Cd3 / bcma / cd38 trispecific antibodies

Abstract

This invention relates to a novel trispecific heterodimeric immunoglobulin. Specifically, this invention relates to a trispecific heterodimeric immunoglobulin targeting human CD3 antigen, human BCMA, and human CD38 antigen. This invention also relates to the use of such novel trispecific heterodimeric immunoglobulins in the treatment of proliferative diseases, particularly cancers such as hematologic malignancies. This invention also relates to the use of novel trispecific antibodies in 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 an epitope on the human CD3 antigen, an epitope on the human BCMA antigen, and an epitope on the human CD38 antigen. The present invention also relates to the use of these novel trispecific heterodimeric immunoglobulins in treating proliferative diseases, particularly cancers such as hematological cancers. Prior Art

[0002] Multispecific antibodies can target one or more immune-related molecules and one or more tumor antigens, recruiting immune cells such as T cells to fight tumors. These antibodies have been developed over the past few decades to meet the needs of cancer immunotherapy. For example, bispecific antibodies targeting CD3 on T cells and a target antigen on the surface of cancer cells can bind T cells to cancer cells, regardless of T cell receptor specificity, co-stimulation, or peptide antigen presentation (see WO2020204708A1). Trispecific T cell-engaging antibodies, which target T cells with two proteins and tumor cells with one, have also shown promising results in the treatment of various cancers, including breast cancer, lymphoma, and multiple myeloma (see US10882922B2). In addition, prior art has also discovered that certain trispecific binding molecules can bind to CD3 or other components of the TCR complex on T cells while also binding to two tumor-associated antigens expressed on cancer cells, thereby achieving the effect of lysing tumor cells (see "US20210163620A1").

[0003] Preclinical and clinical studies have demonstrated that antibodies with multiple specificities and the ability to transduce immune cells can redirect T cells (see 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). Therefore, they are of great importance for the development of new drugs and the treatment of complex diseases, such as inflammatory diseases, autoimmune disorders, and hematological cancers, such as multiple myeloma (MM), for which there is currently no cure. MM is a neoplastic plasma cell disorder characterized by the clonal proliferation of malignant plasma cells in the bone marrow (BM) microenvironment, monoclonal proteins in the blood or urine, and associated organ failure. Multiple myeloma accounts for 1-2% of newly diagnosed cancers and approximately 20% of deaths from hematologic malignancies. The disease is more common in men and African Americans. While recent research into myeloma has led to the development of new treatments and improved survival rates, multiple myeloma remains incurable.

[0004] The diagnosis of multiple myeloma is confirmed not only by the presence of 10% or more replicating plasma cells in BM analysis or by the presence of a plasmacytoma on biopsy, but also by the presence of one or more myeloma-defining events (MDEs). MDEs include the so-called CRAB features (hypercalcemia, renal failure, anemia, or lytic bone lesions) and three specific biomarkers: >60% homologous BM plasma cells, a serum free light chain (sFLC) ratio >100 (if the sFLC level is >100 mg / L), and one or more focal lesions on magnetic resonance imaging (MRI). Several genetic abnormalities in neoplastic plasma cells strongly influence not only the onset of myeloma but also its prognosis.

[0005] The uncontrolled growth of myeloma cells may lead to destruction of bone structure, bone marrow failure, increased and thickened plasma, suppression of normal immunoglobulin production, and kidney damage.

[0006] Currently, symptomatic (active) myeloma is treated aggressively. However, asymptomatic (smoldering) myeloma is treated with observational therapy due to the ineffectiveness of traditional chemotherapy in early treatment. Current research focuses on evaluating whether immunomodulatory drugs can delay the progression of asymptomatic myeloma to symptomatic myeloma. For active myeloma, existing data support initial induction therapy with drugs such as thalidomide, lenalidomide, and / or bortezomib, followed by autologous hematopoietic stem cell transplantation (HSCT) after a significant disease response in patients who can tolerate a conditioning regimen for autologous hematopoietic stem cell transplantation (HSCT). Treatment selection and dosing are determined by the patient's physiologic age (not chronological age) and the presence of coexisting conditions. For example, if a patient has significant complications, such as cardiopulmonary or liver damage, a less intensive treatment regimen may be appropriate due to concerns about treatment-related mortality and the risk of treatment discontinuation.

[0007] Treatment of relapsed / refractory multiple myeloma (RRMM) is particularly challenging due to the diverse nature of relapses and the lack of clear, biologically sound recommendations for rescue therapies at different points in the disease progression. Given that the inherent clonal heterogeneity and genomic instability of plasma cells influence both innate and acquired resistance to therapy, determining the optimal selection and sequencing of therapies is crucial. In recent years, the US Food and Drug Administration (FDA) has approved a number of new drugs for the treatment of 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. Other molecularly targeted therapies targeting specific cell signaling pathways, as well as survival and proliferation control agents (including PI3K / AKT / mTOR inhibitors, Hsp90 inhibitors, cyclin-dependent kinase inhibitors, and kinesin spindle protein inhibitors) are still under development. Despite advances in the management of multiple myeloma, relapse is inevitable for nearly all patients. Each relapse of myeloma is often more aggressive and shortens survival, necessitating the urgent need for complementary therapies.

[0008] In recent years, multiple myeloma patients have benefited from novel treatments targeting multiple myeloma-associated antigens such as CD38 and BCMA.

[0009] CD38 (UniProt P28907) was first identified in 1980 as a surface marker of thymocyte lymphocytes (clusters of differentiation) [see "Lee, HC, ed. (2002). A Natural History of the Human CD38 Gene. Cyclic ADP-Ribose and NAADP. Springer Publishing"]. In 1992, studies also reported it as a surface marker of B cells, monocytes, and natural killer cells (NK cells). Around the same time, CD38 was also reported as a marker of B and T cell activation. Darzalex and sarcoma (SARCLISA), both targeting CD38, are approved for the treatment of multiple myeloma.

[0010] B cell maturation antigen, also known as BCMA, CD269 or TNFRSF17 (UniProt Q02223), belongs to the tumor necrosis receptor superfamily, which is preferentially expressed in differentiated plasma cells [see "Laabi et al. (1992) EMBO J 11(11):3897-3904; and Madry et al. (1998) Int Immunol 10(11):1693-1702"]. BCMA is a non-class I glycosylated transmembrane protein involved in B cell maturation, growth and survival. There are two ligands in the TNF superfamily that use BCMA as their receptor: APRIL (a proliferation-inducing ligand, also known as CD256 or TNFSF13), which has a high affinity for BCMA, and BAFF (also known as THANK, BlyS, B lymphocyte stimulator, TALL-1 or zTNF4), which has a low affinity for BCMA. APRIL and BAFF are structurally similar, but their receptor binding specificities overlap as well as differ. The negative regulator TACI can also bind to both BAFF and APRIL. Ligand binding of APRIL and BAFF to BCMA and / or TACI activates the transcription factor NF-κB, increasing the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1) and downregulating the expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thereby inhibiting apoptosis and promoting survival. This combined action promotes B cell differentiation, proliferation, survival, and antibody production (as examined in Rickert RC et al., Immunol Rev (2011) 244 (1): 115-133). Echoing the above findings, 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 and can treat multiple myeloma, such as teclistamab, can induce T cell-mediated cytotoxicity against BCMA-expressing multiple myeloma cells and / or relapsed or refractory multiple myeloma cells when used alone or in combination with immunotherapeutic drugs such as thalidomide or immunotherapeutic derivatives such as anti-PD-1 antibodies and anti-PD-L1 antibodies (see "US11124577B2").

[0012] While chimeric antigen receptor (CAR) T-cell immunotherapy using bispecific antibodies or T lymphocytes engineered with synthetic chimeric antigen receptors has demonstrated excellent overall response rates, sustained responses beyond two years remain relatively limited (less than 40% progression-free survival for patients treated with idecabtagene vicleucel (see "Munshi, NC et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N. Engl. J. Med. 384, 705–716 (2021)") or teclistamab (see "Moreau, P. et al. Teclistamab in Relapsed or Refractory Multiple Myeloma. N. Engl. J. Med. NEJMoa2203478 (2022) doi:10.1056 / NEJMoa2203478")). Some studies have suggested that relapse is due to target downregulation or insufficient target expression in cell lines (see “Rodríguez-Lobato, LG, Oliver-Caldés, A., Moreno, DF, Fernández de Larrea, C. & Bladé, J. Why Immunotherapy Fails in Multiple Myeloma. Hemato 2, 1–42 (2020)”). This phenomenon of target underexpression in this multiple myeloma population has been observed after treatment with CD38-targeted daratumumab (see "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 (see "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, solutions are needed to address tumor escape, which is often caused by insufficient surface expression of targeted tumor-associated antigens.

[0013] While promising treatments are currently available for hematologic cancers such as multiple myeloma, there remains a need for improvement in therapies, including those based on active cell-mediated antibody therapies. Indeed, despite the expansion of treatment options, resistance to currently available therapies continues to result in a dismal prognosis for patients with multiple myeloma and relapsed / refractory multiple myeloma, necessitating efforts to maximize the therapeutic window. Summary of the Invention

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

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

[0016] The present invention also relates to a trispecific antibody or antibody fragment thereof, comprising at least three binding moieties, at least one of which binds to human CD3, at least one binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a set of heavy chain CDRs having 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 moieties, at least one of which binds to human CD3, at least one binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human BCMA comprises a set of heavy chain CDRs having 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 moieties, at least one of which binds to human CD3, at least one binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety 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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain variable region whose amino acid sequence is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 55; 58; 60; 62 and 66.

[0020] The present invention also relates to a trispecific antibody or antibody fragment thereof, comprising at least three binding moieties, at least one of which binds to human CD3, at least one binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain variable region whose amino acid sequence 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 binds to human BCMA, and at least one 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 whose amino acid sequence is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105 and 108.

[0022] The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds to human CD3, at least one binds to human BCMA, and at least one 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 whose amino acid sequence 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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human BCMA comprises a heavy chain variable region whose amino acid sequence 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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain variable region whose amino acid sequence 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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain variable region whose amino acid sequence is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 593 or 594.

[0026] The present invention also relates to a trispecific antibody or antibody fragment thereof further comprising a light chain variable region of a light chain, the amino acid sequence of which 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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain whose amino acid sequence 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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human BCMA comprises a heavy chain whose amino acid sequence 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 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.

[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 binds to human BCMA, and at least one binds to human CD38, wherein the at least one binding moiety that binds to human CD38 comprises a heavy chain whose amino acid sequence 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 further comprising a light chain whose amino acid sequence is at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1.

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

[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 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; the at least one binding portion that binds to 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; and the at least one binding portion that binds to human CD38 comprises a heavy chain CDR set comprising the amino acid sequences of SEQ ID NO: 236, SEQ ID NO: 362, and SEQ ID NO: The invention also provides a light chain CDR set comprising the amino acid sequence of SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0033] 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 / 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 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 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 antibody fragment thereof comprising a set of three amino acid chains, wherein the amino acid chains comprise 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; 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, wherein the heterodimeric immunoglobulin comprises a first engineered immunoglobulin chain having a first engineered domain; and a second engineered immunoglobulin chain having a second engineered domain, wherein the heterodimeric immunoglobulin is heterodimerized via the first and second engineered domains.

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

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

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

[0046] Specifically, it is used to treat multiple myeloma, relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, active multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer (such as Her2+ breast cancer), prostate cancer, cervical cancer, reproductive center B-cell lymphoma or B-cell acute lymphocytic 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), well-differentiated ovarian serosal epithelial cell carcinoma, and peritoneal cancer.

[0047] The present invention also relates to an epitope located on the extracellular domain of human CD38, which is bound by the trispecific antibodies or antibody fragments thereof disclosed herein.

[0048] More specifically, the present invention also relates to an epitope located 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 determined by X-ray crystallography. Specifically, the resolution determined by X-ray crystallography is at least 5 Å, preferably at least 3.5 Å. In a preferred embodiment, the resolution is approximately 3.4 Å.

[0049] More specifically, the present invention also relates to an epitope located on the extracellular domain of human CD38, which is bound by the above-mentioned antibody 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 determined by X-ray crystallography. Specifically, the resolution as determined by X-ray crystallography is at least 5 Å, preferably at least 3.5 Å. In a preferred embodiment, the resolution is about 3.4 Å.

[0050] The present invention also relates to an epitope located 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 located on the extracellular domain of human CD3 that is bound by the trispecific antibodies or antibody fragments thereof disclosed herein.

[0052] The present invention also relates to an antibody, or antibody fragment, or antigen-binding fragment thereof, which binds to the same epitope on CD3 and / or BCMA and / or CD38 as a reference antibody, or antibody fragment thereof, wherein the reference antibody, or antibody fragment thereof is a trispecific antibody, or antibody fragment thereof, 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: 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.

[0053] The present invention also relates to an antibody or antigen-binding fragment that binds to the same epitope on CD3 as a reference antibody, wherein the reference antibody is a trispecific antibody, 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: 723.

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

[0055] The present invention also relates to an antibody or antigen-binding fragment that binds to an epitope located 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 determined by X-ray crystallography. Specifically, the resolution as determined by X-ray crystallography is at least 5 Å, preferably at least 3.5 Å. In a preferred embodiment, the resolution is about 3.4 Å.

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

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

[0058] The present invention further relates to an antibody or antibody fragment thereof that binds to human CD3, comprising a heavy chain CDR set having 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 of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0059] The present invention further relates to an antibody or antibody fragment thereof that binds to human BCMA, comprising a heavy chain CDR set having 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 further relates to a trispecific heterodimeric antibody comprising a first and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises one or more substitutions selected from the following group: 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 following group: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and the trispecific heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via the first and second engineered CH3 domains. The trispecific heterodimeric antibody comprises at least three binding moieties, each of which binds to a different antigen.

[0061] Specifically, the present invention relates to a trispecific heterodimeric antibody comprising a first 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, and 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, and T411R (EU numbering), wherein the trispecific heterodimeric immunoglobulin or heterodimeric fragment heterodimerizes via the first and second engineered CH3 domains, characterized in that the trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding moieties, each of which binds to a different antigen.

[0062] The present invention discloses antibodies that immunospecifically bind to human BCMA, CD38, and CD3, and antigen-binding fragments thereof. The present invention also discloses polynucleotides encoding the BCMA-specific, CD38-specific, and CD3-specific antibodies and antigen-binding fragments, cells expressing the antibodies and antigen-binding fragments, and related vectors and antibodies and antigen-binding fragments with detectable tags. Furthermore, the present invention provides methods of using the antibodies and antigen-binding fragments. For example, BCMA-specific antibodies and / or CD38-specific antibodies and antigen-binding fragments can be used to diagnose or monitor the progression, improvement, or stabilization of BCMA-expressing and / or CD38-expressing cancers; determine whether a patient should receive cancer treatment; or determine whether a subject has a BCMA-expressing and / or CD38-expressing cancer and is therefore suitable for BCMA-specific anti-cancer therapy and / or CD38-expressing cancer therapy, such as the multispecific antibodies disclosed herein that target BCMA, CD38, and CD3.

[0063] The present invention further provides a multispecific antibody that immunospecifically binds to BCMA, CD38, and CD3, and a multispecific antigen-binding fragment thereof. The present invention also provides polynucleotides encoding the BCMA×CD38×CD3 multispecific antibody, cells expressing the antibody, and related vectors and multispecific antibodies with a detectable label. Furthermore, the present invention provides methods of using the multispecific antibody. For example, the BCMA×CD38×CD3 multispecific antibody can be used to diagnose or monitor the progression, improvement, or stabilization 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 suitable for treatment with a BCMA-specific anti-cancer therapy and / or a CD38-specific anti-cancer therapy, such as the BCMA×CD38×CD3 multispecific antibody of the present invention. Simple diagram description

