization technology is constructed (Skegro et al., (2017) J Biol Chem 292(23):9745-9759 and Stutz et al., (2020) J Biol Chem 295(28):9392-9408, WO2012131555), wherein the BEAT(A) chain is also referred to as BEAT(A) in this article and comprises a first engineered CH3 domain, and the BEAT(B) chain is also referred to as BEAT(B) in this article and comprises a second engineered CH3 domain.

[0267] In a more specific embodiment, the first engineered CH3 domain comprises one or more substitutions selected from the group consisting of: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering); and the second engineered CH3 domain comprises substitutions selected from the group consisting of: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering); in specific aspects, the heterodimeric immunoglobulin or heterodimeric fragment is heterodimerized via the first engineered CH3 domain and the second engineered CH3 domain.

[0268] In a more specific embodiment, the present invention discloses a method based on The invention relates to a trispecific antibody or antibody fragment thereof constructed by the platform, wherein the BEAT(A) CH3 domain comprises one or more substitutions selected from the group consisting of Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and optionally BEAT(A) further comprises a substitution selected from the group consisting of D356E, L358M, N384S, V397M, V42 2I, H435R and Y436F (EU numbering); and the BEAT(B) CH3 domain comprises one or more substitutions selected from the group consisting of 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).

[0269] In certain aspects, the present invention also relates to a trispecific heterodimeric antibody or antibody fragment thereof comprising a first engineered CH3 domain and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises substitutions selected from the group consisting of: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises substitutions selected from the group consisting of: Q347E, Y34 9A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment is heterodimerized by the first engineered CH3 domain and the second engineered CH3 domain, characterized in that the trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding portions, each binding portion binding to a different antigen.

[0270] In certain aspects, the bispecific antibodies of the present invention comprise a variant Fc region comprising at least one amino acid modification relative to the Fc region of a parent antibody, and 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.

[0271] In a specific embodiment of the present invention, BEAT (A) and BEAT (B) have been engineered to increase Fc effector function. More specifically, the CH2 domain of the Fc region has been engineered to include at least one amino acid modification. More specifically, BEAT (A) comprises one or more substitutions at positions selected from the group consisting of 324, 334, 269, 298, 239, 332, and 333; and BEAT (B) comprises one or more substitutions at positions selected from the group consisting of 324, 334, 269, 298, 239, 332, and 333; preferably 324, 334, 269, 289, 298, 333. Even more specifically, BEAT(A) comprises one or more substitutions selected from the group consisting of S324N, K334E, K334A, E269D, S298A, S239D, I332E and E333A; and BEAT(B) comprises one or more substitutions at positions selected from the group consisting of S324N, K334E, K334A, E269D, S289A, K334A, E333A. In certain specific embodiments, BEAT(A) comprises a set of mutations selected from the group consisting of: 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 consisting of 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 the mutation S324N; or the mutations S324N and K334E; or the mutations E269D, S298A, S324N and K334A; or the mutations E269D, S298A, S324N and E334A; or the mutations S298A, S324N and E333A; or the mutations S298A, S324N and K334A; or the mutations S324N and K334E.In a particularly preferred embodiment, BEAT(A) comprises the mutations S239D, I332E and S324N, and BEAT(B) comprises the mutation S324N.

[0272] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or effector functions (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)) of the Fc region.

[0273] 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.

[0274] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fcyl and / or Fcyll binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fcyl and / or Fcyll binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are 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 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233, 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 236; and / or R409K.

[0275] In some embodiments, the binding proteins of the present disclosure include one or more mutations to improve purification, for example, by adjusting the affinity to purification reagents.For example, it is known that if one of the two Fc regions of heterodimer form contains a mutation that reduces or eliminates the binding to protein A, heterodimer binding proteins can be selectively purified from their homodimer form, because heterodimer form will have a higher affinity than any homodimer form for the purification based on protein A, and can be selectively eluted from protein A (see, for example, Smith, EJ. et al. (2015) Sci.Rep.5:17943) by using different pH. In some embodiments, mutations include substitutions at the position corresponding to the position 435 and 436 of the human IgG1 or IgG4 indexed according to EU, wherein amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain and a third polypeptide chain, the second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and wherein only one of the first Fc region and the second Fc region comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F.

[0276] In order to improve the yield of some binding proteins (eg, bispecific or trispecific binding proteins), the CH domain can be altered by BEAT technology, which is described in detail with several examples in International Publication No. WO2012131555.

[0277] In some embodiments, the binding proteins of the present 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 mutation comprises a substitution at a position corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain and a third polypeptide chain, the second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region and / or the 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, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first Fc region and / or the second Fc region is a human IgG1 Fc region.

[0278] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to reduce effector function, such as 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 connected to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions; wherein the third polypeptide chain further comprises a second Fc region connected to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions; wherein the first Fc region and the second Fc region are human IgG1 Fc regions; and wherein the first Fc region and the second Fc region each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitution is L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are 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 CH2 and CH3 immunoglobulin heavy chain constant regions; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant regions; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG4 Fc regions, and wherein the Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A.In some embodiments, the binding protein comprises a second polypeptide chain and a third polypeptide chain, the second polypeptide chain further comprising a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the third polypeptide chain further comprising a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and wherein the first Fc region and the second Fc region each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A.

[0279] 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 Fc region and / or the second Fc region is a human IgG1 Fc region. In some embodiments, the first Fc region and / or the second Fc region is a human IgG4 Fc region. For further description of Fc mutations at position 329, see, e.g., Shields, RL et al. (2001) J. Biol. Chem. 276: 6591-6604 and WO 1999051642.

[0280] In a specific embodiment, the trispecific antibodies or antibody fragments thereof according to the present invention comprise a non-naturally occurring Fc domain comprising L234A / L235A(LALA) / P329A substitutions in the CH2 domain. More specifically, the trispecific antibodies or antibody fragments thereof disclosed herein may comprise an Fc, wherein the Fc may comprise a first BEAT CH3 domain and a second BEAT CH3 domain as described above, or a naturally occurring CH domain as described above, or any other non-naturally occurring CH3 and CH2 domain comprising L234A / L235A(LALA) / P329A substitutions.

[0281] Application of binding proteins

[0282] 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 detecting and quantifying one or more target antigens. The binding protein will bind to one or more target antigens with an affinity suitable for the assay method being employed.

[0283] 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 directly or indirectly produce a detectable signal. For example, the detectable moiety can be a radioisotope, such as 3H, 14C, 32P, 35S, 125I, 99Tc, U1In, or 67Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or fluorescein; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0284] Binding proteins can also be used 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 determined. 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.

[0285] For clinical or research applications, in certain embodiments, the binding protein can be conjugated to a cytotoxic agent. A variety of antibodies coupled 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 agents to antibodies are known in the art; for example, see Parslow, AC et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

[0286] The present disclosure also relates to a test kit comprising binding proteins and other reagents for detecting target antigen levels in biological samples. Such reagents can include detectable labels, closed serum, positive and negative control samples and detection reagents. In some embodiments, the test kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system and / or host cell as described herein. In some embodiments, the test kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container is equipped with a composition, which itself or in combination with another composition can effectively treat, prevent and / or diagnose a disease, and can have a sterile entrance (for example, the container can be an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose and / or treat a selected disease. Alternatively or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0287] In some embodiments, the binding proteins of the present disclosure are administered to patients in need thereof to treat or prevent cancer. In some embodiments, the present disclosure relates to methods for preventing and / or treating proliferative diseases or disorders (e.g., cancer). In some embodiments, the method comprises administering a therapeutically effective amount of a heterodimeric antibody as described herein or a pharmaceutical composition related thereto to a patient. In some embodiments, the present disclosure relates to the use of heterodimeric antibodies as described herein or a pharmaceutical composition related thereto for preventing and / or treating proliferative diseases or disorders (e.g., cancer) in patients in need thereof. In some embodiments, the present disclosure relates to heterodimeric antibodies as described herein or a pharmaceutical composition related thereto for the preparation of a drug for preventing and / or treating proliferative diseases or disorders (e.g., cancer) in patients in need thereof. In some embodiments, the patient is a human. In some embodiments, the binding protein comprises an 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.

[0288] 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, leukemia (such as acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia, acute promyelocytic leukemia, acute myeloblastic leukemia and acute megakaryoblastic leukemia, chronic myeloid leukemia, multiple myeloma, plasma cell 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), Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, myelodysplastic syndrome (MDS), breast cancer such as 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, glioma.

[0289] Binding protein therapeutic compositions and their administration

[0290] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the present disclosure.Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a binding protein or binding protein-drug conjugate mixed with a pharmaceutically or physiologically acceptable formulation selected according to the mode of administration.

[0291] Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.

[0292] Pharmaceutical compositions may contain formulation materials used to alter, maintain, or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrin), proteins (such as serum albumin, gelatin or immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming anti-ionization agents, The pharmaceutical composition may include, but is not limited to, a pharmaceutical composition comprising: a molecule (such as sodium), a preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), a solvent (such as glycerol, propylene glycol, or polyethylene glycol), a sugar alcohol (such as mannitol or sorbitol), a suspending agent, a surfactant, or a wetting agent (such as pluronic; PEG; sorbitol esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol, or tylosaccharate), a stability enhancer (such as sucrose or sorbitol), a tonicity enhancer (such as an alkali metal halide—preferably sodium chloride or potassium chloride—or mannitol sorbitol), a delivery vehicle, a diluent, an excipient, and / or a pharmaceutical adjuvant (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed., A.R. Gennaro, ed., Mack Publishing Company 1990) and subsequent editions, incorporated herein by reference for any purpose).

[0293] The optimal pharmaceutical composition will be determined by a skilled artisan based on, for example, the intended route of administration, delivery form, and desired dosage. Such composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0294] The primary vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. In addition, the binding protein can be formulated as a liquid formulation or a lyophilizate using a suitable excipient.

[0295] Pharmaceutical compositions of the present disclosure can be selected for intravenous or subcutaneous delivery. Alternatively, compositions for inhalation or delivery (such as oral) through the digestive tract can be selected. The preparation of such pharmaceutically acceptable compositions is within the scope of the art.

[0296] When considering parenteral administration, the therapeutic composition employed can be the form of pyrogen-free, parenteral acceptable aqueous solution, which is included in the associated proteins of the expectation in the pharmaceutically acceptable vehicle. The particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the associated proteins are formulated as sterile, isotonic solution, suitably preserve. Another kind of preparation can relate to the preparation of required molecule and reagent, this reagent such as injectable microspheres, biological erodible particles, polymeric compound (such as polylactic acid or polyglycolic acid), pearl or liposome, and this preparation provides control or sustained release of product, and this product can be sent by storage injection then. Hyaluronic acid can also be used, and this can play the effect of promoting circulation duration. Other suitable means for introducing required molecule comprise implantable drug delivery device.

[0297] 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 also be formulated with a propellant for aerosol delivery. In another embodiment, the solution can be atomized.

[0298] It is also contemplated that certain formulations can be administered orally. In one embodiment of the present disclosure, the bound protein administered in this manner can be formulated with or without those carriers typically used to formulate solid dosage forms such as tablets and capsules. For example, the capsule can be designed to release the active portion of the formulation in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to promote the absorption of the bound protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and adhesives can also be used.

[0299] Another pharmaceutical composition can include an effective amount of a binding protein in admixture with a nontoxic excipient suitable for tablet preparation. A unit dosage form solution can be prepared by dissolving the tablet in sterile water or another suitable carrier. 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.