[0064] Figures 1A and 1B show that the anti-human CD3 C1 drug candidate binds to human CD3 and induces T cell activation. Figure 1A evaluates the binding of the anti-CD3 drug candidate to human PBMCs using flow cytometry. Figure 1B also examines the ability of the anti-CD3 drug candidate to induce activation of purified human T cells over a 48-hour period, as measured by upregulation of CD69 on CD4+ T cells, using flow cytometry. Each point represents the mean ± standard deviation of three independent measurements from a single representative experiment. Experimental setup and analysis are described in detail in Example 1. Figure 2 shows the specific binding of anti-CD3-C1 drug candidates and optimized anti-CD3-C1 drug candidates to human CD3. Flow cytometry was used to evaluate the binding of anti-CD3 drug candidates to wild-type HPB-ALL and CD3-depleted cell lines. Each bar represents the geometric mean fluorescence of a single assay tested at 8 nM in a representative experiment. Experimental setup and analysis are described in Example 2. [Figure 3] is a tool molecule format for evaluating the activation propensity of CD3-binding T cells. Figures 4A and 4B show the 2+1 C1-D6 / BCMA drug candidate triggering the strongest tumor cell killing and T cell activation in a redirected lysis assay. Figure 4A shows the ability of the 2+1 CD3xBCMA-dAbx2 drug candidate to trigger T cell-mediated killing of NCI-H929 tumor cells in a redirected lysis assay at a 5:1 effector-to-target ratio, as measured by flow cytometry 48 hours later. Figure 4B shows human T cell activation assessed by flow cytometry in the same assay, as measured by the percentage of CD69 upregulation in CD4 T cells. Each point is the mean + / - standard deviation of two technical replicates from a representative donor. Experimental setup and analysis are described in detail in Example 3. Figure 5 shows the HDX epitope mapping of the C1-D6 binder on hCD3γε. When bound to IgG at AA182-197, YVCYPRGSKPEDANFY (SEQ ID NO: 726), hCD3γε exhibited a significant decrease in deuterium uptake (-10), indicating this epitope as the primary epitope. The differential heatmap in Figure 5 compares hydrogen / deuterium exchange between hsCD3γ (23-103)-hsCD3ε (23-118) and CD3γ (23-103)-hsCD3ε (23-118) and anti-CD3-C1-D6 IgG1 LALA. Figures 6A to 6D show the blockade of human BCMA / human APRIL and human BCMA / human BAFF interactions when anti-BCMA-D3 Fab and anti-BCMA-E6 Fab bind 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 premixed with various concentrations (0 nM, 25 nM, 50 nM, or 200 nM) of anti-BCMA-D3 Fab in electrophoresis buffer was injected onto immobilized human APRIL (Figure 6A) or immobilized human BAFF (Figure 6B). Similarly, 50 nM recombinant BCMA-Fc protein premixed with anti-BCMA-E6 Fab at varying concentrations (0 nM, 25 nM, 50 nM, or 200 nM) in running buffer was injected onto immobilized human APRIL (Figure 6C) or immobilized human BAFF (Figure 6D). The graphs show data expressed as resonance units (RU; Y-axis) versus time (X-axis). Figure 7 shows the specific binding of anti-BCMA-D3 and anti-BCMA-E6 affinity-matured drug candidates to BCMA. Flow cytometry was used to evaluate the binding of anti-BCMA drug candidates to the wild-type and BCMA-depleted NCI-H929 cell lines. Each bar represents the geometric mean fluorescence of a single assay at 10 nM from a representative experiment. Experimental setup and analysis are described in detail in Example 6. Figures 8A to 8C show the binding of anti-CD38-E2 Fab, anti-CD38-B3 Fab, and daratumumab Fab epitopes to human CD38 measured using Octet biofilm interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin SA biosensor. Fab antibody 1 (Ab1) was injected at a concentration of 200 nM in kinetic buffer onto immobilized human CD38 until surface saturation was achieved. Subsequently, a premixed solution of Ab1 and Fab antibody 2 (Ab2) at a final concentration of 200 nM was injected onto the saturated surface (competition phase). The same experimental procedures were followed for a surface saturation control using Ab1 alone at a concentration of 400 nM in the competition phase. The wavelength shift in the graph represents binding to the sensor tip (response, in nm; Y-axis) versus time (X-axis). Figure 8A shows anti-CD38-E2 Fab as Ab1, Figure 8B shows anti-CD38-B3 Fab as Ab1, and Figure 8C shows daratumumab Fab as Ab1. The curves indicate the Ab2 clone name. Figures 9A and 9B are schematic diagrams of the CD3 / BCMA / CD38 trispecific antibody. The CD3 / BCMA / CD38 antibody is composed of three Fabs targeting three different antigens, with the "Fc-distal" Fab domain fused to the N-terminus of the VH domain of the "Fc-proximal" Fab. Figure 9A shows the CD3 / BCMA / CD38 configuration BO (outside the BTB), with the CD38 binder on Arm A, the CD3-binding arm on the outside, Fc-distal on Arm B, and BCMA on the Fc-proximal side of Arm B. Figure 9B shows the CD3 / BCMA / CD38 configuration C, with the CD3-binding arm on Arm A, the BCMA binder located on Arm B, and the CD38 binder located on Arm B, Fc-proximal. Figures 10A to 10D show infrared thermographic images of CD3 / BCMA / CD38 antibodies, including thermal shift analysis, obtained using differential scanning calorimetry (DSC). Figure 10A shows infrared thermographic images and thermal shift analysis of TREAT CD3 / BCMA / CD38-115; Figure 10B shows infrared thermographic images and thermal shift analysis of TREAT CD3 / BCMA / CD38-117; Figure 10C shows infrared thermographic images and thermal shift analysis of TREAT CD3 / BCMA / CD38-118; and Figure 10D shows infrared thermographic images and thermal shift analysis of TREAT CD3 / BCMA / CD38-119. The expansion of each region corresponding to a different shift is indicated in parentheses after the corresponding temperature. Figures 11A to 11D show surface plasmon resonance measurements of hCD3εδ induced by CD3 / BCMA / CD38 antibodies. Biotinylated hCD3εδ (CD3E & CD3D-377H, Creative Biomart, USA) was immobilized on the sensor surface using a modified streptavidin-biotin capture reagent that can be reversely attached to the CAP chip surface. Various concentrations of CD3 / BCMA / CD38 antibodies (as indicated in the figures) in HBS-EP+ buffer were then injected onto the immobilized hCD3εδ. Graphs show data expressed as the number of resonance units (RU; Y-axis) versus time (seconds; X-axis) for the following: Figure 11A for TREAT CD3 / BCMA / CD38-115; Figure 11B for TREAT CD3 / BCMA / CD38-117; Figure 11C for TREAT CD3 / BCMA / CD38-118; and Figure 11D for TREAT CD3 / BCMA / CD38-119. Binding affinity for human CD3εδ was measured using a steady-state affinity model. This model calculates affinity based on a plot of equilibrium response (Req; Y-axis) versus analyte concentration (M; X-axis) using the formula: Req = KA × C × Rmax / (KA × C × n+1). The equilibrium dissociation constant (KD) is the concentration at 50% saturation. Figures 12A to 12D show surface plasmon resonance measurements of human CD38 mediated by CD3 / BCMA / CD38 antibodies. Biotinylated hCD38 (biotinylated human CD38 protein, Avitag™, His Tag, CD8-H82E7, Acrobiosystems, USA) was immobilized on the sensor surface using a modified streptavidin-biotin capture reagent that can be reversely attached to the CAP chip surface. Various concentrations of CD3 / BCMA / CD38 antibodies (as indicated in the figures) in HBS-EP+ buffer were then injected onto the immobilized hCD38. The graphs show data expressed as the number of resonance units (RU; Y-axis) versus time (seconds; X-axis) for the following: Figure 12A for TREAT CD3 / BCMA / CD38-115; Figure 12B for TREAT CD3 / BCMA / CD38-117; Figure 12C for TREAT CD3 / BCMA / CD38-118; and Figure 12D for TREAT CD3 / BCMA / CD38-119. Binding affinities for human CD38 were measured using a 1:1 Langmuir binding model. This model assumes a 1:1 interaction between ligand and analyte and determines the association rate constant (ka), dissociation rate constant (kd), and affinity (KD) for each sample. Using this model, KD is defined as the ratio of the included dissociation rate constant to the association rate constant, i.e., KD = kd / ka. The values ​​measured for the zero-concentration sample are used as reference data. Chi2 and residual values ​​were used to evaluate the quality of fit between the experimental data (sensogram fc2-fc1) and the respective binding models. Solid lines represent measured data, and dashed lines represent simulated fits. KD: equilibrium dissociation constant; ka: association rate constant. Kd: dissociation rate constant. Figures 13A to 13D show surface plasmon resonance measurements of human BCMA using CD3 / BCMA / CD38 antibodies. Biotinylated hBCMA (biotinylated human BCMA / TNFRSF17 protein, His, Avitag™ DMF Filed, BCA-H82E4, Acrobiosystems, USA) was immobilized on the sensor surface using a modified streptavidin-biotin capture reagent that can be reversely attached to the CAP chip surface. Various concentrations of CD3 / BCMA / CD38 antibodies (as indicated in the figures) in HBS-EP+ buffer were then injected onto the immobilized hBCMA. The graphs show data expressed as the number of resonance units (RU; Y-axis) versus time (seconds; X-axis) for the following: Figure 13A for TREAT CD3 / BCMA / CD38-115; Figure 13B for TREAT CD3 / BCMA / CD38-117; Figure 13C for TREAT CD3 / BCMA / CD38-118; and Figure 13D for TREAT CD3 / BCMA / CD38-119. Binding affinities for human CD38 were measured using a 1:1 Langmuir binding model. This model assumes a 1:1 interaction between ligand and analyte and determines the association rate constant (ka), dissociation rate constant (kd), and affinity (KD) for each sample. Using this model, KD is defined as the ratio of the dissociation rate to the association rate, i.e., KD = kd / ka. The values ​​measured for a zero-concentration sample are used as reference data. Chi2 and residual values ​​were used to evaluate the quality of fit between the experimental data (sensogram fc2-fc1) and the respective binding models. Solid lines represent measured data, and dashed lines represent simulated fits. KD: equilibrium dissociation constant; ka: association rate constant. Kd: dissociation rate constant. Figures 14A to 14E show surface plasmon resonance binding of selected TREAT CD3 / BCMA / CD38 to Fc receptors, compared to trastuzumab IgG1. Figure 14A shows CD3 / BCMA / CD38-115, Figure 14B shows CD3 / BCMA / CD38-117, Figure 14C shows CD3 / BCMA / CD38-118, Figure 14D shows CD3 / BCMA / CD38-119, and Figure 14E shows trastuzumab IgG1. [Figure 15] is a graph showing AC-SINS of selected TREAT CD3 / BCMA / CD38. Figures 16A and 16B show binding of a CD3 / BCMA / CD38 drug candidate to primary human CD38-negative T cells. Binding of the anti-CD3 drug candidate to CD38-negative human T cells was assessed by flow cytometry, and the binding curve is shown in Figure 16A. Each point represents the mean ± standard deviation of six measurements from two independent T cell donors in two separate experiments. Figure 16B shows the KD values ​​determined from the binding curves and compared to baseline using paired one-way ANOVA followed by Tukey's HSD post hoc comparisons. # indicates significant difference from 83A10-TCBcv. Experimental setup and analysis are described in detail in Example 18. Figures 17A to 17C show the binding of CD3 / BCMA / CD38 drug candidates to cell lines with variable CD38 and BCMA expression. Flow cytometry was used to evaluate the binding of the candidate drugs CD3 / BCMA / CD38, -118, -119, and 83A10-TCBcv to CD38 and BCMA in KMS-12-BM (shown in Figure 17A), MOLP-8 (shown in Figure 17B), and NCI-H929 (shown in Figure 17C), cell lines with variable CD38 and BCMA expression. Each point represents the mean of three measurements from three separate experiments + / - standard deviation. Experimental setup and analysis are described in detail in Example 18. Figure 18 shows the CD3 / BCMA / CD38-118 drug candidate induced the strongest in vitro cytotoxicity against multiple myeloma cell lines with variable CD38 and BCMA expression. The CD3 / BCMA / CD38 drug candidate and benchmark-triggered T cells were evaluated in a redirected lysis assay at a 5:1 effector-to-target ratio to mediate cytotoxicity against KMS-12-BM (A), MOLP-8 (B), and NCI-H929 (C) tumor cells and measured by flow cytometry 72 hours after inoculation. Half-maximal effective killing concentration (EC50) values ​​were determined from the redirected lysis assay. Each point represents the EC50 value of an individual donor. Means were compared using paired one-way ANOVA followed by Tukey's most significant post hoc comparison. * indicates statistical significance compared to TNB-F2B BEAT Fc. # indicates statistical significance compared to 83A10-TCBcv. + indicates statistically significant differences compared to BEAT CD3 / CD38. Three separate experiments were conducted for each candidate drug, totaling six donors. Experimental setup and analysis are detailed in Example 18. Figure 19 shows the robust T cell activation and proliferation induced by a CD3 / BCMA / CD38 drug candidate, associated with KMS-12-BM cell killing. The ability of CD3 / BCMA / CD38 drug candidates and baseline-triggered T cells to mediate KMS-12-BM cell killing, as well as the resulting T cell activation and proliferation, was assessed in a redirected lysis assay at a 5:1 effector-to-target ratio. CD4+ and CD8+ T cell proliferation and CD25 expression were assessed at endpoint. The EC50 values ​​for activation or proliferation are shown. Each point represents the EC50 value for an individual donor. Three individual experiments were performed for each drug candidate, for a total of six donors. Means were compared using paired one-way ANOVA followed by Tukey's most significant post hoc comparison. * indicates statistical significance compared to TNB-F2B BEAT Fc. # indicates statistical significance compared to 83A10-TCBcv. + indicates statistically significant compared to BEAT CD3 / CD38. Experimental setup and analysis are described in detail in Example 18. Figures 20A to 20E show that the CD3 / BCMA / CD38 candidate drug induces minimal cytokine release with relevant on-target off-tumor activity compared to the positive control and the benchmark 83A10-TCBcv. Cytokine release was assessed in a high-density assay. At the end of the assay, supernatants were stored at -80°C and assessed for interferon-γ (shown in Figure 20A), tumor necrosis factor-α (shown in Figure 20B), IL-2 (shown in Figure 20C), IL-6 (shown in Figure 20D), and IL-10 (shown in Figure 20E) using a multifactor ultrasensitive electrochemiluminescence immunoassay (Meso Scale Discovery (MSD)). Each bar represents the mean ± standard deviation. A total of four PBMC donors were tested in two separate experiments. Dashed lines indicate the positive (PHA) and negative control groups. Experimental setup and analysis are described in detail in Example 18. Figures 21A and 21B show that, compared to daratumumab and mololimab, the CD3 / BCMA / CD38 drug candidate did not bind to erythrocytes. Figure 21A shows flow cytometric assessment of erythrocyte binding of the CD3 / BCMA / CD38 drug candidate and the reference drug. Each point represents the area under the curve (AUC) of the dose-response curve for each donor. Figure 21B shows a paired one-way ANOVA followed by Dunnett's post hoc comparison comparing the mean with daratumumab (*P < 0.05). The experimental setup and analysis are described in detail in Example 18. [Figure 22] The CD3 / BCMA / CD38 candidate drug did not induce hemagglutination. The tendency of the candidate drug CD3 / BCMA / CD38 and the reference to induce hemagglutination was evaluated in a Coombs assay. Each point represents the mean + / - standard deviation of the Coombs of six donors. The experimental setup and analysis are detailed in Example 18. [Figure 23] CD3 / BCMA / CD38_118 exhibited stronger NCI-H929 killing in multi-mode of action killing compared to the anti-CD38 reference. The ability of the CD3 / BCMA / CD38 candidate drug and the reference to trigger NCI-H929 cell lysis was evaluated using a multi-mode of action killing (MmoAK) assay, in combination with human serum and IL-2. Each point represents the mean + / - mean standard error of the percentage of target killing (shown in Figure 23A), the EC50 killing (shown in Figure 23B), and the area under the curve (AUC) of the dose-response curve for each donor (shown in Figure 23C). The means (5 - 8 donors) were compared using the Kruskal-Wallis followed by Dunnett's post-comparison or paired one-way ANOVA test followed by Tukey's post-comparison (*P<0.05, **0.01<P<0.05, ***P<0.01). The experimental setup and analysis are detailed in Example 18. Figures 24A and 24B show that a CD3 / BCMA / CD38 candidate drug inhibited NCI-H929 tumors in a dose-dependent manner, with efficacy exceeding that of the benchmark 83A10-TCBcv at lower doses. On day 10, female NCG mice were subcutaneously implanted with 1x107 NCI-H929 multiple myeloma cells (CD38++ and BCMA++) and intraperitoneally inoculated with 1x107 PBMCs from two healthy human donors (Donor A: n=9 mice / group and Donor B: n=9 mice / group). Mice were randomized on day 0 when the mean tumor volume reached approximately 150 mm3 and treated weekly with intravenous injections on days 0, 7, and 14 for three weeks. Figure 24A shows NCI-H929 tumor growth in NCG mice (n = 9 mice / group) inoculated with PBMCs from donors A (top) or B (bottom) following treatment with 0.5 mg / kg (left), 0.1 mg / kg (center), or 0.02 mg / kg (right). Means ± SEM are shown, including last observation carried forward (LOCF). Figure 24B shows 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 PBMCs from donors A (left) or B (right). Molecular concentrations were determined using a human BCMA-specific antigen capture ELISA (anti-BCMA human IgG quantification method). Values ​​shown are mean ± SD; values ​​within the bars represent mean concentrations, and values ​​above represent fold differences. Figures 25A and 25B show that the CD3 / BCMA / CD38 candidate drug significantly slowed NCI-H929 tumor growth at a dose of 0.1 mg / kg compared to vehicle or CD3 mimic control groups. On day 11, NCG female mice were subcutaneously implanted with 1x107 NCI-H929 multiple myeloma cells (CD38++ and BCMA++) and intraperitoneally inoculated with 1x107 PBMCs from two healthy human donors (Donor A: n=9 mice / group and Donor B: n=9 mice / group). Mice were randomized on day 0, when the mean tumor volume reached approximately 180 mm3, and treated weekly with intravenous injections on days 0, 7, and 14 for three weeks. Figure 25A shows NCI-H929 tumor growth in NCG mice (n=9 mice / group) inoculated with PBMCs from donors A (top) or B (bottom) following treatment at a dose of 0.1 mg / kg. Means ± SEM are shown, including the last observation value extrapolation (LOCF). Figure 25B shows plasma concentrations (ng / mL) of the indicated molecules at the indicated doses on day 10 (for vehicle and CD3 / BCMA / CD38-122) or day 14 (for the remaining groups) after treatment (two intravenous injections). Molecular concentrations were determined using a human BCMA-specific antigen capture ELISA (anti-BCMA human IgG quantification method). Values ​​are shown as mean ± SD. Figures 26A to 26D show the tumor control effects of CD3 / BCMA / CD38-118, -119, and 83A10-TCBcv in NCI-H929 tumors, correlated with T cell infiltration within the tumor microenvironment. Female NCG mice were transplanted with NCI-H929 multiple myeloma cells and intraperitoneally inoculated with PBMCs from two healthy human donors (Donor A and Donor B). On day 0, when the average tumor volume reached approximately 180 mm³, mice were treated with a single intravenous injection of 0.1 mg / kg of the molecule. Tumors and spleens from treated mice were harvested two or six days later and analyzed ex vivo by flow cytometry (n = 2-3 mice / group / time point). Figure 26A is a bar graph showing intratumoral human TCR Ab expression on selected human CD45+ cells at day 2 (left) and day 6 (right) after single-dose treatment. Numbers indicate the percentage of positive cells. Figure legends (from top to bottom): vector, 83A10-TCBcv, CD3 / BCMA / CD38-118, CD3 / BCMA / CD38-119, CD3 / BCMA / CD38-122. Figure 26B is a graph depicting the number of human TCRab cells per gram of tumor on the indicated days and by PBMC donor. Values ​​shown are mean ± SD. Figure 26C is a contour plot showing the expression of human CD69 and human CD25 in tumor-infiltrating CD8+ T cells on day 2 after administration of the indicated molecules. Numbers in the figure represent percentages. Figure 26D is a graph showing the percentage of CD8 T cells positive for human CD69 (left) and human CD25 (right) in the tumor (top) and spleen (bottom) on day 2 after a single dose. Values ​​shown are mean ± SD. Figure 27 shows the induction of human inflammatory cytokine secretion by a CD3 / BCMA / CD38 drug candidate in the tumor microenvironment. Female NCG mice were transplanted with NCI-H929 multiple myeloma cells and intraperitoneally inoculated with PBMCs from two healthy human donors (Donor A and Donor B). On day 0, when the average tumor volume reached approximately 180 mm³, mice were treated with a single intravenous injection of 0.1 mg / kg of the molecule. Tumors were harvested and supernatants obtained 2 or 6 days later. Tumor supernatants were analyzed for cytokine detection using Luminex® with the Cytokine and Chemokine 34-Plex Human Panel (n = 2-3 mice / group / time point). The graph shows the concentration of the indicated cytokine in tumor supernatant on day 2 or 6 after administration of the indicated molecule, normalized to per gram of tumor. Values ​​are mean ± SD. Figures 28A and 28B show that the CD3 / BCMA / CD38 candidate drug demonstrated at least equivalent efficacy to 83A10-TCBcv in controlling BCMA-lowCD38-low KMS-12-BM tumors. NSG female mice were subcutaneously implanted with 1 × 10 KMS-12-BM multiple myeloma cells (CD38-low and BCMA-low) on day 0 and intraperitoneally inoculated with 1 × 10 PBMCs from two healthy human donors (Donor C: n = 8 mice / group and Donor D: n = 8 mice / group). Mice were randomized on day 8, when the mean tumor volume reached approximately 150 mm3, and treated intravenously once weekly on days 9, 16, and 23 for 3 weeks. Figure 28A shows KMS-12-BM tumor growth in NSG mice (n = 8 mice / group) inoculated with PBMCs from donors C (top) or D (bottom) at doses of 0.5 mg / kg (left), 0.1 mg / kg (center), or 0.02 mg / kg (right). Means ± SEM are shown, including the last observation extrapolation (LOCF). Figure 28B shows serum concentrations (ng / mL) of the indicated molecules at the indicated doses on days 23 or 30 after treatment (at trough exposure, before the third dose (day 23) or one week after). Molecular concentrations were determined using a human BCMA-specific antigen capture ELISA (anti-BCMA human IgG quantification method). Values ​​shown are mean ± SD; values ​​in the bars represent mean concentrations, and values ​​above represent fold differences. Figures 29A and 29B show complete regression of KMS-12-BM tumors from one PBMC donor following treatment with a 0.5 mg / kg dose of a CD3 / BCMA / CD38 candidate drug. On day 9, NSG female mice were subcutaneously transplanted with 1x107 KMS-12-BM multiple myeloma cells (CD38low and BCMAlow) and intraperitoneally inoculated with 1x107 PBMCs from two healthy human donors (Donor C: n=8 mice / group and Donor D: n=8 mice / group). Mice were randomized on day 1, when the mean tumor volume reached approximately 120 mm3, and treated intravenously once weekly on days 0, 7, and 14 for three weeks. Figure 29A shows KMS-12-BM tumor growth in NSG mice (n = 8 mice / group) inoculated with PBMCs from donors C (left) or D (right) following treatment at a dose of 0.5 mg / kg. Mean ± SEM is included in the last observation estimate (LOCF). Figure 29B shows serum concentrations (ng / mL) of the indicated molecules in NSG mice (n = 3-4 mice / group) inoculated with PBMCs from donors C (left) or D (right) on day 6 after treatment (after a single intravenous injection). Molecular concentrations were determined using a human BCMA-specific antigen capture ELISA (anti-BCMA human IgG quantification method). Values ​​shown are mean ± SD; values ​​within bars represent mean concentrations. Figures 30A to 30D show the therapeutic efficacy of CD3 / BCMA / CD38 drug candidates as correlated with T cell infiltration and activation within the tumor microenvironment. NSG female mice were transplanted with KMS-12-BM multiple myeloma cells and intraperitoneally inoculated with PBMCs from two healthy human donors (Donor C and Donor D). On day 0, when the average tumor volume reached approximately 120 mm³, they received a single intravenous dose of 0.5 mg / kg of the drug. 