[0300] Other pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including preparations related to proteins bound in a continuous or controlled delivery formulation. Technology for preparing a variety of other continuous or controlled delivery modes, such as liposome carriers, bioerodible microparticles or porous beads and reservoir injections, is also well known to those skilled in the art. Other examples of sustained-release formulations include semipermeable polymer matrices of molded article form, such as films or microcapsules. Sustained-release matrix can include copolymers of polyester, hydrogel, polylactide, L-glutamic acid and γ-ethyl-L-glutamic acid, poly-(2-hydroxyethyl-methacrylate), ethylene vinyl acetate or poly-D (-) -3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0301] Pharmaceutical compositions for in vivo administration generally must be sterile. This can be accomplished by filtration through a sterile filtration membrane. When the composition is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle.

[0302] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that requires reconstitution (e.g., lyophilized) before administration.

[0303] The present disclosure also includes kits for producing single-dose administration units. The kits can each include a first container with a dried protein and a second container with an aqueous formulation. Kits comprising single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilizing syringes) are also included within the scope of the present disclosure.

[0304] The effective amount of the protein-binding pharmaceutical composition to be used in treatment will depend on, for example, the treatment context and purpose. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will therefore vary, in part, depending on the molecule being delivered, the indication for which the protein-binding is used, the route of administration, and the patient's size (body weight, body surface or organ size) and condition (age and general health). Therefore, the clinician can titrate the dosage and modify the route of administration to obtain the best therapeutic effect.

[0305] The frequency of administration depends on the pharmacokinetic parameters of the protein-binding agent in the formulation used. Usually, the clinician will administer the composition until the dosage for achieving the desired effect is reached. Therefore, the composition can be administered over time with a single dose, two or more doses (which may or may not contain the desired molecule of the same amount) or by implantation or catheter continuous infusion. The further refinement of suitable dosage is routinely performed by those of ordinary skill in the art, and within the scope of their conventionally performed tasks. Suitable dosage can be determined by using appropriate dose-response data.

[0306] The route of administration of the pharmaceutical composition is consistent with known methods, for example, oral; subcutaneous; by intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional injection; by sustained release system; or by implant device. When needed, the composition can be administered continuously by bolus injection or by infusion, or by implant device.

[0307] The compositions can also be administered topically by implanting membranes, sponges, or other suitable materials onto which the desired molecule has been absorbed or encapsulated. Where an implant device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, timed-release bolus, or continuous administration.

[0308] Treatment options

[0309] The present disclosure also provides a method for treating multiple myeloma in a subject in need thereof. The method uses a heterodimeric antibody that engages CD3, BCMA and CD38 in such a way that malignant cells are transiently connected to the engaged 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 such a way that target cells are destroyed to the greatest extent possible while reducing unwanted side effects (e.g., uncontrolled cytokine release).

[0310] As used herein, the term "treat" refers to both therapeutic treatment and preventative or prophylactic measures. The antibodies of the present invention are administered to a subject in need thereof; persons in need of treatment include persons having a disorder as well as persons susceptible to a disorder or persons in need of prevention of a disorder. The antibodies of the present invention can be used to treat persons having cancer or persons 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, such as a human subject having a BCMA-expressing cancer and / or a CD38-expressing cancer (such as multiple myeloma, such as relapsed / refractory multiple myeloma). Relapsed myeloma is characterized by a recurrence of the disease after a response. Examples of laboratory and radiological criteria indicative of the disease include, but are not limited to, an increase in serum or urine monoclonal protein (M-protein) of >25%, or a difference of >25% between serum free light chains involved and not involved from the nadir or the development of new plasmacytomas or hypercalcemia. Sonneveld et al., Haematologica. 2016 Apr;101(4):396-406.

[0311] In patients with non-secretory disease, relapse is characterized by an increase in bone marrow plasma cells. The signature of relapsed disease is also characterized by the appearance or recurrence of one or more CRAB criteria or rapid and consistent biochemical relapse. Refractory myeloma is a myeloma that does not respond to treatment. Relapsed / refractory multiple myeloma refers to disease that becomes unresponsive or progresses after therapy or within 60 days of the last treatment in patients who have previously achieved at least a minimal response to previous therapy. Sonneveld, supra; Anderson et al., Leukemia. 2008; 22(2): 231-239.

[0312] The methods of the present disclosure include administering to a subject between about 0.0001 mg / kg to about 200 mg / kg, e.g., between about 0.0005 mg / kg to about 200 mg / kg, or between about 0.001 mg / kg to about 200 mg / kg, or between about 0.0015 mg / kg to about 200 mg / kg, or between about 0.005 mg / kg to about 200 mg / kg, or between about 0.01 mg / kg to about 200 mg / kg, or between about 0.015 mg / kg to about 200 mg / kg, or between about 0.03 mg / kg to about 200 mg / kg, or between about 0.05 mg / kg to about 200 mg / kg, or between about 0.06 mg / kg to about 200 mg / kg, or between about 0.09 mg / kg to about 200 mg / kg, or at about 0.1 mg / kg. In some embodiments, the dosage range is between about 0.1 mg / kg and about 200 mg / kg, or between about 0.12 mg / kg and about 200 mg / kg, or between about 0.15 mg / kg and about 200 mg / kg, or between about 0.18 mg / kg and about 200 mg / kg, or between about 0.2 mg / kg and about 200 mg / kg, or between about 0.3 mg / kg and about 200 mg / kg, or between about 0.36 mg / kg and about 200 mg / kg, or between about 0.5 mg / kg and about 200 mg / kg, or between about 0.6 mg / kg and about 200 mg / kg, or between about 0.7 mg / kg and about 200 mg / kg, or between about 1 mg / kg and about 200 mg / kg, or between about 1.2 mg / kg and about 200 mg / kg, or between about 1.5 mg / kg and about 200 mg / kg. For example, between about 0.0001 mg / kg to about 20 mg / kg, or between about 0.0005 mg / kg to about 10 mg / kg, or between about 0.0005 mg / kg to about 5 mg / kg, or between about 0.0005 mg / kg to about 1.5 mg / kg, or between about 0.0005 mg / kg to about 1.2 mg / kg. The present disclosure also includes dosage ranges including any values ​​between the above disclosed intervals.For example, the trispecific antibodies of the invention are administered to a patient in a single dose or multiple doses selected from the group consisting 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 g, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.36 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 1 mg / kg, at least about 1.2 mg / kg, at least about 1.5 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 7 mg / kg, at least about 10 mg / kg, at least about 12 mg / kg, at least about 16 mg / kg, at least about 50 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 200 mg / kg.

[0313] In various aspects of the method, the dosage is adjusted during the course of treatment. For example, an initial dose is administered to a subject 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 dose of the trispecific antibody at least once during the course of treatment. Alternatively, the dosage can be reduced during the course of treatment such that the amount of the heterodimeric antibody decreases as treatment progresses.

[0314] The present disclosure contemplates a method in which the trispecific antibodies of multiple (i.e., two or more) dosages are administered during treatment. A single dose can be administered at any interval, such as once a week, twice a week, three times a week, four times a week, or five times a week. A single dose can be administered every two weeks, every three weeks, or every four weeks. In other words, in some aspects, a waiting period of at least two weeks has been experienced between administering heterodimeric antibodies to the subject. The waiting time between dosage administrations need not be consistent during the entire treatment period. In other words, the interval between dosages can be adjusted during treatment. In some aspects, the method is included in administering two dosages of heterodimeric antibodies to the subject weekly in the first and second weeks for treatment (i.e., twice a week for the 1st and 2nd weeks), administering a dosage of heterodimeric antibodies to the subject weekly in the third and fourth weeks for treatment (i.e., once a week for the 3rd and 4th weeks), and administering a dosage of heterodimeric antibodies every two weeks from the 5th week to the end of treatment (i.e., a two-week waiting period is present between the dosage from the 5th week to the end of treatment).

[0315] Alternatively, in various aspects, the method comprises administering a dose of the trispecific antibody weekly during weeks 1-4 of treatment, and optionally administering a dose of the trispecific antibody every two weeks starting in week 5 until the end of treatment.

[0316] Multiple doses of the heterodimeric antibody are administered over a treatment period of, for example, three months to about 18 months, or about three months to about 12 months, or about three months to about nine months, or about three months to about six months, or about three months to about eight months, or about six months to about 18 months, or about six months to about 12 months, or about eight months to about 12 months, or about six months to about eight months, or about eight months to about 12 months (e.g., about eight 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, wherein the dosage is twice a week, once a week, once every two weeks, or once every four weeks.

[0317] "Treating" multiple myeloma means achieving any positive therapeutic response to the disease. For example, a positive therapeutic response includes one or more of the following improvements in the disease: (1) a decrease in the number of tumor cells; (2) an increase in tumor cell death; (3) inhibition of tumor cell survival; (4) a decrease in paraprotein production by tumor cells; (5) inhibition (i.e., slowing to some extent, preferably stopping) of tumor growth; (6) an increase in patient survival; and (7) some relief of one or more symptoms associated with the disease or condition. Tumor response can be assessed 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 counting of circulating tumor cells to assess changes in tumor morphology (i.e., total tumor burden, tumor size, etc.). A complete therapeutic response (i.e., absence of clinically detectable disease, normalization of any previously abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF), or abnormal monoclonal protein) is not required; any degree of improvement is contemplated.

[0318] The antibody or its antibody fragment can be administered to the subject in any suitable manner, for example, via intravenous, subcutaneous, intraarterial, intralymphatic, intrathecal, intracerebral, intraperitoneal, intracerebrospinal, intradermal, intraarticular, intrasynovial, oral, topical or inhalation routes, osmotic pumps, drug encapsulation such as nanoparticle encapsulation, surface coating-mediated drug delivery. For example, the antibody can be administered intravenously as a bolus or by continuous infusion over a period of time. In a specific embodiment, the antibody is infused via intravenous infusion, for example, over a period of about 30 minutes to about four hours. Optionally, the infusion time is reduced in subsequent administration. For example, in one embodiment, the first dose of the antibody is administered over a period of about four hours, and subsequent doses are administered over a period of two hours or less. In this regard, the first dose of the antibody is optionally administered over a period of about four hours, the second dose of the antibody is optionally administered over a period of about two hours, and subsequent doses are optionally administered over a period of about 30 minutes. In another specific embodiment, the trispecific antibody of the present invention is administered subcutaneously.

[0319] In some cases, the subject has previously received treatment for a cancer such as multiple myeloma. For example, the subject may have previously been administered an immunomodulatory drug (thalidomide, lenalidomide, pomalidomide), a proteasome inhibitor (such as pomalidomide, bortezomib, or carfilzomib), dexamethasone, doxorubicin, or a combination thereof.

[0320] Optionally, the subject has previously been treated with an anti-CD38 monospecific antibody such as daratumumab In various embodiments, the subject is relapsed or refractory to prior anti-CD38 monospecific antibody therapy. The method may include a waiting period between prior administration of the anti-CD38 monospecific antibody and administration of the heterodimeric antibody.

[0321] Combination therapy

[0322] Optionally, the antibodies of the invention or antibody fragments thereof are part of a treatment regimen that includes administration of one or more other therapeutic agents, radiation therapy, stem cell transplantation, and the like.

[0323] The methods of the present disclosure optionally further comprise administering dexamethasone to the subject.

[0324] Dexamethasone can be administered by any route, such as the routes described herein. Preferably, dexamethasone is administered intravenously or orally. When dexamethasone is administered intravenously, it is optionally administered to the subject within one hour before administration of the antibody. Dexamethasone is optionally administered in an amount of about 8 mg or about 4 mg.

[0325] In various embodiments, the method of the present disclosure also includes administering chemotherapeutics. Non-limiting examples of DNA damaging chemotherapeutics 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, streptozotocin, dacarbazine, methotrexate, mitomycin C and cyclophosphamide); DNA intercalating agents (e.g., cisplatin, oxaliplatin and carboplatin); DNA intercalating agents and free radical generating agents, such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin and hydroxyurea).