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). Figure 30A is a bar graph showing the expression of human TCR Ab in gated human CD45+ cells on day 2 (left) and day 6 (right) after single-dose administration of the drug. Numbers indicate the percentage of positive cells. Figure legends (from top to bottom): vector, 83A10-TCBcv, CD3 / BCMA / CD38-118, CD3 / BCMA / CD38-119, CD3 / BCMA / CD38-122. Figure 30B is a graph depicting the number of human TCRab cells per gram of tumor on the indicated days and by PBMC donor. Values ​​shown are mean ± SD. Figure 30C is a contour plot showing the expression of human CD69 and human CD25 on tumor-infiltrating CD8+ T cells two days after administration of the indicated molecules. Numbers in the figure represent percentages. Figure 30D is a graph showing the percentage of human CD69 (left) and human CD25 (right) positive CD8 T cells in the tumor (top) and spleen (bottom) two days after a single dose. Values ​​shown are mean ± SD. Figure 31 shows the induction of human cytotoxic cytokine secretion by a CD3 / BCMA / CD38 drug candidate in the tumor microenvironment. NSG female mice were transplanted with KMS-12-BM multiple myeloma cells and intraperitoneally inoculated with PBMCs from two healthy human donors (Donor C and Donor D). On day 0, when the average tumor volume reached approximately 120 mm³, they 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 were obtained. Tumor supernatants were analyzed for cytokine detection using the Luminex® Cytokine and Chemokine 34-Plex Human Panel (n = 2-3 mice / group / time point). The graph shows the concentration of the indicated cytokine in tumor supernatant (in ng) on ​​day 2 or day 6 after administration of the indicated molecule, normalized to per gram of tumor. Values ​​are mean ± SD. Figure 32 shows the pharmacokinetic evaluation of CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-119 in TG32 SCID mice. TG32 SCID mice were injected intravenously with a single dose of 5 mg / kg of CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-119, and plasma concentrations of therapeutic drugs were assessed. Data shown are the mean ± SD of 6 mice. Figure 33 shows CD3 / BCMA / CD38-118 binding to CD3-expressing T cells. Flow cytometry was used to assess binding of CD3 / BCMA / CD38-118 to the CD3xBCMA benchmark 83A10-TCBcv, onukinomab, and teixumab on isolated human T cells. Each point represents the mean ± standard deviation of five to six donors from two separate experiments. Experimental setup and analysis are described in Example 22. Figures 34A to 34E show CD3 / BCMA / CD38-118 binding to cell lines displaying variable CD38 and BCMA expression or lacking the antigen. Flow cytometry was used to evaluate CD3 / BCMA / CD38-118 binding to the CD3xBCMA benchmark 83A10-TCBcv, onukinomab, and teixumab on cell lines displaying variable CD38 and BCMA expression, including KMS-12-BM (shown in Figure 34A), MOLP-8 (shown in Figure 34B), NCI-H929 WT (shown in Figure 34C), NCI-H929 BCMA KO (shown in Figure 34D), and NCI-H929 CD38 KO (shown in Figure 34E). Each point represents the mean ± standard deviation of three measurements from three separate experiments. Experimental setup and analysis are described in detail in Example 22. Figures 35A to 35E show that CD3 / BCMA / CD38-118 binds more effectively to cell lines exhibiting variable CD38 and BCMA expression than to control molecules lacking either BCMA or CD38 binders. Flow cytometry was used to assess binding of CD3 / BCMA / CD38-118 and control groups CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-151 on different cell lines exhibiting variable CD38 and BCMA expression: KMS-12-BM (shown in Figure 35A), MOLP-8 (shown in Figure 35B), NCI-H929 WT (shown in Figure 35C), NCI-H929 BCMA KO (shown in Figure 35D), and NCI-H929 CD38 KO (shown in Figure 35E). Each point represents the mean ± standard deviation of three measurements from three separate experiments. Experimental setup and analysis are described in detail in Example 22. Figures 36A to 36E show that CD3 / BCMA / CD38-118 induced the strongest KMS-12-BM killing in vitro in the absence or presence of the respective soluble factors. In a redirected lysis assay, the ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmark 83A10-TCBcv, onukinomab, and teixumab to trigger T cell-mediated KMS-12-BM killing was assessed in the absence or presence of the respective soluble factors at a 5:1 effector-to-target ratio. Flow cytometry was used after 48 hours of culture. The percentage of killing in the absence of antibody and the associated half-maximal effective killing concentration (EC50) values ​​were determined from the redirected lysis assay. Each point represents the EC50 value for an individual donor. Bars represent the data set mean. Means were compared using a mixed-effects model (when some values ​​were missing) and paired one-way ANOVA (when all values ​​were included), followed by Tukey's honestly significant difference post hoc comparison. # p-value < 0.0001 **** CD3 / BCMA / CD38-118 compared to all other soluble factor-containing molecules. Experimental setup and analysis are described in Example 22. Figures 37A to 37E show that CD3 / BCMA / CD38-118 induced the strongest KMS-12-BM killing in vitro in the absence or presence of the respective soluble factors. In a redirected lysis assay, the ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmark 83A10-TCBcv, onukinomab, and teixumab to trigger T cell-mediated KMS-12-BM killing was assessed in the absence or presence of the respective soluble factors at a 5:1 effector-to-target ratio and measured by flow cytometry after 48 hours of culture. Percent cytotoxicity and associated half-maximal effective killing concentration (EC50) values ​​were determined from the redirected lysis assay. Each point represents the EC50 value of an individual donor. Bars represent the data set mean. Means were compared using a mixed-effects model (when some values ​​were missing) and paired one-way ANOVA (when all values ​​were included), followed by Tukey's honestly significant difference post hoc comparison. # p-value < 0.0001 **** CD3 / BCMA / CD38-118 compared to all other soluble factor-containing molecules. Experimental setup and analysis are described in Example 22. Figures 38A and 38B show that CD3 / BCMA / CD38-118 induces the strongest in vitro KMS-12-BM killing in the presence of competing soluble factors. In a redirected lysis assay, the ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmark 83A10-TCBcv, onukinomab, and teixumab to trigger T cell-mediated KMS-12-BM killing was assessed in the absence (left, open white symbols) or presence of a combination of soluble factors (right; soluble BCMA, soluble CD38, and APRIL, solid black symbols) at a 5:1 effector-to-target ratio. Flow cytometry was used after 48 hours of culture. The percentage of killing versus no antibody, the percentage of cytotoxicity, and the associated half-maximal effective killing concentration (EC50) values ​​were determined from the redirected lysis assay. Each point represents the EC50 value for an individual donor. Bars indicate the data set mean. Means were compared using a mixed-effects model (when some values ​​were missing) and paired one-way ANOVA (when all values ​​were included) followed by Tukey's most honestly significant difference post hoc comparison. The experimental setup and analysis are detailed in Example 22. Figures 39A to 39F show that CD3 / BCMA / CD38-118 induced the strongest in vitro killing of all cell lines. In a redirected lysis assay, the ability of CD3 / BCMA / CD38-118 and the CD3xBCMA benchmark 83A10-TCBcv, onukinomab, and teixumab to trigger T cell-mediated killing of multiple myeloma cell lines was assessed using isolated T cells at a 5:1 effector-to-target ratio on KMS-12-BM (shown in Figures 39A and 39D), MOLP-8 (shown in Figures 39B and 39E), and NCI-H929 (shown in Figures 39C and 39F) cells. The results were measured by flow cytometry after 48 hours of culture. Percent killing (shown in Figures 39A, 39B, and 39C) and percent cytotoxicity (shown in Figures 39D, 39E, and 39F) against the non-antibody group were determined from the redirected lysis assay, along with the associated half-maximal effective killing concentration (EC50) values. Each point represents the EC50 value for an individual donor. The bar represents the data group mean. Means were compared using a mixed-effects model (when some values ​​were missing) and a paired one-way ANOVA (when all values ​​were included) followed by Tukey's honestly significant post hoc comparison. Ns: not statistically significant; *: 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 detail in Example 22. Figures 40A to 40F show that CD3 / BCMA / CD38-118 induced higher cytotoxicity across all cell lines compared to bispecific antibody combinations with the same binders. In a redirected lysis assay, isolated T cells were used to assess the ability of CD3 / BCMA / CD38-118, along with controls CD3 / BCMA / CD38-149, CD3 / BCMA / CD38-151, and their combination, to trigger T cell-mediated cytotoxicity of multiple myeloma cell lines at a 5:1 effector-to-target ratio on KMS-12-BM (shown in Figures 40A and 40D), MOLP-8 (shown in Figures 40B and 40E), and NCI-H929 (shown in Figures 40C and 40F). The ability to do so was measured by flow cytometry after 48 hours of culture. Percentages of killing (shown in Figures 40A, 40B, and 40C) and percentages of cytotoxicity (shown in Figures 40D, 40E, and 40F) against the non-antibody group were determined from the redirected lysis assay, along with the associated half-maximal effective killing concentration (EC50) values. Each point represents the EC50 value for an individual donor. Bars represent the data group mean. Means were compared using a mixed-effects model (when some values ​​were missing) and a paired one-way ANOVA (when all values ​​were included) followed by Tukey's honestly significant post hoc comparison. Ns: not statistically significant; *: p = 0.05-0.01; **: p = 0.01-0.001; ***: p < 0.001-0.0001; ****: p < 0.0001. NQ: not quantifiable. Experimental setup and analysis are described in detail in Example 22. Figure 41 shows that CD3 / BCMA / CD38-118 demonstrated statistically superior killing compared to the combination of tektuzumab and daratumumab. In an MmoAK assay, the ability of CD3 / BCMA / CD38-118, along with the benchmark daratumumab, tektuzumab, and the combination, to trigger killing of multiple myeloma cell lines was assessed using human PBMCs at a 5:1 effector-to-target ratio on KMS-12-BM cells. Flow cytometry was used after 48 hours of culture. Two concentrations of CD3 / BCMA / CD38-118 (A, C) and 100 (B, D) pM were used in combination with a single dose of 100 nM daratumumab. After culture, the percentage of killing (A, B) and the percentage of cytotoxicity (C, D) against no antibody were assessed. Each point represents an individual donor. Bars represent the group mean. Means were evaluated using paired one-way analysis of variance followed by Tukey's honestly significant difference post hoc comparison. Ns: not statistically significant; *: 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. Figures 42A to 42D show CD3 / BCMA / CD38-118-induced CD138+ cell killing in multiple myeloma patient samples. BMMCs from four donors (Figure 42A: smoldering multiple myeloma; Figure 42B: newly diagnosed multiple myeloma; Figure 42C: refractory relapsed multiple myeloma; Figure 42D: plasma cell leukemia (PCL)) were cultured in RPMI 10% human serum and 3 ng / ml recombinant human IL-6 and treated for 17-22 hours with various concentrations of CD3 / BCMA / CD38-118, a control (texitumomab), and a negative control (CD3 / BCMA / CD38-085, not shown). Absolute counts of live CD138+ cells were obtained and normalized to the absence of antibody to calculate the percentage of killing compared to the absence of antibody. Each point represents the mean of an individual donor + / - standard error of the mean of two measurements (except for B and C, which were performed in a single run). The experimental setup and analysis are described in detail in Example 22. Figure 43 shows that CD3 / BCMA / CD38-118 induces superior killing of CD138+ cells compared to teixostatin in multiple myeloma patient samples. BMMCs from six to eleven individual donors were cultured in RPMI with 10% human serum and 3 ng / ml recombinant human IL-6 and treated for 17-22 hours with various concentrations of CD3 / BCMA / CD38-118, a CD3xBCMA benchmark (teixostatin), and a negative control (not shown). Absolute counts of live CD138+ cells were obtained and normalized to the no-antibody condition to calculate the percentage of killing compared to the no-antibody condition. Each point represents an individual donor. Bars show the mean + / - standard deviation of the individual donors at the specified concentration. Experimental setup and analysis are described in detail in Example 22. Figures 44A to 44C show that the efficacy of CD3 / BCMA / CD38-118 in controlling KMS-12-BM tumors is dose-dependent, and even high-dose BEAT CD3 / CD38 and teixumab failed to delay tumor progression. On day 9, NSG female mice were subcutaneously implanted with 1x107 NCI-H929 multiple myeloma cells (CD38 low and BCMA low) and intraperitoneally inoculated with 1x107 PBMCs from a healthy human donor (Donor C). Mice were randomized into groups on day 1 when the mean tumor volume reached approximately 187 mm3 and treated intravenously once weekly on days 0, 7, and 14 for three weeks. Figure 44A shows KMS-12-BM tumor growth in NSG mice (n = 8 mice / group) inoculated with donor C PBMCs after treatment with the indicated doses of CD3 / BCMA / CD38-118 (top); BEAT CD3 / CD38 (center); or teixumab (bottom). Mean ± SEM includes the last observed value estimate (LOCF). Figure 44B shows KMS-12-BM tumor growth in NSG mice (n = 8 mice / group) inoculated with donor C PBMCs after treatment with the indicated molecules at doses of 0.5 mg / kg (left) or 0.1 mg / kg (right). Mean ± SEM includes the last observed value estimate (LOCF). Figure 44C shows serum concentrations (ng / mL) of the indicated molecules in NSG mice (n = 4-7 mice / group) inoculated with PBMCs from donor C on days 13, 14, or 21 after treatment with the indicated doses (at trough exposure, before the third dose (days 13 / 14), or one week later). Values ​​shown are mean ± SD; values ​​in the middle of the bars represent mean concentrations, and values ​​above represent fold differences. NIC = non-interpolated concentration. Figures 45A to 45D show that high-dose BEAT CD3 / CD38 and teixumab reduce the tumor microenvironment and peripheral T cell compartment. NSG female mice transplanted with KMS-12-BM multiple myeloma cells and intraperitoneally inoculated with PBMCs from a healthy human donor (Donor C) were treated with the indicated molecules intravenously at the indicated doses on day 0, when the mean tumor volume reached approximately 187 mm³, and then weekly thereafter for 3 weeks. On day 13, tumors and spleens from treated mice were harvested and analyzed ex vivo by flow cytometry (n = 2-3 mice / group). Figure 45A is a grayscale pseudocolor image showing the expression of human and mouse CD45 in live cells within the circled tumor. Numbers indicate the percentage of positive cells. Figure 45B is a contour plot showing the expression of human TCRab in tumor-infiltrating CD45+ cells. Numbers indicate the percentage of positive cells. Figure 45C is a graph showing the number of CD45+ (top) and human TCRab+ (bottom) cells per gram of tumor. Values ​​are shown as mean ± SD. Figure 45D is a graph showing the number of human CD45+ (top) and human TCRab+ (bottom) cells in the spleens of treated mice. Values ​​are shown as mean ± SD. Figures 46A to 46D show that BEAT CD3 / CD38 + IVIG treatment improves T cell pleural tract and enhances efficacy against KMS-12-BM tumors in vivo. On day 9, NCG female mice were subcutaneously transplanted with 1x107 KMS-12-BM multiple myeloma cells and intraperitoneally inoculated with 1x107 PBMCs from a healthy human donor (Donor E). Mice were randomized into groups on day 1, when the mean tumor volume reached approximately 120 mm3. On the same day, either 200 mg / kg of IVIG (Privigen) or PBS was administered intravenously, followed by two weekly injections. On days 0 and 7, vehicle, 0.5 mg / kg of CD3 / BCMA / CD38-118, or 0.5 mg / kg of BEAT CD3 / CD38 were administered intravenously. Figure 46A shows KMS-12-BM tumor growth curves in NSG mice (n = 3-5 mice / group) vaccinated with PBMCs from donor E after treatment with the indicated molecules plus IVIG or without treatment. Values ​​are shown as mean ± SEM. Figure 46B shows KMS-12-BM tumor volume on day 11 (study endpoint) after treatment with the indicated molecules plus IVIG. Values ​​are shown as mean ± SD. Figures 46C and 46D show ex vivo flow cytometric analysis of tumors and spleens harvested from treated mice on day 11 (n = 3-5 mice / group). Figure 46C is a grayscale false-color image showing the expression of human and mouse CD45 in live cells within the circled tumors. Numbers indicate the percentage of positive cells. Figure 46D is a graph showing the number of human CD45+ cells (top) and human TCRab+ cells (bottom) within the circled human CD45+ cells per gram of tumor (Figure 46D) or spleen (Figure 46D (Continued)) of treated mice. Values ​​shown are mean ± SD. Figures 47A to 47C show that the highest doses of CD3 / BCMA / CD38-118, BEAT CD3 / CD38, and tixostat alone induced KMS-12-BM tumor regression when combined with IVIG. On day 9, NSG female mice were subcutaneously implanted with 1x107 KMS-12-BM multiple myeloma cells and intraperitoneally inoculated with 1x107 PBMCs from a healthy human donor (Donor C). Mice were randomized into groups on day -1, when the mean tumor volume reached approximately 147 mm3. On the same day, 200 mg / kg of IVIG (Privigen) was administered intravenously, followed by weekly administration for three weeks (days -1, 6, and 13). On days 0, 7, and 14 (weekly for 3 weeks), vehicle, CD3 / BCMA / CD38-118 at doses of 0.5, 0.1, and 0.02 mg / kg, and teixumab at doses of 2.5, 0.5, and 0.1 mg / kg were administered intravenously. Figure 47A shows KMS-12-BM tumor growth in NSG mice (n = 6-8 mice / group) inoculated with donor C PBMCs after treatment with CD3 / BCMA / CD38-118 (top), BEAT CD3 / CD38 (center), or teixumab (bottom). Means ± SEM are shown, including the last observation value estimate (LOCF). Figure 47B shows KMS-12-BM tumor growth in NSG mice (n = 6-8 mice / group) inoculated with donor C PBMCs at doses of 0.5 mg / kg (left) or 0.1 mg / kg (right). Mean ± SEM is shown, including the last observation extrapolation (LOCF). Figure 47C shows serum concentrations (ng / mL) of the indicated molecules in NSG mice (n = 6-8 mice / group) inoculated with donor C PBMCs at day 21 (at trough exposure, one week after the third dose) after treatment with the indicated doses. Values ​​shown are mean ± SD; values ​​in the middle of the bars represent mean concentrations, and values ​​above represent fold differences. NIC = not interpolated concentration; BLOQ = below the limit of quantification. Figures 48A and 48B compare the competition of CD3 / BCMA / CD38-118 for isatuximab and daratumumab binding to recombinant human CD38 using Octet Bio-Layer Interferometry. Biotinylated human CD38-avi-his protein was loaded onto a streptavidin-SA biosensor. The CD38-loaded biosensor was then immersed in a solution of Antibody 1 in kinetic buffer to achieve surface saturation at a concentration of 200 nM. 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 solution of Antibody 1 alone at a concentration of 400 nM in the competition phase as a surface saturation control. The graph shows changes in sensor tip binding (response, in nm; y-axis) versus time (x-axis), expressed as wavelength shift, for isatuximab (Figure 48A) and daratumumab (Figure 48B) as Antibody 1 (saturating antibody). Antibody names are labeled on the curves. Figure 49 is a schematic diagram of another CD3 / BCMA / CD38 trispecific antibody. This CD3 / BCMA / CD38 antibody is constructed with three Fabs targeting three antigens, with the "Fc-distal" Fab domain fused to the N-terminus of the "Fc-proximal" Fab VH domain. In this CD3 / BCMA / CD38 configuration D, the CD3-binding arm is located on the outer side of the B arm, at the Fc-distal position; the CD38 binder is located on the B arm, at the Fc-proximal position; and the BCMA binder is located on the A arm. Figures 50A to 50C show how binder localization within the CD3 / BCMA / CD38 antibody affects its cytotoxic potency. In a redirected lysis assay, the ability of CD3 / BCMA / CD38-118 and control antibodies CD3 / BCMA / CD38-149, -151, -170, -171, and -172 to trigger T cell-mediated KMS-12-BM tumor cell cytotoxicity was assessed using isolated T cells at a 5:1 effector-to-target ratio. Data shown are the percentage cytotoxicity of CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-171 (shown in Figure 50A); CD3 / BCMA / CD38-151 and CD3 / BCMA / CD38-172 (shown in Figure 50B); and CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-170 (shown in Figure 50C) at different concentrations. Each curve represents a nonlinear logistic 4PL model with variable slope, and symbols represent the mean ± SEM of six T cell donors. Percent killing was compared using paired two-way analysis of variance followed by Stark's multiple comparison test (*: 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 detail in Example 25b. Figures 51A to 51C show how binding site localization within CD3 / BCMA / CD38 antibodies influences on-target T cell activation. High-density PBMC assays were used to measure upregulation of the CD25 activation marker in CD8+ T cells, assessing the potential of CD3 / BCMA / CD38-118 and control antibodies CD3 / BCMA / CD38-149, -151, -170, -171, and -172 to induce on-target T cell activation. The percentages of CD8+ T cells expressing CD25 are shown for: Figure 51A for CD3 / BCMA / CD38-149 and CD3 / BCMA / CD38-171; Figure 51B for CD3 / BCMA / CD38-151 and CD3 / BCMA / CD38-172; and Figure 51C for CD3 / BCMA / CD38-118 and CD3 / BCMA / CD38-170. Each curve represents a nonlinear logistic 4PL model with variable slope, and symbols represent the mean ± SEM of six individual donors. Comparisons of the percentage of CD8+ T cells expressing CD25 were performed using paired two-way analysis of variance followed by Stark's multiple comparison test (*: 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 detail in Example 25b. Figures 52A to 52C show the crystal structures of human CD38 complexed with anti-CD38-B3. Figure 52A shows the overall structure of the anti-CD38-B3 / hsCD38 complex. hsCD38 is in light gray, the Fab heavy chain is in black, and the Fab light chain is in gray. Figure 52B shows the footprint of anti-CD38-B3 on CD38, demonstrating the CD38 residues that interact with B3. Underlined residues indicate residues shared between the B3 epitope and daratumumab. Residues in italics and underlined indicate residues shared between the anti-CD38-B3 epitope and isatuximab. Figure 52C shows a surface representation of the anti-CD38-B3 epitope on CD38, with the side chains of interacting residues from B3 shown as branches. HCDRs are in dark gray, and LCDRs are in gray. Experimental setup and analysis are described in detail in Example 26. Figures 53A to 53C show the binding of anti-CD38-B3, isatuximab, and daratumumab to specific epitopes on CD38. Figure 53A shows the binding pattern of anti-CD38-B3 Fab and daratumumab (PDB 7DHA). Figure 53B shows the binding pattern of anti-CD38-B3 Fab and isatuximab (PDB 4CMH). The light chain of the anti-CD38-B3 Fab clashes with the heavy chain of isatuximab. The clash between the two antibodies is indicated by a dashed oval. Figure 53C shows a surface representation of the three epitopes on CD38 for anti-CD38-B3 (dark gray) and isatuximab (gray). Shared residues between the two epitopes are indicated in light color. Implementation Method

[0065] As used herein, unless otherwise indicated, the following terms should be understood as follows. Unless the context requires otherwise, singular terms shall include plurality 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 combinations of two or more such molecules and the like.

[0066] It should be understood that the aspects and embodiments of the present invention described herein include "including" and "essentially including" the aspects and embodiments.

[0067] As used herein, "polynucleotide" refers to a single- or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms thereof. Such modifications include base modifications such as bromuridine; ribonucleotide modifications such as arabinoside and 2',3'-deoxyribose; and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, selenophosphate, phosphorodiselenoate, anilinothioate, phosphazolylphosphate, and aminophosphate. The term "polynucleotide" specifically encompasses single- and double-stranded forms of DNA.

[0068] An "isolated polynucleotide" is a polynucleotide of genetic, cDNA, or synthetic origin, or any combination thereof, that: (1) is not naturally associated with all or part of a polynucleotide comprising the isolated polynucleotide, (2) is linked to a polynucleotide to which it is not naturally linked, or (3) does not naturally occur as part of a larger sequence.

[0069] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it is normally associated, (2) is substantially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by cells of another species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) is not associated (by covalent or non-covalent interactions) with a portion of a polypeptide with which the "isolated polypeptide" is naturally associated, (6) has been manipulated to become associated (by covalent or non-covalent interactions) with a polypeptide with which the "isolated polypeptide" is not naturally associated, or (7) does not occur in nature. Such an isolated polypeptide may be encoded by synthetically derived genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants that occur in its natural environment and that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0070] The present invention relates to a multispecific binding molecule, such as a multispecific binding protein, such as an antibody or an antibody fragment thereof, comprising at least three binding moieties, at least one of which binds to human CD3, at least one binds to human BCMA, and at least one binds to human CD38.

[0071] As used herein, "binding protein" or "binding molecule" refers to a non-naturally occurring, recombinant, or genetically engineered molecule, such as a protein, such as a non-naturally occurring, recombinant, or genetically engineered antibody, that is capable of specifically binding to at least one target antigen (e.g., the CD38 polypeptide, BCMA polypeptide, or CD3 polypeptide described herein).

[0072] A "recombinant" molecule is one that is prepared, expressed, created, or isolated by recombinant means.

[0073] The terms "antibody" and "immunoglobulin" are used interchangeably herein and include whole antibodies and any antigen-binding fragments or single chains thereof. Naturally occurring antibodies typically comprise tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair comprising a full-length "light" chain (typically with a molecular weight of approximately 25 kDa) and a full-length "heavy" chain (typically with a molecular weight of approximately 50-70 kDa). As used herein, "heavy chain" and "light chain" refer to any immunoglobulin polypeptide with sufficient variable domain sequence to confer specificity for the target antigen. The amino-terminal portions of the light and heavy chains typically have variable domains of approximately 100 to 110 or more amino acids, responsible for antigen recognition. The carboxyl-terminal portion of each chain typically contains constant domains, responsible for the functional role. Therefore, in naturally occurring antibodies, a full-length heavy chain immunoglobulin polypeptide comprises one 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, while a full-length light chain immunoglobulin polypeptide comprises one variable domain (VL) and one 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.

[0074] Human light chains are generally classified as kappa (κ) and lambda (λ), while heavy chains are further classified as mu (μ), delta (δ), gamma (γ), alpha (α), and epsilon (ε), defining the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has several subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into several subclasses, including but not limited to IgA1 and IgA2. In full-length light and heavy chains, the variable and constant domains are typically connected by a "J" region of approximately 12 or more amino acids, while heavy chains also include a "D" region of approximately 10 or more amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated herein by reference in its entirety. The variable regions of each light chain / heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically share the same overall structure, with relatively conserved framework regions (FRs) bounded by three highly variable regions, also known as complementarity-determining regions, or CDRs. The CDRs from the two chains in each pair are typically aligned by the framework regions, which facilitate binding to a specific epitope. Both light and heavy chain variable domains typically comprise, from amino-terminus to carboxyl-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0075] A "CDR set" is a set of three CDRs that appear in a single variable region that is capable of binding an antigen. The exact boundaries of the CDRs are defined differently depending on the system to which they belong. The system described by Kabat (see "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 all variable regions of antibodies, but also provides precise residue boundaries that define the three CDRs mentioned above. The CDRs of Kabat et al. can be called Kabat CDRs. Chothia and colleagues (see "Chothia and Lesk, 1987, J. Mol Biol. 196: 901-17; Chothia et al, 1989, Nature 342: 877-83") found that the Kabat CDRs Specific subsections of the CDRs differ significantly in amino acid sequence but share nearly identical peptide backbone conformations. These subsections are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions are referred to as Chothia CDRs, whose boundaries overlap with those of the Kabat CDRs. (See 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) Other CDR boundary definitions for partially overlapping CDRs have been described. Further CDR boundary definitions may not strictly follow the system described herein; they may be shortened or lengthened based on predictions or experimental findings that specific residues, groups of residues, or even entire CDRs, have no significant effect on antigen binding, yet still partially overlap with Kabat CDRs. While certain embodiments of the present invention utilize Kabat or Chothia definitions of CDRs, the methods employed herein may utilize CDRs defined according to either of these systems.Identifying predicted CDRs using amino acid sequences is a well-known technique in the art, as described, for example, in Martin, AC, "Protein sequence and structure analysis of antibody variable domains," in Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag, Berlin, pp. 33-51 (2010). CDR sequences can also be identified by examining the amino acid sequences of the heavy and / or light chain variable domains using other conventional methods, for example, by comparing the amino acid sequences of other heavy and light chain variable regions to identify highly variable regions. The numbered sequences can be aligned visually or using an alignment program such as that in the CLUSTAL suite, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular modeling is traditionally used to accurately delineate the framework and CDR regions, and sequence-based assignments are refined accordingly. All such alternative definitions are encompassed by the present invention, and the sequences provided in this specification are not intended to exclude CDR sequences defined in alternative ways, which may only include a portion of the CDR sequences provided in the sequence listing. 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.

[0076] As used herein, "antibody fragment" includes a portion of a full-length antibody. Non-limiting examples of such antibody fragments include: (i) a crystallizable fragment (Fc), which is composed of two constant heavy chain fragments, which contain the CH2 and CH3 domains in IgA, IgD, and IgG, and contain the CH2, CH3, and CH4 domains in IgE and IgM, and are paired by disulfide bonds and non-covalent interactions; (ii) an antigen-binding region (Fab), which is composed of VL, CL, VH, and CH1 linked by disulfide bonds; (iii) Fab', which is composed of VL, CL, VH, and CH1 linked by disulfide bonds and contains 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 thiol group; (v) F(ab')2, which is composed of two Fab fragments linked by a disulfide bond in the hinge region; (vi) (i) a variable fragment (Fv), which is composed of a pair of VL and VH chains held together by non-covalent interactions; (ii) a single-chain variable fragment (scFv), which comprises a pair of VL and VH chains held together by a linker; (iii) a trivalent triabody, in which three scFvs, together with VH and VL domains linked by a short peptide, form a trimer; and the like.

[0077] As used herein, "Fc" refers to a molecule derived from the sequence of a non-antigen-binding fragment of an antibody, produced by digestion or other means, and includes both monomeric and multimeric forms, and may have a hinge region. The original immunoglobulin source of native Fc is preferably human, but can be any immunoglobulin. Fc molecules are composed of monomeric polypeptides, which can be linked into dimers or multimers by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between monomeric subunits of a native Fc molecule varies depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4). An example of an Fc is a sulfide-linked dimer produced by papain digestion of IgG. As used herein, "native Fc" is a generic term for monomeric, dimeric, and multimeric forms.

[0078] 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 unable to form disulfide bonds with other heavy chain polypeptides. As used herein, a F(ab) fragment may also comprise a light chain and a heavy chain, wherein the light chain comprises two variable domains separated by an amino acid linker, and the heavy chain comprises two variable domains separated by an amino acid linker and a CH1 domain.

[0079] The F(ab') fragment typically consists of a light chain plus a portion of a heavy chain containing more constant regions (between the CH1 and CH2 domains), allowing the two heavy chains to form interchain disulfide bonds to form the F(ab')2 molecule.

[0080] One embodiment of the present invention provides binding proteins having biological and immunological specificity for one to three target antigens.

[0081] As used herein, "antigen," "target antigen," or "antigenic target" refers to a molecule or portion of a molecule that is capable of being bound by a binding protein and / or, when administered to an animal, produces antibodies capable of binding to a determinant 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 intact antibodies and / or antibody fragments thereof that recognize the target antigen. An antigen is bound by a binding protein, such as an antibody, at an antigen-binding site, also referred to herein as a "binding portion" or "binding domain."

[0082] 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 below. In certain 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.

[0083] Binding proteins, such as antibodies and antibody fragments, can be classified as monovalent or multivalent based on the number of antigen-binding sites. "Valence" as used herein refers to the number of binding sites. A "monovalent binding protein" refers to a binding protein with one antigen-binding site. For example, a monovalent antibody has one antigen-binding site. A "multivalent binding protein" refers to a binding protein with more than one antigen-binding site. For example, a multivalent antibody has more than one antigen-binding site. Non-limiting examples of multivalent binding proteins are bivalent and trivalent. A "bivalent binding protein" refers to a binding protein with two antigen-binding sites. For example, a bivalent antibody has two antigen-binding sites. A "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets. For example, a trivalent antibody has three antigen-binding sites.

[0084] "Single specific binding protein" refers to a binding protein that specifically binds to one antigenic target.

[0085] As used herein, "multispecific binding protein" refers to any binding protein, including antibodies or antibody fragments thereof, that has more than one binding site and can bind to multiple epitopes on the same antigen or multiple antigenic targets.