[0326] Chemotherapeutic agents that disrupt 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 (such as epoxomicin and carfilzomib), beta-lactone inhibitors (such as lactacystin, MLN 519, NPI-0052, Salinosporamide A), compounds that generate dithiocarbamate complexes with metals (such as disulfiram), and certain antioxidants (such as epigallocatechin-3-gallate, catechin-3-gallate, and Salinosporamide A); NF-κB inhibitors, including inhibitors of IκB kinase; antibodies that bind to proteins overexpressed in cancer and thereby downregulate 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, which inhibition downregulates cell replication.

[0327] Treatment regimens may include administration of other antibody therapeutics, such as elotuzumab (a humanized monoclonal antibody 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; rukatumomab (also known as HCD122) and darizumab (also known as SGN-40), which target CD40; and lovotozumab, which targets CD56. For reviews of antibody therapies for the treatment of multiple myeloma, see, for example, 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.

[0328] In some embodiments, the heterodimeric antibody is administered prior to, concurrently with, or after treatment with a proteasome inhibitor (bortezomib, carfilzomib, and ixazomib), an immunomodulatory drug (IMiD) such as the thalidomide derivatives pomalidomide and lenalidomide, the histone deacetylase inhibitor panobinostat, an antibody such as daratumumab, terituzumab, isatuximab, elotuzumab, talquetamab, alnuctamab, belantamab, elranatamab, or any other of the above compounds and / or drugs and / or biotherapeutics (also including venetoclax, mafodotin, seliniexor, dexamethasone, doxorubicin, melflufen, CAR-T cell therapy, and combinations thereof).

[0329] All cited references are expressly incorporated herein by reference in their entirety.While specific embodiments of the present invention have been described above for illustrative purposes, those skilled in the art will appreciate that various changes may be made to the details without departing from the invention described in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0330] Figure 1: Anti-human CD3 C1 candidates bind to human CD3 and induce T cell activation. The binding of anti-CD3 candidates to human PBMCs was evaluated by flow cytometry (A). Simultaneously, 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 is the mean + / - standard deviation of three independent measurements from one representative experiment. The experimental setup and analysis are described in Example 1.

[0331] 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 evaluated by flow cytometry. Each bar represents the geometric mean of fluorescence from one replicate tested at 8 nM from one representative experiment. Experimental setup and analysis are described in Example 2.

[0332] Figure 3 : A format for tool molecules used to assess the T cell activation propensity of CD3 binders.

[0333] Figure 4: 2+1C1-D6 / BCMA candidates trigger the most potent tumor cell killing and T cell activation in a redirected lysis assay. The ability of 2+1CD3xBCMA-dAbx2 candidates to trigger T cell-mediated killing of NCI-H929 tumor cells (A) was assessed in a redirected lysis assay at an effector to target ratio of 5:1 and measured by flow cytometry after 48 hours of incubation. In the same assay, activation of human T cells was assessed by flow cytometry, measured by the percentage of CD69 upregulation on CD4 T cells (B). Each point is the mean + / - standard deviation of two technical replicates from one representative donor. The experimental setup and analysis are described in Example 3.

[0334] Figure 5 : HDX epitope mapping of C1-D6 binders on hCD3ye. hCD3ye showed a significant decrease (-10) in deuterium uptake upon binding IgG at AA182-197 (YVCYPRGSKPEDANFY (SEQ ID NO: 726)), indicating that this is the major epitope. Figure 5 Shown is a differential heat map comparing hydrogen / deuterium exchange of hsCD3γ(23-103)-hsCD3e(23-118) alone with hydrogen / deuterium exchange of hsCD3γ(23-103)-hsCD3e(23-118) and anti-CD3-C1-D6 IgG1 LALA.

[0335] Figure 6: Blockade of human BCMA / human APRIL and human BCMA / human BAFF interactions following binding of anti-BCMA-D3 and anti-BCMA-E6 Fab to human BCMA 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 anti-histidine antibody. A solution of 50 nM recombinant BCMA-Fc protein pre-mixed with varying concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-D3 Fab in running buffer was injected onto A) immobilized human APRIL or B) immobilized human BAFF. Similarly, a solution of 50 nM recombinant BCMA-Fc protein pre-mixed with varying concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-E6 Fab in running buffer was injected onto C) immobilized human APRIL or D) immobilized human BAFF. The graph shows data expressed as resonance units (abbreviated RU; Y-axis) versus time (X-axis).

[0336] 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 evaluated by flow cytometry. Each bar represents the geometric mean of fluorescence from one replicate tested at 10 nM from one representative experiment. Experimental setup and analysis are described in Example 6.

[0337] Figure 8: Epitope binning of human CD38 by anti-CD38-E2 Fab, anti-CD38-B3 Fab, and daratumumab Fab using Octet biolayer interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin SA biosensor. Fab antibody 1 (Ab1) was injected at 200 nM in kinetic buffer onto immobilized human CD38 to achieve surface saturation. A premixed solution of Ab1 and Fab antibody 2 (Ab2) at a final concentration of 200 nM each was then injected onto the saturated surface (competition phase). The same experimental procedure was performed using 400 nM of Ab1 alone in the competition phase as a control for surface saturation. The graph shows binding to the sensor tip as a function of 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. The curves are labeled with the Ab2 clone name.

[0338] Figure 9: Schematic representation of a CD3 / BCMA / CD38 trispecific antibody. The CD3 / BCMA / CD38 antibody was constructed using three Fabs targeting three independent antigens, whereby the "Fc-distal" Fab domain was fused to the N-terminus of the VH domain of the "Fc-proximal" Fab. A) Configuration BO (BTB-outside) CD3 / BCMA / CD38 has a CD38 binder on Arm A, a CD3-binding arm at an Fc-distal position on the outside of Arm B, and BCMA at an "Fc-proximal" position on Arm B, while for B) Configuration C, CD3 / BCMA / CD38 has a CD3-binding arm on Arm A, a BCMA binder at an Fc-distal position on Arm B, and a CD38 binder at an Fc-proximal position on Arm B.

[0339] FIG10 : Thermograms of CD3 / BCMA / CD38 antibodies obtained by differential scanning calorimetry (DSC), including thermal transition analysis. Thermograms and thermal transition analysis for the following: A. TREAT CD3 / BCMA / CD38-115; B. TREAT CD3 / BCMA / CD38-117; C. TREAT CD3 / BCMA / CD38-118; and D. TREAT CD3 / BCMA / CD38-119. The unfolding of the domains corresponding to the different transitions is indicated in parentheses after the corresponding temperature.

[0340] Figure 11: Surface plasmon resonance measurement of hCD3εδ by CD3 / BCMA / CD38 antibodies. Biotinylated hCD3εδ (CD3E & CD3D-377H, Creative Biomart, USA) was immobilized on the sensor surface via a biotin capture reagent (a modified streptavidin that can reversibly attach to the surface of a CAP chip). CD3 / BCMA / CD38 antibodies were injected over the immobilized hCD3εδ in HBS-EP+ buffer at a concentration series (as described in the figure). The figure shows data expressed as resonance units (abbreviated as RU; Y-axis) versus time (s, X-axis) for the following: A. TREAT CD3 / BCMA / CD38-115; B. TREAT CD3 / BCMA / CD38-117; C. TREAT CD3 / BCM A / CD38-118; and D. TREAT CD3 / BCM A / CD38-119. Binding affinity to human CD3εδ was measured using a steady-state affinity model. This model uses a plot of equilibrium response (Req; Y-axis) versus analyte concentration (M; X-axis) to perform affinity calculations according to the following equation: Req = K A ×C×Rmax / (K A ×C×n+1), the concentration at 50% saturation is the equilibrium dissociation constant (K D ).

[0341] Figure 12: Surface plasmon resonance measurement of human CD38 by CD3 / BCMA / CD38 antibodies. TM, His Tag, CD8-H82E7, Acrobiosystems, USA) was immobilized on the sensor surface via a biotin capture reagent (a modified streptavidin that can reversibly attach to the surface of a CAP chip). CD3 / BCMA / CD38 antibodies were injected onto immobilized hCD38 in HBS-EP+ buffer at a concentration series (as described in the figure). The figure shows data expressed as resonance units (abbreviated as RU; Y-axis) versus time (s, X-axis) for the following: A. TREAT CD3 / BCMA / CD38-115; B. TREAT CD3 / BCMA / CD38-117; C. TREAT CD3 / BCMA / CD38-118 and D. TREAT CD3 / BCMA / CD38-119. Binding affinity to human CD38 was measured using a 1:1 Langmuir binding model. The model assumes a 1:1 interaction between the ligand and the analyte, and the association rate constant (k) was determined for each sample. a ), dissociation rate constant (k d ) and affinity (K D ). Using this model, K D is defined as the ratio of the included dissociation rate constant to the association rate constant, i.e., K D =k d / k a The measurement includes a zero concentration sample for reference. 2 The K values ​​and residuals are used to evaluate the quality of fit between the experimental data (sensorgram fc2-fc1) and the individual binding models. The solid line represents the measured data, while the dashed line represents the simulated fit. D : equilibrium dissociation constant; k a : Association rate constant. K d : Dissociation rate constant.

[0342] Figure 13: Surface plasmon resonance measurement of human BCMA by CD3 / BCMA / CD38 antibodies. Biotinylated hBCMA (biotinylated human BCMA / TNFRSF17 protein, His, Avitag TMDMF Filed, BCA-H82E4, Acrobiosystems, USA) was immobilized on the sensor surface via a biotin capture reagent (a modified streptavidin that can reversibly attach to the surface of a CAP chip). CD3 / BCMA / CD38 antibodies were injected onto immobilized hBCMA in HBS-EP+ buffer at a concentration series (as described in the figure). The figure shows data expressed as the relationship between the number of resonance units (abbreviated as RU; Y axis) and time (s, X axis) for the following: A. TREAT CD3 / BCMA / CD38-115; B. TREAT CD3 / BCMA / CD38-117; C. TREAT CD3 / BCMA / CD38-118 and D. TREAT CD3 / BCMA / CD38-119. The binding affinity for human CD38 was measured using a 1:1 Langmuir binding model. The model assumes a 1:1 interaction between the ligand and the analyte, and the association rate constant (k a ), dissociation rate constant (k d ) and affinity (K D ). Using this model, K D It is defined as the ratio of the dissociation rate to the association rate, i.e., K D =k d / k a The measurements included a zero concentration sample for reference. Chi2 values ​​and residual values ​​were used to evaluate the quality of fit between the experimental data (sensorgram fc2-fc1) and the individual binding models. The solid line represents the measured data, while the dashed line represents the simulated fit. K D : equilibrium dissociation constant; k a : association rate constant; k d : Dissociation rate constant.

[0343] Figure 14: Surface plasmon resonance binding of selected TREAT CD3 / BCMA / CD38 to Fc receptors compared to trastuzumab IgG1. (A) CD3 / BCMA / CD38-115, (B) CD3 / BCMA / CD38-117, (C) CD3 / BCMA / CD38-118, (D) CD3 / BCMA / CD38-119 and trastuzumab IgG1.

[0344] Figure 15 : AC-SINS profiles of selected TREAT CD3 / BCMA / CD38.

[0345] Figure 16: CD3 / BCMA / CD38 candidates bind to human primary CD38-negative T cells. Binding of anti-CD3 candidates to CD38-negative human T cells was evaluated by flow cytometry, and the binding curves are plotted in Figure 16. Figure 16A Each point represents the mean + / - standard deviation of six measurements from independent T cell donors from two separate experiments. K values ​​determined from binding curves are plotted. D values ​​and compared them using paired one-way ANOVA followed by Tukey HSD post hoc comparisons Figure 16B The results were compared to the benchmark in Table 1. # indicates significant difference from 83A10-TCBcv. The experimental setup and analysis are described in Example 18.