[0086] "Bispecific binding protein" refers to a binding protein, including an antibody or antibody fragment thereof, that specifically binds to two different antigenic targets. In certain embodiments, a bispecific binding protein, such as an antibody or antibody fragment thereof, binds to two different antigens. In certain embodiments, a bispecific binding protein binds to two epitopes on the same antigen.

[0087] In certain embodiments, a trispecific binding protein, such as an antibody or antibody fragment thereof, binds to three different antigens. In certain embodiments, a trispecific binding protein binds to one, two, or three different epitopes on the same antigen.

[0088] 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).

[0089] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and, in certain embodiments, may possess specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope refers to the region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein specifically binds to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, a binding protein is said to specifically bind to an antigen when the equilibrium dissociation constant (KD) is, for example, <10-8 M, more preferably <10-9 M, and most preferably <10-10 M.

[0090] In a preferred embodiment of the present invention, the antibodies disclosed herein are trivalent, preferably trispecific, and 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.

[0091] "CD3" stands for cluster of differentiation 3, a multimeric protein complex composed of four polypeptide chains: epsilon (epsilon), gamma (gamma), delta (delta), and zeta (zeta). These chains combine to form and function as three pairs of dimers (epsilon gamma, epsilon delta, and zeta). In certain embodiments, the binding proteins of the present invention bind to the extracellular domain of one or more of the CD3 polypeptide chains: CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta. Exemplary CD3 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD3ε (e.g., represented by SEQ ID NO: 610) and the extracellular domain of cynomolgus monkey CD3ε (e.g., represented by SEQ ID NO: 614); the extracellular domain of human CD3δ (e.g., represented by SEQ ID NO: 611) and the extracellular domain of cynomolgus monkey CD3δ (e.g., represented by SEQ ID NO: 613); the extracellular domain of human CD3γ (e.g., represented by SEQ ID NO: 612) and the extracellular domain of cynomolgus monkey CD3γ; the extracellular domain of human CD3ζ and the extracellular domain of cynomolgus monkey CD3ζ.

[0092] "BCMA" stands for 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 certain embodiments, the binding proteins of the present invention 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., represented by SEQ ID NO: 615) and the extracellular domain of cynomolgus macaque BCMA (e.g., represented by SEQ ID NO: 616).

[0093] "CD38" stands for cluster of differentiation 38 polypeptide, a glycoprotein present on the surface of many immune cells. In certain embodiments, the binding proteins of the present invention bind to one or more extracellular domains of CD38 polypeptides. Exemplary CD38 extracellular domain polypeptide sequences include, but are not limited to, the extracellular domain of human CD38 (e.g., represented by SEQ ID NO: 617) and the extracellular domain of cynomolgus monkey CD38 (e.g., represented by SEQ ID NO: 618).

[0094] "T-cell engager antibodies" are binding proteins that target the host immune system, more specifically T cell cytotoxic activity, and tumor target proteins.

[0095] In a preferred embodiment, the present invention provides a trispecific antibody or antibody fragment thereof that binds to human CD3, human BCMA, and human CD38. In a preferred embodiment of the present invention, the antibody is monoclonal.

[0096] In a specific embodiment, the trispecific antibodies of the present invention comprise a common light chain.

[0097] An "isolated" binding protein is one that has been identified and separated and / or recovered from components of its natural environment. Contaminating components of its natural environment are substances that 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 is purified to: (1) greater than 95% by weight of the antibody as determined by the Lowry method, preferably greater than 99% by weight, (2) sufficient to obtain at least 15 residues of N-terminal or intermediate amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. In isolated binding proteins, since at least one component of the binding protein's natural environment is no longer present, this would include binding proteins in situ within recombinant cells.

[0098] As used herein, "substantially pure" or "substantially purified" refers to a compound or substance that is present as a predominant species (i.e., on a molar basis, it is present in a greater proportion than any other individual species in the composition). In some embodiments, a substantially purified fraction refers to a composition in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition comprises at least about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In other embodiments, a substance is purified to a state of substantial homogeneity (contaminating species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0099] A nucleic acid is "isolated" or "substantially purified" after it has been removed from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques. Such standard techniques include alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques known in the art; see, for example, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid of the present invention can be, for example, DNA or RNA, and may or may not contain intronic sequences.

[0100] The dissociation constant (KD) of a binding protein can be determined using methods such as surface plasmon resonance (SPR). Generally, SPR analysis utilizes the 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) based on the surface plasmon resonance (SPR) principle. SPR analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and providing the ligand (target antigen). As used herein, "KD" refers to the dissociation constant of the interaction between a specific binding protein, a binding portion of a binding protein, or an antibody or antibody fragment thereof, and a target antigen and / or antigenic epitope.

[0101] As used herein, "binds to" in reference to a binding protein refers to the ability of the binding protein or antigen-binding fragment thereof to bind to an antigen containing the epitope with a KD of 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 less and / or to bind to the epitope with an affinity at least twice that of the binding protein for a nonspecific antigen.

[0102] As used herein, the term "linker" refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility between the light and heavy chain domains to fold into a dual-variable region immunoglobulin. At the sequence level, linkers can be inserted at the transition region between variable domains or between variable and constant domains. Because the approximate sizes of immunoglobulin domains are well known, identifying interdomain transitions is straightforward. The precise location of interdomain transitions can be determined by locating peptide extensions that do not form secondary structural elements, such as β-sheets or α-helices, based on experimental data, modeling techniques, or secondary structure prediction.

[0103] In certain embodiments, the nucleic acid molecules provided herein comprise a nucleotide sequence encoding a polypeptide chain comprising a binding protein of the present invention. Another embodiment of the present invention provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain comprising such a binding protein. Yet another embodiment of the present invention provides a host cell (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain comprising such a binding protein) for expressing such a binding protein.

[0104] As used herein, "vector" refers to any molecule (e.g., nucleic acid, plasmid) or other vehicle (e.g., virus) used to transfer and / or deliver coding information to a host cell. The term "vector" includes any nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments may be embedded. Another type of vector is a viral vector, in which additional DNA segments may be embedded within the viral genome. Specifically, nucleic acids that promote viral assembly are combined with coding information to facilitate transfer into host cells via virus-like particles. Some vectors are capable of autonomous replication in the host cell to which they are attached (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may integrate into the host cell genome upon introduction into the host cell and thereby be replicated along with the host genome. Some vectors are capable of directing gene expression to overlapping regions with which they are linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally speaking, expression vectors are often used in recombinant DNA technology in the form of plasmids. "Plasmid" and "vector" are used interchangeably herein, as the most commonly used form of plasmid is a vector. However, the present invention is intended to encompass other expression vector forms that provide equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0105] As used herein, "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 the specific subject cell but also encompass the descendants of the designated cell. Because subsequent generations may alter the cell due to mutation or environmental influences, the descendants may not be identical to the parent cell; however, such cells are still considered "host cells" herein. Binding proteins can be expressed using a variety of host cell expression systems, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). To recombinantly express a binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding a binding protein polypeptide chain, such that the polypeptide chain is expressed in the host cell and, preferably, secreted into the host cell culture medium, allowing the binding protein to be recovered from the medium.

[0106] As used herein, the term "transformation" refers to a change in the genetic makeup of a cell. Transformation occurs when a cell is modified to incorporate new DNA. For example, a cell is transformed when it undergoes genetic modification from its native state. Following transformation, the transforming DNA may physically incorporate into the cell's chromosomes and recombine with the cell's DNA, or it may remain as an episomal element without replicating, or it may replicate independently as a plastid. A cell is considered stably transformed when its DNA replicates with cell division. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA by a cell. "Transfection" of a cell occurs when the exogenous DNA enters the cell membrane. Various transfection techniques are known in the art for introducing one or more exogenous DNA molecules into appropriate host cells.

[0107] As used herein, "naturally occurring" means that the substance is found in nature and has not been altered by human manipulation. For example, a polynucleotide or polypeptide that exists in organisms (including viruses), can be isolated from natural sources, and has not been intentionally modified by humans is considered naturally occurring. Conversely, "non-naturally occurring" refers to substances that are not found in nature or have been artificially modified or synthesized.

[0108] As used herein, the twenty known amino acids and their abbreviations follow conventional usage. Suitable components of the polypeptide chain of the binding protein may also be stereoisomers of the twenty known 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. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphatidylserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imidic acids (e.g., 4-hydroxyproline). In the peptide notation used herein, the left side is the amino-terminal direction and the right side is the carboxyl-terminal direction, which is consistent with standard usage and convention.

[0109] Naturally occurring residues can be divided into the following types 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 direction: 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 class for another member of the same class. Non-conservative substitutions may involve exchanging a member of one class for a member of another class.

[0121] Those skilled in the art should be able to identify appropriate variants of the binding protein polypeptide chain using conventional techniques. For example, by targeting regions that are less likely to affect activity, one skilled in the art can identify regions of the polypeptide chain that can be modified without abolishing activity. Alternatively, one skilled in the art can identify residues and portions of the molecule that are conserved among similar polypeptides. Furthermore, even in regions that may affect biological activity or structure, conservative amino acid substitutions can be made without abolishing biological activity or adversely affecting the polypeptide structure.

[0122] When referring to two or more nucleic acid or polypeptide sequences herein, when a percentage is stated as "identical," it refers to two or more identical sequences. Two sequences are said to be "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (e.g., at least 60% identical, e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) when aligned and aligned for maximum correspondence over a specified region (or, if not specified, the entire sequence). Sequences are typically aligned using one sequence as a reference sequence to which test sequences are aligned. Methods for aligning sequences during alignment are well known to those skilled in the art. Non-limiting examples of suitable algorithms for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI); the Clustal Omega algorithm (see “Sievers F, Higgins DG. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 2018 Jan;27(1):135-145.”); and the MUSCULE algorithm (see “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] As used herein, "patient" and "subject" are used interchangeably. "Patient" as used herein includes both human and animal subjects. Animal subjects include all vertebrates, such as mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, and chickens.

[0124] As used herein, the term "treatment" refers to both therapeutic treatment and preventive measures. Those in need of treatment include those who have a disease, those who are predisposed to the disease, or those who wish to prevent the disease. In specific embodiments of the present invention, the disease is characterized by expression, such as overexpression, of BCMA, and / or expression, such as overexpression, of CD38, including cancer and non-cancerous diseases such as gastrointestinal diseases, nervous system diseases, and lung diseases (see "Szlasa W at al., Targeting CD38 in Neoplasms and Non-Cancer Diseases. Cancers. 2022 Aug 28;14(17):4169"); for example, the antibodies or antibody fragments thereof according to the present invention can be used to treat B-cell malignancies and autoimmune diseases. In other specific embodiments, the antibodies or antibody fragments thereof of the present invention can be used to treat humans with cancer or humans at high risk of cancer, or to improve cancer symptoms in human subjects. The antibodies 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 using the antibodies or antibody fragments of the present invention include leukemias, such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myeloid leukemia, acute myeloid 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, and tumors, active multiple myeloma, plasma cell leukemia, lymphoma, germline B-cell lymphoma or B-cell acute lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, myelodysplastic syndrome (MDS), breast cancer (e.g., Her2+ breast cancer), prostate cancer, cervical cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), well-differentiated ovarian serosal epithelial cell carcinoma, peritoneal cancer, smoldering myeloma, and glioma.

[0125] The present invention also relates to a trispecific antibody or antibody fragment thereof for use as a medicine. Specifically, it is used to treat the aforementioned diseases, especially cancer and autoimmune diseases, and more specifically, hematological cancers, including but not limited to the aforementioned hematological cancers. 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 in treating the aforementioned diseases; specifically, the treatment of cancer and autoimmune diseases, and more specifically, the treatment of hematological cancers, including but not limited to those listed above. In a preferred embodiment, the present invention relates to the use of the trispecific antibodies in 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 diseases, comprising administering a therapeutically effective amount of a trispecific antibody of the present invention to a patient in need thereof. Specifically, the present invention also relates to methods for treating hematological cancers, comprising administering a therapeutically effective amount of a trispecific antibody of the present invention or an antibody fragment thereof to a patient in need thereof. More specifically, the hematological cancer is selected from the list including, but not limited to, the above-mentioned. In a preferred embodiment, the present invention relates to methods for treating multiple myeloma, including relapsed multiple myeloma, refractory multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, and active multiple myeloma, comprising administering a therapeutically effective amount of a trispecific antibody of the present invention or an antibody fragment thereof to a patient in need thereof.

[0128] The present invention also relates to pharmaceutical compositions comprising the trispecific antibodies and one or more pharmaceutically acceptable excipients or carriers. When preparing pharmaceutical compositions or sterile compositions comprising the trispecific antibodies or antibody fragments thereof, the MBM preparation can be combined with one or more pharmaceutically acceptable excipients or pharmaceutically acceptable carriers.

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

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

[0131] When referring to a pharmaceutical composition comprising one or more binding proteins, "effective amount" and "therapeutically effective amount" mean an amount or dosage sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount refers to an amount of the binding protein sufficient to inhibit, for a period of time, one or more clinically defined pathological processes of the condition being treated. The effective amount may vary depending on the specific binding protein used, as well as a variety of factors and conditions related to the patient being treated and the severity of the condition. For example, if the binding protein is administered in vivo, factors such as the patient's age, weight, and health, as well as dose-response curves and toxicity data from preclinical animal studies, should be taken into consideration. Determining the effective or therapeutically effective amount of a particular pharmaceutical composition is within the skill of those skilled in the art.

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

[0133] Anti-CD3 binding protein

[0134] Some aspects of the present invention relate to binding proteins comprising an antigen-binding site that binds to a CD3 polypeptide (e.g., human and cynomolgus monkey CD3 polypeptides). In certain embodiments, the antigen-binding domains and / or binding proteins of the present invention are "cross-reactive" with human and cynomolgus monkey CD3 polypeptides.

[0135] In certain embodiments, the binding proteins described herein are monospecific, bispecific, trispecific, or multispecific and / or monovalent, bivalent, trivalent, or multivalent, and comprise a binding moiety that binds to CD3, for example, human CD3.

[0136] In certain embodiments, the antibodies or antibody fragments of the invention comprise a binding portion that binds to human CD3, comprising a heavy chain CDR set having 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: 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: 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 more specific embodiments, the binding portion that binds to human CD3 also comprises the set of CDRs of a common light chain having 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 having 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 also comprises the common light chain CDRs, wherein the light chain has 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 the light chain CDR set having 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 whose amino acid sequence 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 also comprises a light chain variable region of a light chain whose amino acid sequence 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 whose amino acid sequence 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 more specific embodiments, the binding portion that binds to human CD3 also comprises a light chain variable region of a light chain whose amino acid sequence 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 to human CD3 comprises a heavy chain variable region whose amino acid sequence 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 to human CD3 comprises a heavy chain whose amino acid sequence 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 also comprises a light chain whose amino acid sequence 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 to human CD3 comprises a heavy chain variable region whose amino acid sequence 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; and a light chain whose amino acid sequence 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 an antibody or antibody fragment thereof having multiple specificities (eg, bispecific or trispecific) and being multivalent (eg, bivalent or trivalent), 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 antigen targets: A2AR, APRIL, ATPase, BAFF, BAFFR, BCMA, B1YS, 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-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MCP-1b), CCL21 (also known as MCP-1c), CCL22 (also known as MCP-1c), CCL23 (also known as MIP-1a), CCL24 (also known as MIP-1b), CCL25 (also known as MCP-1c), CCL26 (also known as MCP-1b), CCL27 (also known as MCP-1c), CCL28 (also known as MCP-2c), CCL29 (also known as MIP-2b), CCL30 (also known as MIP-1c), CCL31 (also known as MIP-1d), CCL32 (also known as MIP-1b), CCL33 (also known as MIP-1c), CCL34 (also known as MIP-1b), CCL35 (also known as MIP-1c), CCL36 (also known as MIP-1b), CCL37 (also known as MIP-1c), CCL38 (also known as MCP-1 (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, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, GPRC5D, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNa, IgE, IGF1R, IL2Rbeta, IL1, IL1RAP, ILILIA, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, RAP, IL9R, IL10, rIL10, IL12, IL13, IL13Ral, 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, 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 certain embodiments, one or more of the above antigenic targets are human antigenic targets. In a preferred embodiment, the antibodies or antibody fragments thereof of the present invention 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.

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

[0145] In a specific aspect, the present invention relates to an antibody or antibody fragment thereof that binds to human CD3, comprising a heavy chain CDR set having 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: 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, the light chain having the amino acid sequence of SEQ ID NO: 1;Specifically, the light chain CDR group comprises the amino acid sequences of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0146] In a preferred embodiment, the antibody or antibody fragment that binds to human CD3 comprises a heavy chain CDR set having 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 of SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0147] In a preferred embodiment, the present invention discloses an antibody comprising the aforementioned binding portion that binds to human CD3, wherein the antibody is monoclonal.

[0148] Anti-BCMA binding protein

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

[0150] In certain embodiments, the binding proteins disclosed herein are monospecific, bispecific, trispecific, or multispecific and / or monovalent, bivalent, trivalent, or multivalent, and comprise a binding moiety that binds to BCMA, such as human BCMA.

[0151] In certain embodiments, the antibodies or antibody fragments of the invention comprise a binding portion that binds to human BCMA, comprising a heavy chain CDR set having 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. NO: 468; SEQ ID NO: 217, SEQ ID NO: 343 and SEQ ID NO: 469; SEQ ID NO: 218, SEQ ID NO: 344 and SEQ ID NO: 470; SEQ ID NO: 219, SEQ ID NO: 345 and SEQ ID NO: 471; SEQ ID NO: 220, SEQ ID NO: 346 and SEQ ID NO: 472; SEQ ID NO: 221, SEQ ID NO: 347 and SEQ ID NO: 473; SEQ ID NO: 222, SEQ ID NO: 348 and SEQ ID NO: 474; 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 more specific embodiments, the binding portion that binds to human BCMA also comprises a common light chain CDR set having 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.