[0346] Figure 17: CD3 / BCMA / CD38 candidates bind to cell lines showing variable expression of CD38 and BCMA. Candidates CD3 / BCMA / CD38 candidates (-118, -119) and 83A10-TCBcv benchmarks were evaluated for binding to CD38 and BCMA by flow cytometry on different cell lines showing variable expression of CD38 and BCMA, namely KMS-12-BM (A), MOLP-8 (B) and NCI-H929 (C). Each point is the mean + / - standard deviation of three measurements from three separate experiments. The experimental setup and analysis are described in Example 18.

[0347] Figure 18 Figure 3: The CD3 / BCMA / CD38-118 candidate induces the most potent in vitro killing of multiple myeloma cell lines displaying variable expression of CD38 and BCMA. The ability of the CD3 / BCMA / CD38 candidate and benchmark to trigger T cell-mediated killing of KMS-12-BM (A), MOLP-8 (B), and NCI-H929 (C) tumor cells was assessed in a redirected lysis assay at an effector to target ratio of 5:1 and measured by flow cytometry after 72 hours of incubation. The half-maximal effective killing concentration (EC 50 ) values ​​were determined by redirected lysis assay. Each point represents the EC of an individual donor 50 Values ​​were compared using paired one-way ANOVA followed by Tukey HSD post hoc comparisons. * indicates statistical significance compared to TNB-F2B BEAT Fc. # indicates statistical significance compared to 83A10-TCBcv. + indicates statistical significance compared to BEAT CD3 / CD38. Three independent experiments were performed for each candidate, with a total of six donors. Experimental setup and analysis are described in Example 18.

[0348] Figure 19: CD3 / BCMA / CD38 candidates induce strong T cell activation and proliferation associated with KMS-12-BM killing. The ability of CD3 / BCMA / CD38 candidates and benchmarks to trigger T cell-mediated killing of KMS-12-BM cells and thus trigger T cell activation and proliferation was assessed in a redirected lysis assay at a 5:1 effector to target ratio. At the endpoint, CD4 + and CD8 + T cell proliferation and CD25 expression. This figure shows that activated or proliferating EC 50 Each point represents the EC of an individual donor. 50 Values. Three independent experiments were performed for each candidate, with a total of 6 donors. Means were compared using paired one-way ANOVA followed by Tukey HSD post hoc comparisons. * indicates statistical significance compared to TNB-F2B BEAT Fc. # indicates statistical significance compared to 83A10-TCBcv, and + indicates statistical significance compared to BEAT CD3 / CD38. Experimental setup and analysis are described in Example 18.

[0349] Figure 20: Compared with the positive control and benchmark 83A10-TCBcv, the CD3 / BCMA / CD38 candidate induces minimal cytokine release associated with on-target off-tumor activity. The release of cytokines was evaluated in a high-density assay. At the endpoint, the supernatant was stored at -80 ° C and the cytokine release of IFN-γ (A), TNF-α (B), IL-2 (C), IL-6 (D) and IL-10€ cytokines was evaluated using mesoscale discovery (MSD) assay. Each bar is the mean + / - standard deviation. A total of 4 PBMC donors were tested in two independent experiments. The dotted lines show positive (PHA) and negative controls. The experimental setup and analysis are described in Example 18.

[0350] Figure 21: CD3 / BCMA / CD38 candidates do not bind to erythrocytes compared to daratumumab and mololimab. Binding of candidate CD3 / BCMA / CD38 candidates and benchmarks to erythrocytes was evaluated by flow cytometry (A). Each point represents the area under the curve (AUC) of the dose-response curve for each donor. Mean values ​​were compared using paired one-way ANOVA followed by Dunnett post hoc comparisons with daratumumab (*P<0.05) (B). The experimental setup and analysis are described in Example 18.

[0351] Figure 22: CD3 / BCMA / CD38 candidates do not induce hemagglutination. The tendency of CD3 / BCMA / CD38 candidates and benchmarks to induce hemagglutination was evaluated in the Coombs assay. Each point represents the mean + / − standard deviation of Coombs from six donors. The experimental setup and analysis are described in Example 18.

[0352] Figure 23: CD3 / BCMA / CD38_118 showed excellent NCI-H929 killing in multiple killing modes compared to the anti-CD38 benchmark. The ability of CD3 / BCMA / CD38 candidates and benchmarks to trigger NCI-H929 cell lysis was evaluated in a multiple killing modes (MmoAK) assay in the presence of human serum and IL-2. Each point represents the mean + / − mean standard error of the percentage of target killing (A), the EC 50 (B) and the area under the curve (AUC) of the dose-response curve for each donor (C). The Kruskal-Wallis, followed by Dunn's post-comparison, or paired one-way ANOVA test, followed by Tukey's post-comparison, was used to compare the means (5-8 donors) (*P < 0.05, **0.01 < P < 0.05, ***P < 0.01). The experimental setup and analysis are described in Example 18.

[0353] Figure 24: CD3 / BCMA / CD38 candidates control NCI-H929 tumors in a dose-dependent manner and have higher potency than the benchmark 83A10-TCBcv at lower doses. NCG female mice were subcutaneously implanted with 1 × 10 7 NCI-H929 MM tumor cells (CD38++ and BCMA++) on day -10 and intraperitoneally inoculated with 1 × 10 7 PBMC from 2 healthy human donors (Donor A: n = 9 mice / group, Donor B: n = 9 mice / group). On day 0 when the mean tumor volume reached approximately 150 mm 3Mice were randomized at 4 hr and treated with intravenous injections once a week on days 0, 7, and 14 for 3 weeks. A. Growth of NCI-H929 tumors after treatment with 0.5 mg / kg dose (left), 0.1 mg / kg dose (middle), or 0.02 mg / kg dose (right) in NCG mice (n=9 mice / group) inoculated with donor A (top) or donor B (bottom) PBMCs. Mean ± SEM is shown with last observation carried forward (LOCF). B. Plasma concentrations (ng / mL) of the indicated molecules at the indicated doses on day 13 or 14 after treatment (after two intravenous injections) in NCG mice (n=3 mice / group) inoculated with donor A (left) or donor B (right). Molecular concentrations were determined using human BCMA-specific antigen capture ELISA (a method for quantifying anti-BCMA human IgG). Mean ± SD is shown, the values ​​within the bars represent the mean concentrations, and the values ​​above represent the fold differences.

[0354] Figure 25: CD3 / BCMA / CD38 candidate significantly delayed the growth of NCI-H929 tumors at a dose of 0.1 mg / kg compared to vehicle or CD3 sham control. NCI female mice were implanted subcutaneously with ×10 7 NCI-H929 MM tumor cells (CD38++ and BCMA++) were intraperitoneally inoculated with 1×10 7 PBMCs. On day 0, when the average tumor volume reached approximately 180 mm 3 Mice were randomized at 4 hr and treated with intravenous injections once a week on days 0, 7, and 14 for 3 weeks. A. NCI-H929 tumor growth after treatment with a 0.1 mg / kg dose in NCG mice (n=9 mice / group) inoculated with donor A (top) or donor B (bottom) PBMCs. Mean ± SEM is shown with last observation carried forward (LOCF). B. Plasma concentrations (ng / mL) of the indicated molecules on day 10 (for vehicle and CD3 / BCMA / CD38-122) or day 14 (for the remaining groups) after treatment (after two intravenous injections) in NCG mice (n=3 mice / group) inoculated with donor A (top) or donor B (bottom) PBMCs. Molecular concentrations were determined using human BCMA-specific antigen capture ELISA, a method for quantifying anti-BCMA human IgG. Mean ± SD is shown.

[0355] Figure 26: The effectiveness of CD3 / BCMA / CD38-118, -119, and 83A10-TCBcv in controlling NCI-H929 tumors correlates with the extent of T cell infiltration in the tumor microenvironment.3 At , NCG female mice implanted with NCI-H929 MM tumor cells and intraperitoneally inoculated with PBMCs from 2 healthy human donors (donor A and donor B) were treated with a single intravenous dose of 0.1 mg / kg of the molecule. After 2 or 6 days, the tumors and spleens of the treated mice were harvested and analyzed ex vivo by flow cytometry (n=2-3 mice / group / time point). A. Histograms show the expression of human TCRαβ in gated human CD45+ cells on day 2 (left) and day 6 (right) after a single dose in the tumor. The numbers indicate the percentage of positive cells. Legend from top to bottom: vehicle, 83A10-TCBcv, CD3 / BCMA / CD38-118, CD3 / BCMA / CD38-119, CD3 / BCMA / CD38-122. B. The figure depicts the number of human TCRαβ cells per gram of tumor at the indicated days and PBMC donors. Mean ± SD is shown. C. Contour plots showing the expression of human CD69 versus human CD25 in tumor-infiltrating CD8+ T cells on day 2 after dosing with the indicated molecules. Numbers in the graph indicate percentages. D. Graphs showing the percentage of CD8 T cells positive for human CD69 (left) and human CD25 (right) in tumors (top) and spleens (bottom) on day 2 after a single dose. Mean ± SD is shown.

[0356] Figure 27 :CD3 / BCMA / CD38 candidate induces secretion of human inflammatory cytokines in the tumor microenvironment. On day 0, when the average tumor volume reached approximately 180 mm 3 At 1 , NCG female mice implanted with NCI-H929MM tumor cells and intraperitoneally inoculated with PBMCs from two healthy human donors (donor A and donor B) were treated with a single intravenous dose of 0.1 mg / kg of the molecule. After 2 or 6 days, the tumors of the treated mice were harvested and the tumor supernatants were recovered. The tumor supernatants were analyzed for cytokine detection using Luminex with a cytokine and chemokine 34-Plex human kit (n=2-3 mice / group / time point). The graph shows the concentration of the indicated cytokines in the tumor supernatant normalized per gram of tumor on day 2 or day 6 after administration of the indicated molecules. Mean ± SD is shown.

[0357] Figure 28: CD3 / BCMA / CD38 candidates are at least as effective as 83A10-TCBcv in controlling BCMA-lowCD38-low KMS-12-BM tumors. 1×10 7 KMS-12-BM MM tumor cells (CD38 low and BCMA low) were intraperitoneally inoculated with 1×10 7PBMCs. On day 8, when the average tumor volume reached approximately 150 mm 3 Mice were randomized at 4 hr and treated with intravenous injections once weekly on days 9, 16, and 23 for 3 weeks. A. KMS-12-BM tumor growth in NSG mice (n=8 mice / group) inoculated with PBMCs from donors C (top) or D (bottom) after treatment with a dose of 0.5 mg / kg (left), 0.1 mg / kg (middle), or 0.02 mg / kg (right). Mean ± SEM is shown with last observation carried forward (LOCF). B. Serum concentrations (ng / mL) of the indicated molecules at the indicated doses on day 23 or 30 after treatment (at trough exposure, before the third dose (day 23) or one week later) in NSG mice (n=3-6 mice / group) inoculated with PBMCs from donors C (top) or D (bottom). Molecular concentrations were determined using a human BCMA-specific antigen capture ELISA, a method for quantifying anti-BCMA human IgG. Mean ± SD are shown, values ​​within the bars represent mean concentrations, and values ​​above represent fold differences.