[0152] In more specific embodiments, the binding portion that binds to human BCMA comprises a heavy chain CDR set having 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 having SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486. In more specific embodiments, the binding portion that binds to human CD3 also 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. 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 to human BCMA comprises a heavy chain variable region of a heavy chain whose amino acid sequence 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 more specific embodiments, the binding portion that binds to human BCMA also comprises a light chain variable region of a light chain whose amino acid sequence 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 to human BCMA comprises a heavy chain variable region whose amino acid sequence 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 to 109 further comprises the mutation N82aS. In more specific embodiments, the binding portion that binds to human BCMA also comprises a light chain variable region of a light chain whose amino acid sequence 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 to human BCMA comprises a heavy chain variable region of a heavy chain whose amino acid sequence 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 more specific embodiments, the binding portion that binds to human BCMA also comprises a light chain variable region of a light chain whose amino acid sequence 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 to human BCMA comprises a heavy chain variable region whose amino acid sequence 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 more specific embodiments, the binding portion that binds to human BCMA also comprises a light chain variable region of a light chain whose amino acid sequence 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 to human BCMA comprises a heavy chain variable region whose amino acid sequence 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 moiety that binds to human BCMA comprises a heavy chain whose amino acid sequence 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 more specific embodiments, the binding moiety that binds to human BCMA also comprises a light chain whose amino acid sequence 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 moiety that binds to human BCMA comprises a heavy chain whose amino acid sequence 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 to 109 further comprises the mutation N82aS. In more specific embodiments, the binding moiety that binds to human BCMA also comprises a light chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.

[0160] Preferably, the binding portion that binds to human BCMA comprises a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 101, 105, and 108; and a light chain whose amino acid sequence 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 whose amino acid sequence 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 amino acid substitution N82aS; and a light chain whose amino acid sequence 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.

[0162] The present invention relates to an antibody or antibody fragment thereof having multiple specificities (e.g., bispecific or trispecific) and being multivalent (e.g., bivalent or trivalent), 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 antigen targets: A2AR, APRIL, ATPase, BAFF, BAFFR, BCMA, B1YS, 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-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MCP-1b), CCL21 (also known as MCP-1c), CCL22 (also known as MCP-1c), CCL23 (also known as MIP-1a), CCL24 (also known as MIP-1b), CCL25 (also known as MCP-1c), CCL27 (also known as MCP-1c), CCL28 (also known as MCP-2c), CCL29 (also known as MIP-2b), CCL30 (also known as MIP-1d), CCL31 (also known as MIP-1b), CCL32 (also known as MIP-1c), CCL33 (also known as MIP-1b), CCL34 (also known as MIP-1b), CCL35 (also known as MIP-1c), CCL36 (also known as MIP-1b), CCL37 (also known as MIP-1c), CCL38 (also known as MCP-1b), CCL39 (also known as MIP- (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, alpha receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD47, CD48, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PD-L2), CD274 (also known as PD-L1), CD275 (also known as B7H2), CD276 (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, extracellular nucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, 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, rIL10, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as the IL25 receptor), 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 IL33 receptor), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1), XCL1, and XCL2. In certain embodiments, one or more of the above antigenic targets are human antigenic targets. In a preferred embodiment, the antibodies or antibody fragments thereof of the present invention 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 to BCMA, such as human BCMA.

[0164] In a specific aspect, the present invention relates to an antibody or antibody fragment thereof that binds to human BCMA, comprising a heavy chain CDR set having 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. NO: 468; SEQ ID NO: 217, SEQ ID NO: 343 and SEQ ID NO: 469; SEQ ID NO: 218, SEQ ID NO: 344 and SEQ ID NO: 470; SEQ ID NO: 219, SEQ ID NO: 345 and SEQ ID NO: 471; SEQ ID NO: 220, SEQ ID NO: 346 and SEQ ID NO: 472; SEQ ID NO: 221, SEQ ID NO: 347 and SEQ ID NO: 473; SEQ ID NO: 222, SEQ ID NO: 348 and SEQ ID NO: 474; 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 sequences 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 having 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 a preferred embodiment, the present invention discloses an antibody comprising the aforementioned binding portion that binds to human BCMA, wherein the antibody is monoclonal.

[0167] Anti-CD38 binding protein

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

[0169] In certain embodiments, the binding proteins described herein are monospecific, bispecific, trispecific, or multispecific and / or are monovalent, bivalent, trivalent, or multivalent, and comprise a binding portion that binds to CD38, for example, human CD38.

[0170] In certain embodiments, the antibodies or antibody fragments of the invention comprise a binding portion that binds to human CD38, comprising a heavy chain CDR set having 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. NO: 371 and SEQ ID NO: 497; SEQ ID NO: 246, SEQ ID NO: 372 and SEQ ID NO: 498; SEQ ID NO: 247, SEQ ID NO: 373 and SEQ ID NO: 499; SEQ ID NO: 248, SEQ ID NO: 374 and SEQ ID NO: 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: and SEQ ID NO: 237, SEQ ID NO: 363, and SEQ ID NO: 700. In a more specific embodiment, the binding portion that binds to human CD38 also comprises the set of CDRs of a common light chain having 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 more specific embodiments, the binding portion that binds to human CD38 comprises a heavy chain CDR set having 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; and 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 CD3 also comprises a common light chain CDR set, wherein the light chain has 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 to human CD38 comprises a heavy chain variable region of a heavy chain whose amino acid sequence 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 to human CD38 also comprises a light chain variable region of a light chain whose amino acid sequence 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 whose amino acid sequence 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 more specific embodiments, the binding portion that binds to human CD38 also comprises a light chain variable region of a light chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. The present invention also relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, at least one of which binds 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 whose amino acid sequence 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 moiety that binds to human CD38 comprises a heavy chain whose amino acid sequence 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 moiety that binds to human BCMA also comprises a light chain whose amino acid sequence 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 to human CD38 comprises a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 an antibody or antibody fragment thereof having multiple specificities (eg, bispecific or trispecific) and being multivalent (eg, bivalent or trivalent), comprising any of the above-mentioned binding portions that bind to human CD38.

[0177] In any of the above trispecific binding proteins, the target antigen other than CD38 can be any of the following exemplary antigen targets: A2AR, APRIL, ATPase, BAFF, BAFFR, BCMA, B1YS, 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-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MCP-3b), CCL22 (also known as MCP-3b), CCL23 (also known as MIP-3c), CCL24 (also known as MIP-3d), CCL25 (also known as MCP-3d), CCL26 (also known as MCP-3d), CCL27 (also known as MCP-3d), CCL28 (also known as MCP-3d), CCL29 (also known as MIP-3d), CCL30 (also known as MIP-3d), CCL31 (also known as MIP-3d), CCL32 (also known as MIP-3d), CCL33 (also known as MIP-3d), CCL34 (also known as MIP-3d), CCL35 (also known as MIP-3d), CCL36 (also known as MIP-3d), CCL37 (also known as MIP-3d), CCL38 (also known as MCP-3d), CCL (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), 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, extracellular nucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, 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, rIL10, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as the IL25 receptor), 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 IL33 receptor), 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 certain 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 binds to BCMA, and at least one binds to CD38.

[0178] The present invention also relates to any of the above-mentioned monospecific antibodies or antibody fragments thereof comprising a binding portion that binds to CD38, such as human CD38.

[0179] In a specific aspect, the present invention relates to an antibody or antibody fragment thereof that binds to human BCMA, comprising a heavy chain CDR set having 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: 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: and SEQ ID NO: 237, SEQ ID NO: 363 and SEQ ID NO: 700; 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.

[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 having 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 a preferred embodiment, the present invention discloses an antibody comprising the aforementioned binding portion that binds to human CD38, wherein the antibody is monoclonal.

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

[0183] In certain aspects, the present invention relates to a trivalent, bispecific antibody or antibody fragment thereof comprising at least two binding moieties, at least one of which binds to human CD3 and at least one of which binds to BCMA, preferably at least two of which bind to BCMA.

[0184] In a specific embodiment of the present invention, the trivalent bispecific anti-CD3 and anti-BCMA antibody is a heterodimeric immunoglobulin, specifically constructed using the above-mentioned BEAT® heavy chain (Hc) heterodimerization technology (see "Kegro et al., (2017) J Biol Chem 292(23): 9745-9759 and Stutz et al., (2020) J Biol Chem 295(28): 9392-9408, WO2012131555"), which comprises a BEAT (A) chain, also referred to herein as BTA, and a BEAT (B) chain, also referred to herein as BTB. More specifically, the trivalent bispecific anti-CD3 and anti-BCMA antibody system was designed by combining a Fab-type CD3 binder with a BCMA bi-dAb on the silent BEAT Fc backbone (see "LALA mutations, Hezareh et al., 2001, J Virol, 75(24): 12161-12168").

[0185] In a specific embodiment of the present invention, the trivalent bispecific anti-CD3 and anti-BCMA antibody comprises a BTA chain having an amino acid sequence of SEQ ID NO: 515, a BTB chain having an amino acid sequence of SEQ ID NO: 516, and a common light chain having an amino acid sequence of SEQ ID NO: 1; or a BTA chain having an amino acid sequence of SEQ ID NO: 509, a BTB chain having an amino acid sequence of SEQ ID NO: 510, and a common light chain having an amino acid sequence of SEQ ID NO: 1; or a BTA chain having an amino acid sequence of SEQ ID NO: 513, a BTB chain having an amino acid sequence of SEQ ID NO: 514, and a common light chain having an amino acid sequence of SEQ ID NO: 1; or a BTA chain having an amino acid sequence of SEQ ID NO: 511, a BTB chain having an amino acid sequence of SEQ ID NO: 512, and a common light chain having an amino acid sequence of SEQ ID NO: 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 having the amino acid sequence of SEQ ID NO: 1; or a BTA chain having the amino acid sequence of SEQ ID NO: 517, a BTB chain having the amino acid sequence of SEQ ID NO: 518, and a common light chain having 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 proteins

[0187] 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 binds to human BCMA, and at least one binds to human CD38. These antibodies are also referred to herein as CD3 / BCMA / CD38 antibodies. 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, the antibody is monoclonal. The binding moieties of the present invention that bind to CD3, BCMA, or CD38 are as described above.

[0188] Trispecific antibodies are a group of genetically 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, specifically constructed using the TREAT® trispecific antibody technology based on the BEAT® platform. The BEAT® platform utilizes the protein-protein interface of the α-β constant regions of the T cell receptor (TCR) to replace the protein-protein interface of the CH3 domain pair of the antibody Fc region. The resulting BEAT® interface favors the formation of heterodimeric bispecific or trispecific antibodies (bsAbs or TriAbs) over homodimeric contaminants. Furthermore, the CD3 / BCMA / CD38 antibody utilizes a Fab CD38 binding arm as the Fab unit, a Fab BCMA binding arm, and a Fab CD3 binding arm. All three share a common light chain, thus addressing the light chain mispairing issue commonly seen in bsAbs and even more frequently encountered in TriAbs platforms. Additional engineering can also be used to simplify and accelerate the purification process, maintain the natural conformation of the antibody structure, stabilize it, and retain binding to the native Fc receptor (FcRn) to maximize the serum half-life of CD3 / BCMA / CD38 antibodies (see "Skegro et al., J Biol Chem, 2017, 292(23):9745-9759; Stutz et al., J Biol Chem, 2020, 295(28):9392-9408").

[0189] The CD3-, BCMA-, or CD38-binding moiety of the present invention may be any of the CD3-, BCMA-, or CD38-binding moieties described above.

[0190] In certain embodiments, the at least one binding portion that binds to 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; and the at least one binding portion that binds to 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: 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 present invention comprise at least one binding portion that binds to human CD3, comprising a heavy chain CDR set having SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433; at least one binding portion that binds to BCMA, comprising a heavy chain CDR set having SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486; and at least one binding portion that binds to human CD38, comprising a heavy chain CDR set having 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 particularly preferred embodiments, the at least one binding portion that binds to human CD38 comprises a heavy chain CDR set having the amino acid sequences 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 having the amino acid sequence of SEQ ID NO: 1.

[0193] More specifically, the present invention relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, wherein at least one binds to human CD3, at least one binds to human BCMA, and at least one 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 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.

[0194] Still more specifically, the present invention relates to a trispecific antibody or antibody fragment thereof comprising at least three binding moieties, wherein at least one binds to human CD3, at least one binds to human BCMA, and at least one 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 having SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set having 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 having SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0195] The present invention also relates to an antibody or antibody fragment or antigen-binding fragment thereof that binds to the same epitope on CD3 and / or BCMA and / or CD38 as a reference antibody, wherein the reference antibody is a trispecific antibody comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain CDR set having SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and the at least one binding moiety that binds to BCMA comprises a heavy chain CDR set having SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: and 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 having SEQ ID NO: 239, SEQ ID NO: 365 and SEQ ID NO: 491, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0196] The present invention also relates to an antibody or antibody fragment or antigen-binding fragment thereof that binds to the same epitope on CD3 and / or BCMA and / or CD38 as a reference antibody, wherein the reference antibody is a trispecific antibody comprising at least three binding moieties, at least one of which binds to human CD3, at least one of which binds to human BCMA, and at least one of which binds to human CD38, wherein the at least one binding moiety that binds to human CD3 comprises a heavy chain CDR set having SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723; and the at least one binding moiety that binds to BCMA comprises a heavy chain CDR set having SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: and 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 having SEQ ID NO: 236, SEQ ID NO: 362 and SEQ ID NO: 712, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: 722 and SEQ ID NO: 723.

[0197] In certain aspects of the invention, the trispecific antibody comprises at least one binding portion that binds to human CD3, comprising a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62, and 66; and at least one binding portion that binds to human BCMA, comprising a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 93, 101, 105 and 108; and at least one binding portion that binds to human CD38 comprises a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122 and 111. In a more specific aspect, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 1.

[0198] In certain aspects of the invention, the trispecific antibody comprises at least one binding portion that binds to human CD3, comprising a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62, and 66; and at least one binding portion that binds to human BCMA, comprising a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 103, 122, and 111. In a more specific embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 1.

[0199] In certain aspects of the invention, the trispecific antibody comprises at least one binding portion that binds to human CD3, comprising a heavy chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62, and 66; and at least one binding portion that binds to human BCMA, comprising a heavy chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 93, 101, 105 and 108; and at least one binding portion that binds to human CD38 comprises a heavy chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122 and 111. In a more specific aspect, the trispecific antibody or antibody fragment thereof further comprises a light chain having SEQ ID NO: 1.

[0200] In certain aspects of the invention, the trispecific antibody comprises at least one binding portion that binds to human CD3 comprising a heavy chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 55; 58; 60; 62, and 66; and at least one binding portion that binds to human BCMA comprising a heavy chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 93, 101, 105, and 108. SEQ ID NOs: 93, 101, 105, and 108 further comprise the substitution N82aS; and at least one binding portion that binds to human CD38 comprises a heavy chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110; 113, 122, and 111. In a more specific embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain having the 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, comprising a heavy chain variable region of a heavy chain, the amino acid sequence of which is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; and at least one binding portion that binds to human BCMA, comprising a heavy chain variable region of a heavy chain, the amino acid sequence of which 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: and at least one binding portion that binds to human CD38 comprises a heavy chain variable region whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 110 and 113. In a more specific embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region having 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, comprising a heavy chain variable region of a heavy chain, the amino acid sequence of which is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; and at least one binding portion that binds to human BCMA, comprising a heavy chain variable region of a heavy chain, the amino acid sequence of which 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 SEQ ID NO: 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 comprises a heavy chain variable region whose amino acid sequence 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 NO: 110 and 113. In a more specific embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 1.

[0203] Still more specifically, the trispecific antibodies or antibody fragments thereof of the present invention comprise at least one binding portion that binds to human CD3, comprising a heavy chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; and at least one binding portion that binds to human BCMA, comprising a heavy chain whose amino acid sequence 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: and at least one binding portion that binds to human CD38 comprises a heavy chain whose amino acid sequence 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 identical to SEQ ID NO: 113. In a more specific aspect, the trispecific antibody or antibody fragment thereof further comprises a light chain having the amino acid sequence of SEQ ID NO: 1.

[0204] More specifically, the trispecific antibodies or antibody fragments thereof of the present invention comprise at least one binding portion that binds to human CD3, comprising a heavy chain whose amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 55; and at least one binding portion that binds to human BCMA, comprising a heavy chain whose amino acid sequence 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 SEQ ID NO: 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 comprises a heavy chain whose amino acid sequence 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 NO: 110 and 113, preferably identical to SEQ ID NO: 113. In a more specific embodiment, the trispecific antibody or antibody fragment thereof further comprises a light chain having 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, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 108; and at least one binding portion that binds to human CD38, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 113; and a light chain having 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, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, comprising a heavy chain having 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, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 113; and a light chain having 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, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 108; and at least one binding portion that binds to human CD38, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 110; and a light chain having 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, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 55; at least one binding portion that binds to human BCMA, comprising a heavy chain having 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, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 110; and a light chain having 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, comprising a heavy chain variable region whose amino acid sequence 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, comprising a heavy chain variable region whose amino acid sequence 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: and at least one binding portion that binds to human CD38 comprises a heavy chain variable region whose amino acid sequence 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 the group consisting of SEQ ID NO: 593 and 594, preferably identical to SEQ ID NO: 594. In a more specific aspect, the trispecific antibody or antibody fragment thereof further comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 1.

[0210] The present invention also relates to an antibody or antigen-binding fragment that binds to an epitope located on human CD3, wherein the amino acid sequence of the epitope on CD3 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] Specifically, the present invention also relates to an antibody, or antibody fragment thereof, or antigen-binding fragment thereof, that binds to the same epitope on CD3 as a reference antibody, or antibody fragment thereof, wherein the reference antibody, or antibody fragment thereof, comprises 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, and 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 having SEQ ID NO: 181, SEQ ID NO: 307, and SEQ ID NO: 433, and a light chain CDR set having 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 having SEQ ID NO: 234, SEQ ID NO: 360, and SEQ ID NO: 486, and a light chain CDR set having 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 having SEQ ID NO: 239, SEQ ID NO: 365, and SEQ ID NO: 491, and a light chain CDR set having SEQ ID NO: 721, SEQ ID NO: 722, and SEQ ID NO: 723.

[0212] The present invention also relates to an epitope located on human CD3, the amino acid sequence of which 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 an antibody or antigen-binding fragment that binds to an epitope located on human CD38, wherein the epitope of 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 determined by X-ray crystallography. Specifically, the resolution as determined by X-ray crystallography is at least 5 Å, preferably at least 3.5 Å. In a preferred embodiment, the resolution is about 3.4 Å.