[0358] Figure 29: Treatment with 0.5 mg / kg of CD3 / BCMA / CD38 candidate resulted in complete regression of KMS-12-BM tumors from one PBMC donor. 1×10 7 KMS-12-BM MM tumor cells (CD38 low and BCMA low) were intraperitoneally inoculated with 1×10 7 PBMCs. On day -1, when the average tumor volume reached approximately 120 mm 3 Mice were randomized at 4 hr and treated with intravenous injections once a week on days 0, 7, and 14 for 3 weeks. A. KMS-12-BM tumor growth after treatment with a dose of 0.5 mg / kg in NSG mice (n=8 mice / group) inoculated with donor C (left) or donor D (right) PBMCs. Mean ± SEM is shown with last observation carried forward (LOCF). B. Serum concentrations (ng / mL) of the indicated molecules on day 6 after treatment (after one intravenous injection) in NSG mice (n=3-4 mice / group) inoculated with donor C (left) or donor D (right) PBMCs. Molecular concentrations were determined using a human BCMA-specific antigen capture ELISA, a method for quantifying anti-BCMA human IgG. Mean ± SD is shown, and the values ​​within the bars represent the mean concentrations.

[0359] Figure 30: The therapeutic efficacy of CD3 / BCMA / CD38 candidates is correlated with the extent and activation state of T cell infiltration in the tumor microenvironment.3 At 14:15 PM, NSG female mice implanted with KMS-12-BM MM tumor cells and inoculated intraperitoneally with PBMCs from two healthy human donors (Donor C and Donor D) were treated with a single intravenous dose of 0.5 mg / kg of the molecule. Two or six days later, tumors and spleens from treated mice were harvested and analyzed ex vivo by flow cytometry (n=2-3 mice / group / time point). A. Histogram showing gated human CD45 expression in tumors at day 2 (left) and day 6 (right) after a single dose. + A. Expression of human TCR α β in cells. The numbers indicate the percentage of positive cells. Legends from top to bottom: vehicle, 83A10-TCBcv, CD3 / BCMA / CD38-118, CD3 / BCMA / CD38-119, CD3 / BCMA / CD38-122. B. The figure depicts the number of human TCR α β cells per gram of tumor when indicating the number of days and PBMC donors. Mean ± SD is shown. C. The contour plot shows the expression of human CD69 to human CD25 in tumor-infiltrating CD8+ T cells on the second day after administration with the indicated molecules. The numbers in the figure indicate percentages. D. The figure shows the % of human CD69 (left) and human CD25 (right) positive CD8 T cells in the tumor (top) and spleen (bottom) on the second day after a single dose. Mean ± SD is shown.

[0360] Figure 31 :CD3 / BCMA / CD38 candidate induces secretion of human cytotoxic cytokines in the tumor microenvironment. On day 0, when the average tumor volume reached approximately 120 mm 3 At 14 days post-administration, NSG female mice implanted with KMS-12-BM MM tumor cells and intraperitoneally inoculated with PBMCs from two healthy human donors (Donor C and Donor D) were treated with a single intravenous dose of 0.5 mg / kg of the molecule. Two or six days later, tumors from treated mice were harvested and tumor supernatants recovered. Tumor supernatants were analyzed for cytokine detection using Luminex with a cytokine and chemokine 34-Plex human kit (n=2-3 mice / group / time point). The graph shows the concentration (in ng) of the indicated cytokines in tumor supernatants normalized per gram of tumor at day 2 or day 6 after dosing with the indicated molecule. Mean ± SD is shown.

[0361] Figure 32 : Pharmacokinetic evaluation of CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-119 in TG32 SCID mice. TG32 SCID mice were injected IV with a single 5 mg / kg dose of CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-119, and plasma TREAT concentrations were assessed. Data are plotted as the mean + / - SD of 6 mice.

[0362] Figure 33 : CD3 / BCMA / CD38-118 binding to CD3-expressing T cells. Binding of CD3 / BCMA / CD38-118 to the CD3xBCMA benchmark 83A10-TCBcv, Anoukinomab, and Terituzumab was evaluated by flow cytometry on isolated human T cells. Each point is the mean + / - standard deviation of five to six donors from two separate experiments. The experimental setup and analysis are described in Example 22.

[0363] Figure 34: CD3 / BCMA / CD38-118 binds to cell lines that display variable expression of CD38 and BCMA or lack one antigen. The binding of CD3 / BCMA / CD38-118 to the CD3xBCMA benchmark 83A10-TCBcv, Anouketuzumab, and Terituzumab was evaluated by flow cytometry on different cell lines that display variable expression of CD38 and BCMA, namely KMS-12-BM (A), MOLP-8 (B), NCI-H929 WT (C), NCI-H929 BCMA KO (D), and NCI-H929 CD38 KO€. Each point is the mean + / - standard deviation of three measurements from three separate experiments. The experimental setup and analysis are described in Example 22.

[0364] Figure 35: CD3 / BCMA / CD38-118 shows higher binding to cell lines displaying variable expression of CD38 and BCMA compared to control molecules lacking BCMA or CD38 binders. The binding of CD3 / BCMA / CD38-118 and control, CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-151 was analyzed by flow cytometry on different cell lines displaying variable expression of CD38 and BCMA, namely KMS-12-BM (A), MOLP-8 (B), NCI-H929 WT (C), NCI-H929 BCMA KO (D), NCI-H929 CD38-118 and control, CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-151. Evaluation was performed on . Each point is the mean + / - standard deviation of three measurements from three separate experiments. The experimental setup and analysis are described in Example 22.

[0365] Figure 36: CD3 / BCMA / CD38-118 induces the most potent KMS-12-BM killing in vitro in the absence or presence of individual soluble factors. The ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmark 83A10-TCBcv, Anoukinomab, and Terituzumab to trigger T cell-mediated killing of KMS-12-BM was assessed in a redirected lysis assay at an effector to target ratio of 5:1 in the absence or presence of individual soluble factors and measured by flow cytometry after 48 hours of incubation. The percentage of killing and the associated half-maximal effective killing concentration (EC) to no Ab were calculated. 50 ) values ​​were determined by reorientation analysis. Each point represents the EC of an individual donor 50 Values. The bars show the mean of the data set. Means were compared using a mixed-effects model (when some values ​​were missing) or paired one-way ANOVA (when all values ​​were included) followed by Tukey HSD post hoc comparisons. #p value <0.0001 for CD3 / BCMA / CD38-118 relative to all other molecules with soluble factors. Experimental setup and analysis are described in Example 22.

[0366] Figure 37: CD3 / BCMA / CD38-118 induces the most potent KMS-12-BM killing in vitro in the absence or presence of individual soluble factors. The ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmark 83A10-TCBcv, Anoukinomab, and Terituzumab to trigger T cell-mediated killing of KMS-12-BM was assessed in a redirected lysis assay at an effector to target ratio of 5:1 in the absence or presence of individual soluble factors and measured by flow cytometry after 48 hours of incubation. Percent cytotoxicity and the associated half-maximal effective killing concentration (EC 50 ) values ​​were determined by reorientation analysis. Each point represents the EC of an individual donor 50 Values. The bars show the mean of the data set. Means were compared using a mixed-effects model (when some values ​​were missing) or paired one-way ANOVA (when all values ​​were included) followed by Tukey HSD post hoc comparisons. #p value <0.0001 for CD3 / BCMA / CD38-118 relative to all other molecules with soluble factors. Experimental setup and analysis are described in Example 22.

[0367] Figure 38: CD3 / BCMA / CD38-118 induces the most potent KMS-12-BM killing in vitro in the absence of competing soluble factors. The ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmarks 83A10-TCBcv, Anouketuzumab, and Terituzumab to trigger T cell-mediated killing of KMS-12-BM was assessed in a redirected lysis assay at a 5:1 effector to target ratio in the presence (left, open symbols) or absence of a combination of soluble factors (right; soluble BCMA, soluble CD38, and APRIL, all black symbols) and measured by flow cytometry after 48 hours of incubation. Percent killing, percent cytotoxicity, and associated half-maximal effective killing concentration (EC) were compared to the absence of Ab. 50 ) values ​​were determined by reorientation analysis. Each point represents the EC of an individual donor 50 The bars show the mean values ​​of the data sets. Mean values ​​were compared using a mixed-effects model (when some values ​​were missing) or paired one-way ANOVA (when all values ​​were included) analysis, followed by Tukey HSD post hoc comparisons. The experimental setup and analysis are described in Example 22.

[0368] Figure 39: CD3 / BCMA / CD38-118 induces the most potent in vitro killing of all cell lines. The ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmarks 83A10-TCBcv, Anouketuzumab, and Terituzumab to trigger T cell-mediated killing of multiple myeloma cell lines was assessed using T cells isolated on KMS-12-BM (A, D), MOLP-8 (B, E), and NCI-H929 (C, F) cells in a redirected lysis assay at an effector to target ratio of 5:1 and measured by flow cytometry after 48 hours of incubation. Percent killing (A, B, C), percent cytotoxicity (D, E, F), and associated half-maximal effective killing concentration (EC) to no Ab. 50 ) values ​​were determined by reorientation analysis. Each point represents the EC of an individual donor 50 Values. Lines show the mean of the data set. Means were compared using a mixed-effects model (when some values ​​were missing) or paired one-way ANOVA (when all values ​​were included) followed by Tukey HSD post hoc comparisons. Ns: no statistical difference; *: p = 0.05-0.01; **: p = 0.01-0.001; ***: p = 0.001-0.0001; ****: p < 0.0001. The experimental setup and analysis are described in Example 22.

[0369] Figure 40: CD3 / BCMA / CD38-118 induces higher killing efficacy against all cell lines compared to the combination of bispecific antibodies and the same binder. The ability of CD3 / BCMA / CD38-118 and controls, CD3 / BCMA / CD38-149, CD3 / BCMA / CD38-151 and a combination of the two to trigger T cell-mediated killing of multiple myeloma cell lines was assessed using T cells isolated on KMS-12-BM (A, D), MOLP-8 (B, E) and NCI-H929 (C, F) cells in a redirected lysis assay with an effector to target ratio of 5: 1 and measured by flow cytometry after 48 hours of incubation. The percentage of killing (A, B, C), percentage of cytotoxicity (D, E, F) and the associated half-maximal effective killing concentration (EC) to no Ab were compared. 50 ) values ​​were determined by reorientation analysis. Each point represents the EC of an individual donor 50 Values. The bars show the mean of the data set. Means were compared using a mixed-effects model (when some values ​​were missing) or paired one-way ANOVA (when all values ​​were included) followed by Tukey HSD post hoc comparisons. Ns: not statistically different; *: p = 0.05-0.01; **: p = 0.01-0.001; ***: p 0.001-0.0001; ****: p < 0.0001. NO: not quantifiable. The experimental setup and analysis are described in Example 22.

[0370] Figure 41 :CD3 / BCMA / CD38-118 showed statistically superior killing of the combination of terituzumab and daratumumab. The ability of CD3 / BCMA / CD38-118 and benchmark, daratumumab, terituzumab and the combination of the two to trigger killing of multiple myeloma cell lines was assessed using human PBMC on KMS-12-BM cells in MmoAK assay with an effector to target ratio of 5: 1 and measured by flow cytometry after 48 hours of incubation. Two concentrations of CD3 / BCMA / CD38-118 or terituzumab were evaluated with a single dose of 100nM daratumumab (A, C) and 100pM (B, D). After incubation, the killing percentage (A, B) and cytotoxicity percentage (C, D) without Ab were assessed. Each point represents a separate donor. The bar shows the mean value of the data set. Mean values ​​were compared using paired one-way ANOVA analysis followed by Tukey HSD post hoc comparisons. Ns: no statistical difference; *: p = 0.05-0.01; **: p = 0.01-0.001; ***: p 0.001-0.0001; ****: p < 0.0001. The experimental setup and analysis are described in Example 22.

[0371] Figure 42: CD3 / BCMA / CD38-118 induces killing of CD138+ cells in MM patient samples. BMMCs from four separate donors (A, smoldering MM; B, newly diagnosed MM; C, r / r MM; D, plasma cell leukemia (PCL)) were cultured in RPMI 10% human serum, 3ng / ml recombinant human IL-6 and treated with varying concentrations of CD3 / BCMA / CD38-118, a benchmark (terituzumab) and a negative control (CD3 / BCMA / CD38-085, not shown) for 17-22 hours. The absolute counts of live CD138+ cells were extracted and normalized to antibody-free conditions to calculate the killing percentage compared to antibody-free conditions. Each point represents the mean + / - standard error of the mean of 2 repeated measurements of a single donor (except B and C, where simplification was used). The experimental setup and analysis are described in Example 22.

[0372] Figure 43 :CD3 / BCMA / CD38-118 induces CD138+ cell killing that is superior to terituzumab in MM patient samples. BMMCs from six to eleven individual donors were cultured in RPMI 10% human serum, 3ng / ml recombinant human IL-6 and treated for 17-22 hours with varying concentrations of CD3 / BCMA / CD38-118, CD3xBCMA benchmark (terituzumab) and negative controls (not shown). The absolute counts of live CD138+ cells were extracted and normalized to antibody-free conditions to calculate the killing % compared to antibody-free conditions. Each point represents a single donor. The bars show the mean + / - standard deviation for different donors at a specific concentration. The experimental setup and analysis are described in Example 22.

[0373] Figure 44: Although CD3 / BCMA / CD38-118 controls KMS-12-BM tumors in a dose-dependent manner, BEATCD3 / CD38 and terituzumab fail to delay tumor progression at high doses. 1×10 7 KMS-12-BM MM tumor cells (CD38 低 And BCMA 低 ) and were inoculated intraperitoneally with 1×10 7 PBMCs. On day -1, when the average tumor volume reached approximately 187 mm 3Mice were randomized at 4 hr and treated with intravenous injections once weekly on days 0, 7, and 14 for 3 weeks. A. KMS-12-BM tumor growth in NSG mice (n=8 mice / group) inoculated with donor C PBMCs after treatment with CD3 / BCMA / CD38-118 (top), BEAT CD3 / CD38 (middle), or terituzumab (bottom) at the indicated doses. Mean ± SEM is shown with last observation carried forward (LOCF). B. KMS-12-BM tumor growth in NSG mice (n=8 mice / group) inoculated with donor C PBMCs after treatment with the indicated molecules at a dose of 0.5 mg / kg (left) or 0.1 mg / kg (right). Mean ± SEM is shown with last observation carried forward (LOCF). C. Serum concentrations (ng / mL) of the indicated molecules at the indicated doses on day 13, day 14, or day 21 (at trough exposure, before the third dose (day 13 / 14) or one week later) in NSG mice inoculated with donor CPBMCs (n=4-7 mice / group). Mean ± SD is shown, values ​​within the bars represent mean concentrations, and values ​​above represent fold differences. NIC = no interpolation concentration.

[0374] Figure 45: High doses of BEAT CD3 / CD38 and terituzumab induce a reduction in T cell compartments within the tumor microenvironment and in the periphery. At day 0, when the mean tumor volume reached approximately 187 mm 3 NSG female mice implanted with KMS-12-BM MM tumor cells and intraperitoneally inoculated with PBMCs from a healthy human donor (donor C) were treated intravenously with the indicated molecule at the indicated dose, then once a week for 3 weeks. On day 13, the tumors and spleens of the treated mice were harvested and analyzed ex vivo by flow cytometry (n=2-3 mice / group). A. The grayscale pseudocolor image shows the expression of human CD45 versus mouse CD45 in gated live cells in the tumor. The numbers in the figure indicate the percentage of positive cells. B. The contour map shows the expression of human TCRab in tumor-infiltrating CD45+ cells. The numbers in the figure indicate the percentage of positive cells. C. The figure depicts the number of human CD45+ (top) and human TCRab+ (bottom) cells per gram of tumor. Mean ± SD is shown. D. The figure depicts the number of human CD45+ (top) and human TCRab+ (bottom) cells in the spleen of the treated mice. Mean ± SD is shown.

[0375] Figure 46: Treatment with BEAT CD3 / CD38+IVIG rescues the T cell compartment and improves in vivo efficacy against KMS-12-BM tumors. NSG female mice were implanted subcutaneously with 1×10 7 KMS-12-BM MM tumor cells were intraperitoneally inoculated with 1×107 PBMCs. On day -1, when the average tumor volume reached approximately 120 mm 3 Mice were randomized and injected intravenously with 200 mg / kg of IVIG (Privigen) or PBS on the same day, then twice a week. Vehicle, 0.5 mg / kg of CD3 / BCMA / CD38-118, or 0.5 mg / kg of BEAT CD3 / CD38 were administered (intravenously) on days 0 and 7. A. KMS-12-BM tumor growth curves after treatment with the indicated molecules in the presence or absence of IVIG in NSG mice (n=3-5 mice / group) inoculated with donor E PBMCs. Mean ± SEM are shown. B. Tumor volume of KMS-12-BM tumors on day 11 (study endpoint) after treatment with the indicated molecules ± IVIG. Mean ± SD are shown. C, D. On day 11, tumors and spleens of treated mice were harvested and analyzed ex vivo by flow cytometry (n=3-5 mice / group). C. Grayscale pseudocolor image shows the expression of human CD45 versus mouse CD45 in gated live cells in the tumor. The numbers in the graph indicate the percentage of positive cells. D. The graph depicts the percentage of positive cells per gram of tumor in treated mice ( Figure 45D ) or spleen ( Figure 45D (Continued) Gating on human CD45 in live cells + Number of cells (top) and gated human CD45 + Human TCRab in cells + Number of cells (bottom). Mean ± SD is shown.

[0376] Figure 47: In the presence of IVIG, CD3 / BCMA / CD38-118, BEAT CD3 / CD38, and Terituzumab all induced regression of KMS-12-BM tumors at the highest dose. NSG female mice were implanted subcutaneously with 1×10 7 KMS-12-BM MM tumor cells were intraperitoneally inoculated with 1×10 7 PBMCs. On day -1, when the average tumor volume reached approximately 147 mm 3Mice were randomized and injected intravenously with 200 mg / kg of IVIG (Privigen) on the same day and then weekly for 3 weeks (days -1, 6, and 13). Vehicle, CD3 / BCMA / CD38-118, and BEAT CD3 / CD38 (0.5 mg / kg, 0.1 mg / kg, and 0.02 mg / kg) and Terituzumab (2.5 mg / kg, 0.5 mg / kg, and 0.1 mg / kg) were administered (iv) on days 0, 7, and 14 (once weekly for 3 weeks). A. KMS-12-BM tumor growth after treatment with CD3 / BCMA / CD38-118 (top), BEAT CD3 / CD38 (middle), or Terituzumab (bottom) at the indicated doses in NSG mice inoculated with donor C PBMCs (n=6-8 mice / group). Mean ± SEM is shown with last observation carried forward (LOCF). B. KMS-12-BM tumor growth after treatment with the indicated molecules at a dose of 0.5 mg / kg (left) or 0.1 mg / kg (right) in NSG mice (n = 6-8 mice / group) inoculated with donor C PBMCs. Mean ± SEM is shown with last observation carried forward (LOCF). C. Serum concentrations (ng / mL) of the indicated molecules at the indicated doses on day 21 (at trough exposure, 1 week after the third dose) in NSG mice (n = 6-8 mice / group) inoculated with donor C PBMCs. Mean ± SD is shown, the values ​​within the bars represent the mean concentration, and the values ​​above represent the fold difference. NIC = no interpolated concentration; BLOQ = below limit of quantitation.

[0377] Figure 48: Competition of CD3 / BCMA / CD38-118 with isatuximab and daratumumab for binding to recombinant human CD38 using Octet biolayer interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin SA biosensor. The CD38-loaded biosensor was immersed in a 200 nM solution of Antibody 1 in kinetic buffer to achieve surface saturation. The saturated biosensor was then immersed in a premixed solution of Antibody 1 and Antibody 2 at a final concentration of 200 nM each (competition phase). The same experimental procedure was performed using a 400 nM solution of Antibody 1 alone in the competition phase as a control for surface saturation. The graph shows the binding to the sensor tip as a function of wavelength shift (response, in nm; y-axis) versus time (x-axis) for A) isatuximab and B) daratumumab as Antibody 1 (saturating antibody), respectively. The curves are labeled with the antibody names.

[0378] Figure 49Schematic diagram of an additional CD3 / BCMA / CD38 trispecific antibody. The CD3 / BCMA / CD38 antibody is constructed using three Fabs targeting three independent antigens, whereby the "Fc-distal" Fab domain is fused to the N-terminus of the VH domain of the "Fc-proximal" Fab. In this configuration D, the CD3 / BCMA / CD38 binding arm is located on the outer Fc-distal position of the B arm, the CD38 binder is located on the "Fc-proximal" position of the B arm, and the BCMA binder is located on the A arm.

[0379] Figure 50: The positioning of the conjugate in the CD3 / BCMA / CD38 antibody affects their cytotoxic killing efficacy. The ability of CD3 / BCMA / CD38-118 and control antibodies CD3 / BCMA / CD38-149, -151, -170, -171 and -172 to trigger T cell-mediated killing of KMS-12-BM tumor cells was assessed in a redirected lysis assay using isolated T cells at a 5:1 effector to target ratio. The percentage of cytotoxicity at different concentrations is shown: CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-171, A; CD3 / BCMA / CD38-151 and CD3 / BCMA / CD38-172, B; CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-170, C. Each curve is a nonlinear logistic 4PL model with variable slope, and the symbols represent the mean + / - SEM of six individual T cell donors. Paired two-way ANOVA analysis, followed by Sidak's multiple comparison test, was used to compare the percentage of killing (*: p = 0.05-0.01; **: p = 0.01-0.001; ***: p 0.001-0.0001; ****: p < 0.0001). Experimental settings and analysis are described in Example 25b.

[0380] Figure 51: The positioning of the conjugate in the CD3 / BCMA / CD38 antibody affects their on-target off-tumor T cell activation. The tendency of CD3 / BCMA / CD38-118 and control antibodies CD3 / BCMA / CD38-149, -151, -170, -171 and -172 to induce on-target off-tumor T cell activation was evaluated by measuring the upregulation of the CD25 activation marker on CD8+ T cells in a high-density PBMC assay. The percentage of CD8+ T cells expressing CD25 is shown for the following items: CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-171, A; CD3 / BCMA / CD38-151 and CD3 / BCMA / CD38-172, B; CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-170, C. Each curve is a nonlinear logistic 4PL model with variable slope, and the symbols represent the mean + / - SEM of six individual donors. Paired two-way ANOVA analysis, followed by Sidak's multiple comparison test, was used to compare the percentages of CD8+ T cells expressing CD25 (*: p = 0.05-0.01; **: p = 0.01-0.001; ***: p 0.001-0.0001; ****: p < 0.0001). Experimental setup and analysis are described in Example 25b.

[0381] Figure 52: Crystal structure of human CD38 in complex with anti-CD38-B3. (A) Overall structure of the anti-CD38-B3 / hsCD38 complex. hsCD38 is shaded light grey, the Fab heavy chain is shaded black, and the Fab light chain is shaded grey. (B) Footprint of anti-CD38-B3 on CD38, highlighting the CD38 residues that interact with B3. The underlined residues correspond to residues shared between the epitopes of B3 and daratumumab. The italicized and underlined residues correspond to residues shared between the epitopes of anti-CD38-B3 and isatuximab. (C) Surface representation of the anti-CD38-B3 epitope on CD38, with side chains of the interacting residues from B3 shown as sticks. The HCDRs are dark grey and the LCDRs are grey. The experimental setup and analysis are described in Example 26.