[0214] The present invention also relates to an epitope located 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 determined by X-ray crystallography. Specifically, the epitope is determined by X-ray crystallography to a resolution of at least 5 Å, preferably at least 3.5 Å. In a preferred embodiment, the resolution is approximately 3.4 Å.

[0215] The present invention also provides full-length antibodies and antibody fragments and / or binding portions that bind to human CD3 and / or bind to human BCMA and / or bind to human CD38. Such antibody fragments include, but are not limited to, (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of the VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody, (iv) dAb fragments consisting of a single variable domain (see "Ward ES et al., (1989) Nature, 341: 544-546"), (v) F(ab')2 fragments, which are bivalent fragments comprising two linked Fab fragments, and (vi) single-chain Fv molecules (scFv) in which the VH domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site (see "Bird RE et al., (1988) Science 242: 423-426" and "Huston JS et al., (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-83), (vii) bispecific single chain Fv dimers (see PCT / US92 / 09965), (viii) dimers or trimers, which are multivalent or multispecific fragments constructed by genetic fusion (see Tomlinson I & Hollinger P (2000) Methods Enzymol. 326: 461-79 and WO94 / 13804 and Holliger P et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-48), and (ix) scFv genetically fused to the same or different antibodies (see 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 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 an antibody fragment, such as a Fab region. As used herein, "Fab," "Fab region," or "Fab domain" includes a polypeptide comprising VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region alone or in the context of a full-length antibody or antibody fragment.

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

[0218] According to a specific embodiment, in the trispecific antibody, 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. According to another embodiment, in the trispecific antibody, 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.

[0219] According to another embodiment, in the trispecific antibody, 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. According to yet another embodiment, in the trispecific antibody of the present invention, 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.

[0220] According to another embodiment, in the trispecific antibody, the at least one binding moiety that binds to human CD3 is fused at the C-terminus to the N-terminus of the at least one binding moiety that binds to human CD38. According to yet another embodiment, in the trispecific antibody of the present invention, the at least one binding moiety that binds to human CD3 is fused at the N-terminus to the C-terminus of the at least one binding moiety that binds to human CD38.

[0221] In a specific aspect, the binding moieties are fused to each other via a linker, such as a peptide linker.

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

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

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

[0225] According to a particular aspect of the present invention, the binding moiety fused to the antibody is fused via a peptide linker. Preferably, the linker sequence is selected from SEQ ID NOs: 604 and 605. Other preferred peptide linkers are listed in the "Linkers" section below.

[0226] Preferably, the trispecific antibody of the present invention comprises a set of three amino acid chains whose sequence 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 a 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: SEQ ID NO: 540, SEQ ID NO: 541 and SEQ ID NO: 1; SEQ ID NO: 542, SEQ ID NO: 543 and SEQ ID NO: 1; SEQ ID NO: 544, SEQ ID NO: 545 and SEQ ID NO: 1; SEQ ID NO: 546, SEQ ID NO: 547 and SEQ ID NO: 1; and SEQ ID NO: 548, SEQ ID NO: 549 and SEQ ID NO: 1 have the same amino acid sequence within the group.

[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 of the present invention comprises a set of three amino acid chains, the amino acid sequences of which are SEQ ID NO: 546, SEQ ID NO: 547 and SEQ ID NO: 1.

[0229] Nucleic Acids

[0230] Standard recombinant DNA methods are used to construct polynucleotides encoding the polypeptides that form the binding proteins; these polynucleotides are incorporated into recombinant expression vectors; and these vectors are introduced into host cells. See, for example, "Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.)." Enzymatic reactions and purification techniques may be performed according to manufacturer's instructions, as commonly practiced in the art, or as described herein. Unless otherwise specifically defined, nomenclature used in connection with, and laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well-known or customarily employed in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and patient treatment.

[0231] Other aspects of the present invention relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. In certain embodiments, the sequences of the isolated nucleic acid molecules are 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 invention relate to polynucleotide kits. In certain embodiments, one or more polynucleotides are a vector (e.g., an expression vector). Uses of the kits include producing one or more binding proteins described herein, such as bi- or tri-specific binding proteins of the present invention. In certain embodiments, the kits comprise one, two, three, or four polynucleotides.

[0233] In certain embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct transcription of the nucleic acid sequence encoding the binding protein. A promoter refers to a nucleic acid control sequence that directs the 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 coding sequence if the promoter affects the transcription or expression of the coding sequence. Examples of promoters include, but are not limited to, promoters from viral genomes (e.g., polyoma virus, fowl pox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, polyomaviruses, such as simian virus 40 (SV40) and the like), from heterologous eukaryotic promoters (e.g., actin promoter, which is an immunoglobulin promoter derived from a heat shock promoter, and the like), cytomegalovirus immediate early enhancer / chicken β-actin CAG-promoter (see "Niwa et al., Gene 108(2): 193-9, 1991"), phosphoglycerate kinase (PGK)-promoter, tetracycline-inducible promoter (see "Masui et al., Nucleic Acids Res. 33:e43, 2005"), lac system, The tip system, tac system, trc system, the major operator and promoter region of phage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast α-mating factor. The polynucleotide encoding the binding protein of the present invention can be controlled by a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or readily identified by one skilled in the art.

[0234] In certain embodiments, the isolated nucleic acid is incorporated into a vector. In certain embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably linked to the polynucleotide to be expressed. "Regulatory sequences" include 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 (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, and the like), and enhancer sequences from eukaryotic cells (e.g., the SV40 enhancer (bp 100-270) located on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer located on the late side of the replication origin, the adenovirus enhancer, and the like). Examples of suitable vectors include plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, variola virus, Sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). Expression vectors can 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 the host are known in the art and can be used to transfect any target cell.

[0235] Other aspects of the present invention 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 certain 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 certain 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 certain 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 certain 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 certain embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. One or more vectors of the vector system may be any of the vectors described herein. In certain embodiments, the one or more vectors are expression vectors.

[0236] Isolated host cells

[0237] Other aspects of the present invention relate to isolated host cells comprising one or more isolated polynucleotides, polynucleotide kits, vectors, and / or vector systems described herein. In certain embodiments, the host cell is a bacterial cell (e.g., an Escherichia coli cell). In certain embodiments, the host cell is a yeast cell (e.g., a Saccharomyces cerevisiae cell). In certain embodiments, the host cell is an insect cell. Examples of insect host cells include Drosophila melanogaster cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Fall Armyworm cells (e.g., Sf21 or Sf9 cells). In certain embodiments, the host cell is a mammalian cell. Examples of mammalian host cells can include human embryonic kidney cells (e.g., 293 or 293 cells and its variant 293T that have been recloned for culture in suspension), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human hepatocytes (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 (such as Hep G2) and myeloma cells (such as NS0 and Sp2 / 0 cells).

[0238] Other aspects of the present invention relate to methods for producing any of the binding proteins described herein. In certain embodiments, the methods comprise a) culturing a host cell (e.g., any host cell described herein) containing 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 specific conditions to express proteins are well known to those skilled in the art. Cell culture is the process of growing cells under controlled conditions in an artificial environment. "Cell culture," "culture," or "host cell culture" refers 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 composition of the cell culture medium, bioreactor operating parameters, nutrient supply, and incubation time. The composition of the culture medium is optimized to achieve desired cell viability and proliferation; 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 different ways. In batch culture, all necessary nutrients are contained in the initial culture medium, which gradually depletes and accumulates waste products. In fed-batch culture, additional feed medium is added to prevent nutrient depletion and prolong the culture period. In contrast, in perfusion-based culture, fresh medium containing nutrients is continuously supplied to the cells in culture, and cellular waste products are removed by flowing the supplementary medium into the bioreactor. Those skilled in the art will recognize the importance of culture time; it must be long enough for the cells to produce consistent amounts of product. However, excessive culture time can compromise cell viability.

[0240] Methods for isolating proteins from cultured host cells are well known to those skilled in the art. These methods include, for example, chromatography techniques. "Chromatography" or "chromatography" refers to the separation of compounds in a mixture based on their ability to interact statically with a chromatographic resin, resulting in retention or elution. Chromatographic techniques are well known in the art. For example, ion exchange chromatography can separate ions and polar molecules based on charge differences. Examples of ion exchange chromatography techniques include cation exchange layers and anion exchange layers. Affinity chromatography relies on the specific interaction between proteins and immobilized ligands. Examples of ligands that can be used to purify antibodies via affinity chromatography include protein A and protein G. Protein A is a cell wall protein isolated from Staphylococcus aureus that binds to the Fc region of immunoglobulins (but not to the antigen-binding site). Here, "protein A chromatography" refers to affinity chromatography in which the ligand that binds to the antibody is protein A. "Protein A" includes native protein A, recombinant protein A, and its analogs or derivatives. Protein analogs and derivatives retain their binding activity for antibodies (e.g., IgG Fc). Protein G is a cell wall protein isolated from group G Streptococcus. Its N-terminus is divided into an albumin-binding domain, and its C-terminus is divided into an IgG-binding domain and a cell wall-binding domain. Herein, "protein G chromatography" refers to an affinity chromatography method in which the antibody-binding ligand is protein G. "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 (e.g., protein A affinity chromatography) followed by one or more ion exchange chromatography and / or size exclusion chromatography steps.

[0241] Antibody structure

[0242] In specific 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 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, such as 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 a preferred embodiment, 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, or the binding moiety that binds to human BCMA is fused at the N-terminus of the binding moiety that binds to human CD38.

[0247] In a preferred embodiment, the linked binding moieties are fused to each other via a linker, such as a peptide linker.

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

[0249] In a more preferred embodiment, 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 via a peptide linker of SEQ ID NO: 605, or 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 via a peptide linker of SEQ ID NO: 604.

[0250] In specific embodiments of the present invention, the antibody binding portion disclosed herein is an antibody fragment selected from the non-limiting list of "antibody fragments" described above. In more specific embodiments, the binding portion that binds to human CD3 and / or the binding portion that binds to human BCMA and / or the binding portion that binds to human CD38 is a Fab domain.

[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, such as the Fab domain located between the antibody Fc domain and the second binding moiety (e.g., the second Fab domain), is referred to as "Fc-proximal," while the second binding moiety is referred to as "Fc-distal."

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

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

[0254] In a specific embodiment of the present invention, a trispecific antibody or antibody fragment thereof comprises three binding moieties: one that binds to human CD3, one that binds to human BCMA, and one that binds to human CD38. These three binding moieties are Fab fragments, and the antibody is constructed using TREAT® trispecific antibody technology based on the aforementioned BEAT® platform. In a specific embodiment, the binding moiety that binds to human BCMA and the binding moiety that binds to human CD38 are located on the same arm of the BEAT antibody. More specifically, the binding moiety that binds to the human BCMA arm is located distal to the Fc region, while the binding moiety that binds to human CD38 is located proximal to the Fc region. In a more specific embodiment, the binding moiety that binds to human BCMA and the binding moiety that binds to human CD38 are fused via a flexible linker; preferably, the linker comprises the amino acid sequence of SEQ ID NO: 604. In a preferred embodiment, the binding moiety that binds to human CD3 and the binding moiety that binds to human BCMA are located on the same arm of the BEAT antibody. More specifically, the binding moiety that binds to the human CD3 binding arm is located distal to the Fc region, while the binding moiety that binds to human BCMA is located proximal to the Fc region. In a further embodiment, the binding moiety that binds to human CD3 and the binding moiety that binds to human BCMA are fused via a flexible linker; preferably, the linker comprises the amino acid sequence of SEQ ID NO: 605.

[0255] Linker

[0256] In certain embodiments, the linkers of the present invention, such as L1, L2, L3, and L4, range in length from zero amino acids (length = 0) to about 100 amino acids, or less than 100, 50, 40, 30, 20, or 15 amino acids. The 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 they can all have different amino acid sequences.

[0257] Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues; a peptide with five glycine residues; a peptide with six glycine residues; a peptide with seven glycine residues; and a peptide with eight glycine residues. Other combinations of amino acid residues, such as the peptides GGGT or GGGGS, or repeats of these peptides, such as the peptides GGGGS GGGGS GGGGS, may also be used. In a preferred embodiment, the linker used to link different binding moieties in the present invention has the amino acid sequence of SEQ ID NO: 604 or 605, or the amino acid sequence of SEQ ID NO: 604 or 605, including one or more conservative substitutions thereof.

[0258] The above examples are not intended to limit the scope of the present invention in any way. Linkers suitable for use in binding proteins may also include randomly selected amino acids, such as valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline. For detailed descriptions of linker sequences, see, for example, WO2012135345 and international application PCT / US2017 / 027488.

[0259] The degree of identity and sequence of amino acid residues in a linker may vary depending on the type of secondary structural elements required for the linker to function. For example, glycine, serine, and alanine confer maximum linker flexibility. Certain combinations of glycine, proline, threonine, and serine are suitable for linkers requiring greater rigidity and extensibility. Depending on the desired properties, any amino acid residue may serve as a linker and, if desired, may be combined with other amino acid residues to construct larger peptide linkers.

[0260] Fc region and constant domain

[0261] The present invention also relates to a trispecific antibody comprising an Fc region. In certain embodiments, the binding proteins of the present invention comprise antibody fragments, including but not limited to antibody Fab, (Fab')2, Fab'-SH, Fv, or scFv fragments. In certain embodiments, the binding proteins of the present invention comprise antibody fragments having an Fc region, including but not limited to antibody Fab, (Fab')2, Fab'-SH, Fv, or scFv fragments.

[0262] In certain embodiments, the binding proteins of the present invention comprise a full-length antibody heavy chain or polypeptide chain having an Fc region. In certain embodiments, the Fc region is a human Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region. In certain embodiments, the Fc region comprises the antibody hinge (CH1), CH2, CH3, and optionally, the CH4 domain. In certain embodiments, the Fc region is a human IgG1 Fc region. In certain embodiments, the Fc region is a human IgG2 Fc region. In certain embodiments, the Fc region is a human IgG3 Fc region. In certain embodiments, the Fc region is a human IgG4 Fc region. In certain embodiments, the Fc region comprises one or more mutations described herein.

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

[0264] In certain embodiments, the binding proteins of the present invention include one or two Fc variants. As used herein, "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a binding site for the rescue receptor FcRn (the naive Fc receptor). Exemplary Fc variants and their interactions with rescue receptors are known in the art. Therefore, the term "Fc variant" may include molecules or sequences that are humanized from a non-human native Fc. Furthermore, the native Fc contains certain regions that may be removed because they possess structural features or biological activities that are not required for the mimetic antibody binding proteins of the present invention. Thus, the term "Fc variant" encompasses a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues are modified and wherein said Fc sites or residues affect or are 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 rescue receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0265] As used herein, "parent antibody" or "parent immunoglobulin" includes unmodified antibodies, which may be modified to produce variants. The parent antibody may be a naturally occurring antibody, a non-naturally occurring antibody, or a variant or modified version of a naturally occurring antibody. The parent antibody may comprise the antibody itself, a composition containing the parent antibody, or an amino acid sequence encoding the parent antibody. In preferred embodiments of the present invention, the parent antibody comprises an Fc region. More specifically, the Fc region of the parent antibody of the present invention is a human IgG1, IgG2, IgG3, or IgG4 Fc region; in certain embodiments, the Fc region of the parent antibody of the present invention is a modified or unmodified IgG1 Fc region.

[0266] In a specific embodiment, the antibody of the present invention is a heterodimeric antibody or antibody fragment thereof having a modified Fc, wherein the Fc comprises a first and a second modified CH3 domain, wherein the first modified CH3 domain comprises a substitution that facilitates heterodimerization. In a specific embodiment of the present invention, the antibody is constructed using the above-mentioned BEAT® heavy chain (Hc) heterodimerization technology (see "Skegro et al., (2017) J Biol Chem 292(23): 9745-9759", "Stutz et al., (2020) J Biol Chem 295(28): 9392-9408" and "WO2012131555"), wherein the BEAT (A) chain is also referred to herein as BEAT (A) and comprises the first modified CH3 domain, and the BEAT (B) chain is also referred to herein as BEAT (B) and comprises the second modified CH3 domain.

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

[0268] In a more specific embodiment, the present invention discloses a trispecific antibody or antibody fragment thereof constructed based on the BEAT® platform, wherein the CH3 domain of BEAT(A) comprises one or more substitutions selected from the following group: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and BEAT(A) may optionally further comprise a substitution selected from the following group: D356E, L358M, N384S, V422I, H435R, and Y436F (EU numbering); and BEAT(B) The CH3 domain comprises one or more substitutions selected from the following group: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (EU numbering), and BEAT(b) may optionally further comprise the substitution D401Q (EU numbering).

[0269] In certain aspects, the present invention also relates to a trispecific heterodimeric antibody or antibody fragment thereof having a first and a second engineered CH3 domain, wherein the first engineered CH3 domain comprises a substitution selected from the group consisting of: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (EU numbering), and the second engineered CH3 domain comprises a substitution 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 via the first and second engineered CH3 domains, characterized in that the trispecific heterodimeric antibody or antibody fragment thereof comprises at least three binding moieties, each binding to a different antigen.

[0270] In certain aspects, the bispecific antibodies of the present invention comprise a variant Fc region having at least one amino acid modification that differs from the Fc region of the parent antibody, such that the antibody comprising the variant Fc region exhibits a different efficacious function than the parent antibody. More specifically, the CH2 domain of the Fc region comprises at least one amino acid modification.

[0271] In certain embodiments of the present invention, BEAT(A) and BEAT(B) are genetically engineered to enhance Fc function. More specifically, the CH2 domain of the Fc region is engineered to include at least one amino acid modification. More specifically, BEAT(A) has one or more substitutions at positions selected from the group consisting of 324, 334, 269, 298, 239, 332, and 333; and BEAT(B) has one or more substitutions at positions selected from the group consisting of 324, 334, 269, 298, 239, 332, and 333. Preferably, these positions include 324, 334, 269, 289, 298, and 333. Still more specifically, BEAT(A) has one or more substitutions at a position selected from the group consisting of S324N, K334E, K334A, E269D, S298A, S239D, I332E, and E333A; and BEAT(B) has one or more substitutions at a position selected from the group consisting of S324N, K334E, K334A, E269D, S289A, K334A, E333A. In certain embodiments, BEAT(A) comprises a group of mutations selected from the following groups: 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 embodiments, BEAT(B) comprises a group 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 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 certain preferred embodiments, BEAT(A) comprises mutations S239D, I332E, and S324N, and BEAT(B) comprises mutation S324N.