[0382] Figure 53: Anti-CD38-B3, isatuximab, and daratumumab bind to different epitopes on CD38. (A) Binding pattern of anti-CD38-B3 Fab and daratumumab (PDB 7DHA). (B) Binding pattern of anti-CD38-B3 Fab and isatuximab (PDB 4CMH). The light chain of the anti-CD38-B3 Fab sterically clashes with the heavy chain of isatuximab. The steric clash of the two antibodies is indicated by the dashed ellipse. (C) Surface representation of the epitopes of anti-CD38-B3 (dark gray) and isatuximab (gray) on CD38, where residues common to both epitopes are highlighted.

[0383] Example 1: Generation of anti-CD3 antibodies

[0384] Materials and methods

[0385] Recombinant human CD3γε protein (SEQ ID NO: 606)

[0386] According to published information (Arnett et al., 2004, PNAS, 101(46):16268-16273), the CD3γ-CD3ε complex (comprising residues 23-103 of CD3γ, a linker, and residues 23-118 of CD3ε, hereinafter referred to as recombinant human CD3γε protein) was expressed as a fusion protein in an E. coli expression system. The protein was refolded from the inclusion complex and purified by affinity chromatography (using affinity resin generated with the OKT3 monoclonal antibody) and size exclusion chromatography.

[0387] Using EZ-link TM Sulfo-NHS-LC-Biotin No-Weigh TM Recombinant human CD3γε protein was biotinylated using the Format kit (ThermoFisher Scientific, catalog number: A39257). Briefly, recombinant human CD3γε protein was dialyzed against acetate buffer, pH 5.0, at 4°C and then incubated on ice for 2 hours with a 50-fold molar excess of biotin. The biotinylated recombinant human CD3γε protein was then dialyzed against PBS at 4°C.

[0388] Transient expression of full-length human CD3εδ, human C3εγ, and cynomolgus monkey C3εδ in the CHO-S cell line

[0389] Human codon-optimized extracellular domain sequences of human CD3ε (Uniprot ID P07766 residues 1-126), human CD3δ (Uniprot ID P04234 residues 1-105), human CD3γ (Unipro ID P09693 residues 1-116), cynomolgus monkey CD3ε (Uniprot ID Q95LI5 residues 1-105), and cynomolgus monkey CD3δ (Uniprot ID Q95LI8 residues 1-117) were each fused to the extracellular domain, full transmembrane, and a portion of the cytoplasmic domain of mouse CD80 (Uniprot ID Q00609 residues 237-306) to generate constructs comprising the sequences provided in SEQ IDs: 610-614. For protein expression, plasmids (SEQ ID NO: 611 and SEQ ID NO: 612; SEQ ID NO: 610 and SEQ ID NO: 612 or SEQ ID NO: 613 and SEQ ID NO: 614) were co-transfected into suspension-adapted CHO-S cells (cGMP banked, Invitrogen, catalog number: A1136401) using polyethyleneimine (PEI; Polysciences). Briefly, cells were prepared at 2 million cells / ml in CD CHO (Gibco). Cells were then transfected with the DNA-PEI mixture at 37°C. Four hours after transfection, cell cultures were diluted 1:1 in PowerCHO 2 (Lonza) supplemented with 4mM L-glutamine and incubated at 37°C, 5% CO2, and 80% humidity with orbital shaking. The expression of the target antigen was assessed by monitoring the expression of the eGFP reporter protein using a fluorescence microscope.

[0390] Generation of CD3ε knockout (KO) HPB-ALL cell bank

[0391] As described in Ran et al. (Ran et al., 2013, Cell, 154: 1380-1389), by using clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 technology targeting the first exon of the CD3 gene, HPB-ALL CD3 KO cell line was derived from original HPB-ALL cells (DSMZ, catalog number: ACC483, batch 15). HPB-ALL cell line was transfected using Neon electroporator (Invitrogen Life Technologies) according to the manufacturer's instructions. After transfection, cells were stained with anti-CD3 antibodies (OKT3 clones, ThermoFisher, catalog number: 14-0037-85) and CD3 knockout cells were sorted using Melody FACS sorter (BD) to generate CD3 knockout cell libraries. The lack of CD3 expression was further confirmed by flow cytometry using Qifikt (DAKO, catalog number: K007811-8).

[0392] Library generation

[0393] The library used herein is synthetic in origin, and its diversity is limited to heavy chain (CDR-H1, CDR-H2 and CDR-H3) and fixed Vκ3-15 / Jκ1 light chain (SEQ ID NO:1). The library contains 4 different sub-libraries based on VH1-69, VH3-23, VH3-15 and VH3-53 antibody germlines. CDRs have been randomized using trimeric oligonucleotides. Primers for diversifying CDR-H1 and CDR-H2 were designed for each sub-library, and the diversity of the germline specificity at Kabat residues 27-35 and 80-58 was encoded respectively. CDR-H3 was randomized using an oligonucleotide library encoding 15 CDR-H3 lengths (6-20) and the length-specificity at Kabat residues 95-102. The diversified scFv fragments were pooled to mimic the natural CDR-H3 length distribution and cloned into pNGLEN (an in-house modified pUC119 phagemid vector), and the resulting ligation reaction was electroporated into E. coli TG1 cells. Each sublibrary had a length between 1.2 × 10 10 With 1.7×10 10 The diversity among the four sub-libraries reached 5.6×10 10 total diversity.

[0394] Library Selection

[0395] The panning strategy consisted of four rounds of selection using recombinant CD3 protein, or two rounds of selection using recombinant CD3 protein followed by two or three rounds of selection using CD3-expressing cells.

[0396] Purified phage particles (10 12 Plaque forming units) were blocked in 1 ml of phosphate buffered saline (PBS) containing 3% (w / v) skim milk (3% MPBS) at room temperature (RT) for 1 hour. 2×100 μl magnetic Dynabeads were blocked under the same conditions. TM MyOne TM Streptavidin Cl beads (Invitrogen, catalog number: 65002). Phage was exhausted for 1 hour at room temperature for pre-blocked beads. Another aliquot of pre-blocked beads was incubated at room temperature for 30 minutes with 100nM biotinylated recombinant human CD3εδ protein (Creative Biomart, catalog number: CD3E & CD3D-377H) or internally produced biotinylated recombinant human CD3γε protein (SEQ ID NO: 606). Then, the exhausted phage was incubated at room temperature for 2 hours with pre-coated beads. The beads were washed five times with PBS (PBS-Tween 0.1%) containing 0.1% (v / v) Tween, and washed twice with PBS. Phage was eluted with 100mM triethylamine for 10 minutes at room temperature and neutralized with Tris-HCl 1M pH 8. The eluted phage was used to infect 10ml exponentially growing Escherichia coli TG1 cells. Infected cells were grown in 2YT medium at 37°C and 100 revolutions per minute (RPM) for 1 hour, then spread on 2YTAG (2YT medium supplemented with 100 μg / ml ampicillin and 2% glucose) agar plates and incubated overnight (ON) at 30°C. Colonies were scraped from the plates into 10ml 2YT and 15% glycerol (v / v) was added and stored at -80°C. TG1 cells from glycerol stock were grown in 2YTAG medium at 37°C and 240RPM until the OD at 600nm reached 0.5. Cells were then superinfected with M13K07 helper phage using a multiplicity of infection (MOI) of 10 at 37°C and 100RPM for 1 hour. The culture medium is then replaced with 2YTAK (2YT culture medium supplemented with 100 μg / ml ampicillin and 50 μg ml kanamycin), and the cells are further cultured overnight at 30°C and 280RPM. Alternatively, the eluted phage is used to infect 2ml of exponentially growing Escherichia coli TG1 cells. The infected cells are grown at 37°C and 100RPM for 1 hour in 2YT culture medium, and then grown at 37°C and 240RPM for 1 hour in 2YT culture medium supplemented with 2% glucose. The cells are then superinfected with the M13K07 helper phage produced as described above. The next day, 10 μl of the cell-free supernatant containing phage is used for a subsequent round of selection.

[0397] For cell-based panning, 2-10 × 10 7 Non-expressing cells (CHO, HEK or CD3ε knockout HPB-ALL) and 2-10×10 7 Cells expressing CD3ε (HPB-ALL or CHO cells transiently expressing the membrane-bound extracellular domain of cynomolgus monkey CD3ε (SEQ ID NO: 613) and CD3δ (SEQ ID NO: 614)) were blocked with PBS / BSA 3% supplemented with 0.1% azide to prevent receptor internalization for 1 hour at room temperature. Phage were deselected against non-expressing cells for 1 hour at room temperature. The deselected phage were then incubated with cells expressing CD3ε for 2 hours at room temperature. To remove nonspecific phage, the cells were washed four times with PBS-Tween 0.1% and twice with PBS. The phage were eluted with citric acid 76mM, pH 2.0 for 10 minutes at room temperature and neutralized with Tris-HCl 1M pH 8. The eluted phage were used for exponentially growing E. coli TG1 cells. Phage amplification was performed as described above.

[0398] scFv screening by flow cytometry

[0399] scFv clones were evaluated by flow cytometry for binding to CHO cells transiently expressing the membrane-bound extracellular domains of human CD3ε (SEQ ID NO: 610) and CD3δ (SEQ ID NO: 611) or CD3γ (SEQ ID NO: 612). Individual E. coli colonies from the fourth or fifth round of screening were picked and grown in 2YT medium supplemented with 100 μg / ml ampicillin and 0.1% glucose in 96-well deep-well plates. scFv expression was induced by adding 0.02 mM IPTG and incubating overnight at 30°C and 260 RPM. The cells were centrifuged and periplasmic extracts were obtained by resuspending the bacterial pellet in TES buffer (50 mM Tris-HCl pH 8; 1 mM EDTA pH 8; 20% sucrose) and then incubating on ice for 30 minutes. Cell debris was removed by centrifugation, and the supernatant containing the scFv was used for flow cytometry experiments. CD3-expressing and non-transfected CHO cells were seeded in microtiter plates at a density of 105 cells / well. Next, the plates were centrifuged to remove the cell supernatant, and 100 μl of periplasmic extract diluted 1:1 in PBS containing 3% (w / v) bovine serum albumin (PBS-BSA 3%) was added to each well, and the plates were further incubated at 4°C for 30 minutes. The cells were then washed with PBS-BSA 3% and incubated with biotin-chicken anti-c-Myc antibody (Gallus Immunotech catalog number: ACMYC-B) diluted 1:200 in PBS-BSA 3% for 30 minutes at 4°C. Next, the cells were washed with PBS-BSA 3% and incubated with streptavidin APC (eBioscience, catalog number: 17-4317) diluted 1:100 in PBS-BSA 3% for 30 minutes at 4°C. Finally, cell fluorescence was measured using a FACSCalibur flow cytometer (BD biosciences).

[0400] IgG1 LALA expression

[0401] The cDNA encoding the constant regions of the different antibodies was synthesized by GENEART AG (Regensburg, Germany) and modified using standard molecular biology techniques. The PCR products were digested with appropriate DNA restriction enzymes, purified, and ligated into a modified pcDNA3.1 plasmid (Invitrogen) containing a CMV promoter and bovine hormone polyadenylation (poly(A)). The expression vector also contained oriP, the origin of plasmid replication for the Epstein-Barr virus, and a mouse VJ2C leader peptide for secretion of the encoded polypeptide chain. To reformat the scFv library clones into human IgG1 LALA (human IgG1 with L234A and L235A substitutions, EU numbering) format, each scFv clone in its phage library vector was used to amplify its individual VH cDNA by PCR. The VH PCR product was then cloned upstream of the cDNA encoding the human IgG1 heavy chain CH1 domain in the modified pcDNA 3.1 vector described above, while the fixed Vκ3-15 / Jκ1 light chain (SEQ ID NO: 1) was cloned upstream of the cDNA encoding the human κ constant light chain domain in the modified pcDNA 3.1 vector described above.

[0402] For IgG1 LALA expression, equal amounts of heavy and light chain vectors were co-transfected into suspension-adapted HEK293-EBNA cells (ATCC, catalog number CRL-10852) using linear polyethyleneimine (PEI) (Polysciences Inc., catalog number: 24314). Typically, cells were prepared at 8 million cells / ml in RPMI 1640 (Biowest; catalog number: L0501) supplemented with 0.1% Pluronic F-68 (ThermoScientific, catalog number: 24040032). Cells were then transfected with a DNA-PEI mixture. Four hours after transfection, cell culture was diluted 1: 1 in BalanceCD HEK293 (Irvine Scientific, catalog number: 91165) supplemented with 4mM L-glutamine and incubated at 37°C, 5% CO2, and 80% humidity under orbital shaking for 5 days. The cell-free culture supernatant containing the recombinant protein was prepared by centrifugation followed by filtration and used for further purification. Protein A affinity resin (Repligen, catalog number: CA-PRI-0100) was used to purify the IgG1LALA protein. The affinity resin was added to the filtered culture supernatant and incubated overnight at 4°C with gentle mixing. The next day, the resin beads were collected in a Poly-Prep chromatography column (Bio-Rad Laboratories), washed with PBS, and the recombinant protein was eluted with an acidic buffer (usually glycine 0.1M pH 3). After neutralization with 1 / 10 volume of Tris-HCl pH 8, the formulation buffer was exchanged into PBS.

[0403] Screening of anti-CD3 IgG1 LALA clones on CD3-expressing cells

[0404] Screening of anti-CD3 antibodies generated as human IgG1-LALA included selective binding studies to HPB-ALL wild-type and CD3 knockout (KO) HPB-ALL cell lines, as well as binding studies to primary isolated PBMCs using flow cytometry.

[0405] Binding to HPB-ALL and CD3ε knockout HPB-ALL cells

[0406] The cells were plated at 1×10 5 Cells / well were plated in 96-well round-bottom plates (TPP, catalog number: 92097). The plates were centrifuged at 350 g for 3 minutes at 4 ° C, and the cells were resuspended in 100 μl of cold FACS buffer (PBS IX, supplemented with 2.5% heat-inactivated fetal bovine serum, 2mM EDTA, and 0.05% sodium azide) containing increasing concentrations of anti-CD3 IgG1-LALA antibodies. The stained cells were incubated at 4 ° C for 30 minutes, washed twice with FACS buffer at 350 g for 3 minutes each, and resuspended in 100 μl goat polyclonal anti-human IgG PE secondary antibody (ThermoFisher Scientific, catalog number: 12-4998-82) diluted 1 / 200 in FACS buffer for 30 minutes. The cells were then washed twice and resuspended in 100 μl of FACS buffer containing 1 / 2000 diluted SYTOX Green dead cell stain (ThermoFisher Scientific, catalog number: S34860). Samples were collected on a CytoFlex instrument (Beckman Coulter). Cells were gated based on the size of the FSC versus SSC ratio, and debris, doublets, and dead cells were excluded. FlowJo software was used to analyze the PE-geometric mean (geometric mean) fluorescence intensity of the cells. Only samples showing at least 200 cells in the final gate were considered in the further steps of analysis. Prism software (GraphPad) was finally used to draw PE-geometric mean (geometric mean) fluorescence intensity values, and the binding status is summarized in Table 1.

[0407] CD4 + Binding of human T cells

[0408] Human PBMCs were harvested from buffy coats / apheresis filtrates obtained from the Blood Transfusion Center of La Chaux-de-Fonds / Bern (Switzerland) using ficoll density gradient separation and frozen on the day of harvest. The day before the assay, cells were thawed in pre-warmed complete RPMI medium (RPMI supplemented with 10% heat-inactivated fetal bovine serum, 1% glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin / streptomycin) and plated at 1×10 6 On the day of the assay, cells were plated at 1×10 5 Cells / well were plated in 96-well round-bottom plates. The plates were centrifuged at 350 g for 3 minutes at 4 ° C, and the cells were resuspended in 100 μl of cold FACS buffer containing a serial dilution of anti-CD3 IgG1-LALA antibody starting from 800 nM and diluted 3 or 10 times. The cells were incubated at 4 ° C for 30 minutes, washed twice with FACS buffer at 350 g for 3 minutes each, and resuspended in 100 μl of a mixture of goat polyclonal anti-human IgG PE secondary antibody (ThermoFisherScientific, catalog number: 12-4998-82, 1 / 200) and a co-staining antibody for human CD4-AlexaFluor700 (ThermoFisher Scientific, catalog number: 56-0048-82, 1 / 100) diluted in FACS buffer for 30 minutes. The cells were then washed twice and resuspended in 100 μl of FACS buffer containing 1 / 2000 dilution of SYTOX Green dead cell stain (ThermoFisherScientific, catalog number: S34860). Samples were collected on a CytoFlex instrument (Beckman Coulter). Cells were gated based on the size of FSC versus SSC, and debris, doublets, and dead cells were excluded. Finally, the PE-geometric mean (geometric mean) fluorescence intensity of the CD4+ T cell population was analyzed using FlowJo software. Finally, the PE-geometric mean (geometric mean) fluorescence intensity values ​​were plotted using Prism software (GraphPad), as shown in Figure 5. Figure 1A The binding status is summarized in Table 1.

[0409] Screening of anti-CD3 IgG1LALA clones for T cell activation

[0410] To evaluate the potential of anti-CD3 antibodies generated as human IgG1-LALA to induce T cell activation, additional screening was performed using a T cell activation assay. Serial dilutions of anti-CD3 IgG1-LALA antibodies starting at 200 nM and diluted 3 or 10-fold were coated in PBS in 96-well flat-bottom plates (TPP, catalog number: 92096). The plates were then incubated overnight at 4°C. Frozen isolated human T cells were thawed in prewarmed complete RPMI medium and plated at 1×10 6 On the day of the assay, the plate was washed twice in PBS and primary T cells were harvested, counted, and expressed as 1×10 5 Cells / well (100 μl) were plated. After incubation for 48 hours at 37°C, 5% CO2, the plate was centrifuged (350 g for 5 minutes) and the cells were transferred to a 96-well round-bottom plate, washed and resuspended in 100 μl of an antibody mixture against human CD4-PE-eFluor610 (ThermoFisherScientific, catalog number: 61-0049-42, 1 / 200), CD8-AlexaFluor700 (BioLegend, catalog number: 344724, 1 / 100), CD25-PE, and CD69-PE-Cy7 (ThermoFisher Scientific, catalog number: 12-0259-42 and 25-0699-42, 1 / 100) diluted in FACS buffer and incubated for 30 minutes at 4°C. The cells were then washed twice and resuspended in 100 μl of FACS buffer containing SYTOX Green dead cell stain (ThermoFisherScientific, catalog number: S34860) diluted 1 / 2000. Samples were collected on a CytoFlex instrument (Beckman Coulter). Cells were gated based on the size of the FSC versus SSC, and debris and doublets were excluded. Finally, cells that were negative for viability staining were gated and analyzed for CD4 + The PE-geometric mean (GEM) fluorescence intensity of the population was evaluated by + T cells were gated for CD69 positive expression to determine T cell activation. Finally, Prism software (GraphPad) was used to plot CD4 + The percentage of activated CD69+ T cells is depicted in Figure 1B, and the binding status is summarized in Table 1. A total of eight independent experiments were performed.

[0411] Results and Conclusions

[0412] Phage display panning and scFv screening by flow cytometry

[0413] A phage display library based on an immobilized Vκ3-15 / Jκ1 light chain (SEQ ID NO: 1) was panned against CD3ε, and the phage display output was screened by flow cytometry. scFv clones that showed specific binding to CHO cells expressing CD3ε were sequenced, and the unique sequence was expressed as IgG1 LALA for further characterization.

[0414] Screening of anti-CD3 IgG1 ...

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 according to claim 1, further comprising a common light chain.

3. The trispecific antibody or antibody fragment thereof according to any of the preceding claims, wherein the at least one binding portion that binds to human CD3 comprises a heavy chain CDR set comprising an amino acid sequence 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.

4. The trispecific antibody or antibody fragment thereof according to any one of the preceding claims, wherein the at least one binding portion that binds to human BCMA comprises a heavy chain CDR set comprising an amino acid sequence 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.

5. The trispecific antibody or antibody fragment thereof according to any of the preceding claims, wherein at least one binding portion that binds to human CD38 comprises a heavy chain CDR set comprising an amino acid sequence comprising the following items: 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 of the preceding claims, wherein at least one binding portion 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 consisting of SEQ ID NO: 55, 58, 60, 62 and 66.

7. The trispecific, multispecific antibody or antibody fragment thereof according to any of the preceding claims, wherein the at least one binding portion that binds to human BCMA 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: 93, 101, 105 and 108, or an amino acid sequence selected from the group comprising SEQ ID NOs: 93, 101, 105 and 108, wherein the amino acid sequence further comprises the amino acid substitution N82aS.

8. The trispecific antibody or antibody fragment thereof according to any of the preceding claims, wherein at least one binding portion 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 consisting of SEQ ID NOs: 110, 113, 122 and 111.

9. The trispecific antibody or antibody fragment thereof according to any of the preceding claims, wherein the at least one binding portion that binds to human CD3 comprises a heavy chain CDR set comprising an amino acid sequence of SEQ ID NO: 181, SEQ ID NO: 307 and SEQ ID NO: 433, and a light chain CDR set comprising an amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723; the at least one binding portion that binds to human BCMA comprises a heavy chain CDR set comprising an amino acid sequence of SEQ ID NO: 234, SEQ ID NO: 360 and SEQ ID NO: 486, and a light chain CDR set comprising an amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723; and the at least one binding portion that binds to human CD38 comprises a heavy chain CDR set comprising an amino acid sequence of SEQ ID NO: 239, SEQ ID NO: 365 and SEQ ID NO: 491, and a light chain CDR set comprising an amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: The light chain CDR group of the amino acid sequence of NO:

723. 10 . The trispecific antibody or antibody fragment thereof according to 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 claims 1 to 10, wherein the at least one binding moiety that binds to human BCMA is fused at the 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 according to claim 1, wherein the at least one binding moiety that binds to human BCMA is fused at the C-terminus to the N-terminus of the at least one binding moiety that binds to human CD3 via a peptide linker, or wherein the at least one binding moiety that binds to human BCMA is fused at the C-terminus to the N-terminus of the at least one binding moiety that binds to human CD38 via a peptide linker, or wherein the at least one binding moiety that binds to human BCMA is fused at the 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 the preceding claims, wherein the trispecific antibody or antibody fragment thereof comprises a non-naturally occurring Fc domain.

14. The trispecific antibody or antibody fragment thereof according to any one of the preceding claims for use as a medicament.

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

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 syndrome (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 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 human CD3, one binding portion that binds BCMA and one binding portion that binds CD38 according to claim 9.

18. An epitope on the extracellular domain of human CD38 which is bound by the antibody or antibody fragment thereof as claimed in any one of the preceding claims and comprises 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 an amino acid sequence 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 containing the amino acid sequence of SEQ ID NO:721, SEQ ID NO:722 and SEQ ID NO:

723.

20. An antibody or antibody fragment thereof 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: 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 light chain CDR set containing the amino acid sequence of SEQ ID NO:721, SEQ ID NO:722 and SEQ ID NO:

723.

21. A trispecific heterodimeric antibody or antibody fragment thereof, comprising a first engineered CH3 domain and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises a substitution comprising the group consisting of: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises a substitution comprising the group consisting of: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment is heterodimerized by the first engineered CH3 domain and the second engineered CH3 domain, characterized in that The trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding moieties, wherein each binding moiety binds to a different antigen.

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