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

[0273] In certain 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 certain embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In certain 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 certain embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0274] In certain embodiments, the Fc region is a human IgG4 Fc region having one or more mutations that reduce or eliminate Fcyl and / or Fcyll binding. In certain embodiments, the Fc region is a human IgG4 Fc region having one or more mutations that reduce or eliminate Fcyl and / or Fcyll binding without affecting FcRn binding. In certain embodiments, the Fc region is a human IgG4 Fc region having amino acid substitutions at positions corresponding to positions 228 and / or 409 of human IgG4 according to the EU index. In certain embodiments, the amino acid substitutions are S228P and / or R409K. In certain embodiments, the Fc region is a human IgG4 Fc region having amino acid substitutions at positions corresponding to positions 234 and / or 235 of human IgG4 according to the EU index. In certain embodiments, the amino acid substitutions are F234A and / or L235A. In certain embodiments, the Fc region is a human IgG4 Fc region having amino acid substitutions at positions corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU index. In certain embodiments, the amino acid substitutions are S228P, F234A, L235A, and / or R409K. In certain embodiments, the Fc region is a human IgG4 Fc region having amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In certain embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In certain embodiments, the Fc region is a human IgG4 Fc region having amino acid mutations at positions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In certain embodiments, the amino acid substitutions are S228P; E233P, F234V, L235A and a deletion at 236; and / or R409K.

[0275] In certain embodiments, the binding proteins of the present invention comprise one or more mutations that improve purification, for example by modulating affinity for purification reagents. For example, if one of the two Fc regions of a heterodimeric binding protein is known to contain a mutation that reduces or eliminates binding to Protein A, it can be selectively purified away from its homodimeric form because the heterodimeric form has an affinity more intermediate than either homodimeric form for Protein A-based purification and can be selectively eluted from Protein A using different pH values ​​(see, e.g., Smith, EJ et al. (2015) Sci. Rep. 5: 17943). In certain embodiments, the mutations comprise substitutions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, where the amino acid substitutions are H435R and Y436F. In certain embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, wherein the first Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the binding protein further comprises a third polypeptide chain further comprising a second Fc region linked to CH1, wherein the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and wherein only one of the first and second Fc regions has 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] To improve the production of certain binding proteins (e.g., bispecific or trispecific binding proteins), the CH domain can be altered using BEAT technology. For details and examples of this technology, please refer to International Publication No. WO2012131555.

[0277] In certain embodiments, the binding proteins of the present invention comprise one or more mutations that increase serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1):346-56). In certain embodiments, the mutations comprise substitutions 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 certain embodiments, the binding protein comprises a 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; and a 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. The first and / or second Fc regions comprise amino acid substitutions 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 certain embodiments, the binding proteins of the present invention comprise knob and hole mutations and one or more mutations for improving serum half-life. In certain embodiments, the first and / or second Fc regions are human IgG1 Fc regions.

[0278] In certain embodiments, the binding proteins of the invention comprise one or more mutations that reduce a function, such as Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In certain 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 domains; 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 domains; wherein the first and second Fc regions are Fc regions of human IgG1; and wherein the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In certain embodiments, the Fc regions of the second and third polypeptide chains are Fc regions of human IgG1, and 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, the amino acid substitutions being L234A and L235A. In certain embodiments, the second polypeptide chain further comprises 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; wherein the third polypeptide chain further comprises 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; wherein the first and second Fc regions are Fc regions of human IgG1; and wherein the first and second Fc regions each have an amino acid substitution at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In certain embodiments, the Fc region of the second and third polypeptide chains is a human IgG1 Fc region, wherein the Fc region each has 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 certain embodiments, the Fc region of the second and third polypeptide chains is a human IgG4 Fc region, wherein the Fc region each has 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 certain embodiments, the binding protein comprises a 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, and a 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 and second Fc regions each have amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

[0279] In certain embodiments, the binding proteins of the present invention comprise knob and hole mutations and one or more mutations that reduce efflux function. In certain embodiments, the first and / or second Fc regions are human IgG1 Fc regions. In certain embodiments, the first and / or second Fc regions are human IgG4 Fc regions. For more information on Fc mutations at position 329, see, for example, Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604 and WO 1999051642.

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

[0281] Application of binding proteins

[0282] The antibodies or antibody fragments of the present invention can be used to detect and quantify one or more target antigens using any known method, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays. The binding protein will bind to the one or more target antigens with an affinity appropriate for the assay used.

[0283] In diagnostic applications, in certain embodiments, the binding protein is labeled with a detectable moiety. The detectable moiety can be any moiety capable of directly or indirectly generating a detectable signal. For example, the detectable moiety can be a radioisotope, such as 3H, 14C, 32P, 35S, 125I, 99Tc, U1ln, or 67Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0284] The binding proteins can also be used for in vivo imaging. By administering a binding protein labeled with a detectable moiety to an animal, preferably into the bloodstream, the presence and location of the labeled antibody in the host can be determined. The binding protein can be labeled with any moiety that can be detected in an animal by nuclear magnetic resonance (NMR), radiology, or other detection methods known in the art.

[0285] In clinical or research applications, in certain embodiments, the binding protein can be conjugated to a cytotoxic drug. Currently, a variety of antibodies coupled to cytotoxic agents (i.e., antibody-drug conjugates) are used to deliver cytotoxic payloads to specific tumor cells. Cytotoxic drugs and linkers for conjugating them to antibodies are known in the art; see, for example, Parslow, AC et al. (2016) Biomedicines 4:14 and Kalim, M. et al. (2017) Drug Des. Devel. Ther. 11:2265-2276.

[0286] The present invention also relates to a kit comprising a binding protein and other reagents, which can be used to detect the abundance of a target antigen in a biological sample. The reagents may include a detectable label, blocking serum, positive and negative control samples, and a detection reagent. In certain embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In certain embodiments, the kit comprises a container and a label or package insert provided on or attached to the container. Suitable containers include bottles, tubes, syringes, IV solution bags, and the like. The container can be made of various materials, such as glass or plastic. The composition can be contained in the container alone or in combination with another composition useful for treating, preventing, and / or diagnosing a condition, and the container has a sterile access port (for example, the container can be an IV solution bag or a vial with a stopper pierceable by a hypodermic needle). In certain embodiments, the label or package insert states that the composition is used to prevent, diagnose, and / or treat a selected condition. Additionally or alternatively, 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. Other materials beneficial from a commercial or user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.

[0287] In certain embodiments, the binding proteins of the present invention are administered to a patient in need of treatment or prevention of cancer. In certain embodiments, the present invention relates to a method for preventing and / or treating a proliferative disease or condition (e.g., cancer). In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a heterodimeric antibody of the present invention or a pharmaceutical composition related thereto. In certain embodiments, the present invention relates to the use of a heterodimeric antibody of the present invention or a pharmaceutical composition related thereto for preventing and / or treating a proliferative disease or condition (e.g., cancer) in a patient in need thereof. In certain embodiments, the present invention relates to the use of a heterodimeric antibody of the present invention or a pharmaceutical composition related thereto for the manufacture of a composition for preventing and / or treating a proliferative disease or condition (e.g., cancer) in a patient in need thereof. In certain embodiments, the patient is a human. In certain embodiments, the binding protein comprises one antigen-binding site that binds to a T cell surface protein and another antigen-binding site that binds to the extracellular domain of a human CD38 polypeptide. In certain 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 such cancers include, but are not limited to, multiple myeloma, leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic leukemia, and acute myeloid leukemia), chronic myeloid leukemia, multiple myeloma, plasma cell leukemia, lymphoma, breast cancer (e.g., Her2+ breast cancer), prostate cancer, cervical cancer, germline B-cell lymphoma or B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, myelodysplastic syndrome (MDS), breast cancer (e.g., Her2+ breast cancer), prostate cancer, cervical cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), well-differentiated ovarian serosal epithelial cell carcinoma, peritoneal cancer, smoldering myeloma, and glioma.

[0289] [Binding protein therapeutic composition and its administration]

[0290] Therapeutic compositions or pharmaceutical compositions comprising the binding protein also fall within the scope of the present invention. These therapeutic compositions or pharmaceutical compositions may comprise a therapeutically effective amount of the binding protein or binding protein-drug complex admixed with a pharmaceutically or physiologically acceptable formulation suitable for the mode of administration.

[0291] Preferably, the acceptable formulation materials are non-toxic to the recipient at the dosage concentrations employed.

[0292] The pharmaceutical composition may contain formulation materials used to modify, maintain or preserve, for example, pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate, or release, absorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other sugars (e.g., glucose, maltose, or dextran), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants, or wetting agents (e.g., polyols; PEG; sorbitan esters; polysorbates, such as polysorbate 20 or polysorbate 80; polyethylene glycol octylphenyl ether; trishydroxymethylaminomethane; lecithin; cholesterol, or tetrabutylamyl), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, or mannitol or sorbitol), delivery vehicles, diluents, excipients, and / or adjuvants (see, e.g., "REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990)), and its subsequent editions, which are incorporated herein by reference for any purpose).

[0293] Those skilled in the art can determine the optimal pharmaceutical composition based on factors such as the intended route of administration, delivery form, and desired dosage regimen. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0294] The primary carrier or vehicle of the pharmaceutical composition can be aqueous or non-aqueous. For example, suitable carriers or vehicles for injection can be water, saline, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are also suitable carriers. Furthermore, the binding protein can be formulated as a liquid formulation or lyophilized using appropriate excipients.

[0295] The pharmaceutical compositions of the present invention may be administered intravenously or subcutaneously. Alternatively, the compositions may be delivered via the digestive tract by inhalation, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0296] If parenteral administration is desired, the therapeutic composition can be a combination of the desired binding protein in a pharmaceutically acceptable carrier in a pyrogen-free, parenterally suitable aqueous solution. A particularly suitable carrier for parenteral injection is sterile distilled water, to which the binding protein is added to form a sterile, isotonic solution and then stored appropriately. Another formulation involves combining the desired molecule with an agent that provides controlled or sustained release, such as injectable microparticles, bioerodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), microbeads, or liposomes, and administering the molecule via a long-acting depot injection. Hyaluronic acid can also be used to extend the duration of the circulatory system. Other suitable methods for introducing the desired molecule include implantable drug delivery devices.

[0297] In one embodiment, the pharmaceutical composition can be formulated as an inhalation formulation. For example, the binding protein can be formulated as a dry powder for inhalation. Alternatively, the binding protein can be formulated as an inhalation solution and combined with a propellant for administration as a spray. In another embodiment, the solution can be aerosolized.

[0298] The present invention can also be implemented as an oral dosage form. In one embodiment of the present invention, the binding protein administered in this manner can be formulated into a pharmaceutical formulation with or without carriers commonly used in the compounding of solid pharmaceutical formulations, such as tablets and capsules. For example, the capsule can be designed to release the active portion of the formulation while the drug is in the gastrointestinal tract, avoiding pre-systemic degradation and thereby ensuring maximum bioavailability. Alternatively, other agents may be added to enhance absorption of the binding protein. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet-disintegrating agents, and binding agents may also be used.

[0299] Another pharmaceutical composition comprises mixing an effective amount of a protein with a nontoxic excipient suitable for tablet manufacture. The tablets can be dissolved in sterile water or another suitable carrier to produce a single-dose formulation. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or bicarbonate, lactose, or calcium phosphate; binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0300] Other pharmaceutical compositions of the present invention will be readily apparent to those skilled in the art, including formulations involving sustained or controlled release of binding proteins. Technologies for achieving various other sustained or controlled release methods, such as liposome carriers, bioerodible microparticles or porous microbeads, and long-acting depot injectables, are also known to those skilled in the art. Other examples of sustained-release formulations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and γ-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate copolymers, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared using a variety of methods known in the art.

[0301] Pharmaceutical compositions for in vivo administration generally must be sterile. This can be achieved by filtering through a sterile filter membrane. If the composition is lyophilized, it can be sterilized in this manner before and after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Furthermore, parenteral compositions are typically contained in containers with sterile access ports, such as intravenous solution bags or vials with stoppers pierceable by hypodermic needles.

[0302] Once formulated into a dosage form, the pharmaceutical composition can be stored in sterile vials in the form of a solution, suspension, colloid, emulsion, solid, or dehydrated or lyophilized powder. These preparations can be stored in a ready-to-use form or in a form that requires reconstitution prior to administration (e.g., lyophilization).

[0303] The present invention also includes kits for making single-dose dosing units. The kits may include a first container containing the dried protein and a second container containing the aqueous formulation. The present invention also includes kits with single- and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

[0304] The effective amount of the binding protein drug composition used therapeutically depends on factors such as the therapeutic context and purpose. Those skilled in the art will recognize that the appropriate therapeutic dose depends, in part, on the delivered molecule, the indication for which the binding protein is used, the route of administration, and the patient's size (weight, body or organ size) and condition (age and general health). Accordingly, clinicians can adjust the dose and vary the route of administration to achieve optimal therapeutic effect.

[0305] The frequency of dosing depends on the pharmacokinetic parameters of the binding protein in the formulation used. Clinicians typically administer the composition until a dose that provides the desired effect is achieved. Thus, the composition may be administered as a single dose, or as two or more doses (each containing the same or different amounts of the desired molecule) over time, or as a continuous infusion via an implantable device or catheter. Fine-tuning the appropriate dosage is routinely performed by those skilled in the art and is within their usual tasks. Appropriate dose-response data can be used to assist in determining the appropriate dosage.

[0306] The pharmaceutical composition is administered according to known methods, such as orally; subcutaneously; intravenously, intraperitoneally, intracerebrally (intracerebral), intraventricularly, intramuscularly, intraocularly, intraarterially, intrahilarly, or intralesionally; via a continuous drug delivery system; or via an implantable device. If desired, the composition may also be administered by rapid or continuous infusion or via an implantable device.

[0307] The composition can also be administered topically via a film, cotton wool, or other suitable material that has been pre-absorbed or encapsulated with the desired molecule. If an implantable device is used, it can be implanted in any appropriate tissue or organ, and delivery of the desired molecule can be achieved via diffusion, timed injection, or continuous administration.

[0308] Treatment options

[0309] The present invention also provides a method for treating multiple myeloma in a subject in need thereof. This method utilizes heterodimeric antibodies that bind to CD3, BCMA, and CD38, allowing malignant cells to rapidly bind to bound T cells, thereby inducing T cell-mediated killing of the bound malignant cells. The method of the present invention utilizes heterodimeric antibodies that bind to CD3, BCMA, and CD38 to effectively destroy target cells while minimizing adverse side effects (e.g., uncontrolled cytokine release).

[0310] As used herein, "treatment" refers to both therapeutic and / or preventative measures. The antibodies of the present invention are administered to a subject in need thereof; subjects in need of treatment include those already suffering from a disease, those predisposed to developing a disease, or those in whom a disease is to be prevented. The antibodies of the present invention can be used to treat or ameliorate cancer in humans, as well as to prevent cancer in human patients. The antibodies of the present invention or their fragments are administered to a subject in need thereof, such as a human subject suffering from a BCMA-expressing cancer and / or a CD38-expressing cancer, such as multiple myeloma, for example, relapsed / refractory multiple myeloma. Relapsed myeloma is characterized by a relapse after a previous response to treatment. Laboratory and radiological criteria indicative of disease include, but are not limited to, an increase in serum or urine monoclonal protein (M protein) >25%, a difference of >25% from nadir in affected and unaffected serum free light chains, respectively, the development of a new plasmacytoma, or hypercalcemia. See Sonneveld et al., Haematologica. 2016 Apr; 101(4): 396-406.

[0311] Relapse in patients with nonsecretory disease is characterized by an increase in bone marrow plasma cells. Signs of relapse also include 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 a patient who previously had at least some response to a previous treatment and then lost response or had worsening disease while receiving treatment or within 60 days of the last treatment. See "Sonneveld, supra; Anderson et al., Leukemia. 2008;22(2):231-239."

[0312] The methods of the present invention comprise administering to a subject a dose of between about 0.0001 mg / kg and about 200 mg / kg, for example, between about 0.0005 mg / kg and about 200 mg / kg, or between about 0.001 mg / kg and about 200 mg / kg, or between about 0.0015 mg / kg and about 200 mg / kg, or between about 0.005 mg / kg and about 200 mg / kg, or between about 0.01 mg / kg and about 200 mg / kg, or between about 0.015 mg / kg and about 200 mg / kg, or between about 0.03 mg / kg and about 200 mg / kg, or between about 0.05 mg / kg and about 200 mg / kg, or between about 0.06 mg / kg and about 200 mg / kg, or between about 0.09 mg / kg and about 200 mg / kg, or between about 0.1 mg / kg and about 200 mg / kg, or between about 0.12 mg / kg to about 200 mg / kg, or between about 0.15 mg / kg to about 200 mg / kg, or between about 0.18 mg / kg to about 200 mg / kg, or between about 0.2 mg / kg to about 200 mg / kg, or between about 0.3 mg / kg to about 200 mg / kg, or between about 0.36 mg / kg to about 200 mg / kg, or between about 0.5 mg / kg to about 200 mg / kg, or between about 0.6 mg / kg to about 200 mg / kg, or between about 0.7 mg / kg to about 200 mg / kg, or between about 1 mg / kg to about 200 mg / kg, or between about 1.2 mg / kg to about 200 mg / kg, or between about 1.5 mg / kg to 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 invention also includes any dosage range of any value between the above intervals.For example, the trispecific antibodies of the present invention are administered to a patient in a single or multiple doses at a dose 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, 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, or at least about 200 mg / kg.

[0313] In various aspects of the methods, the dosage can be adjusted continuously during the course of treatment. For example, a subject can receive the same initial dose in one or more administrations and then receive a higher dose in one or more subsequent administrations. In other words, the present invention encompasses increasing the dose of the trispecific antibody at least once during the course of treatment. Alternatively, the dosage can be decreased during the course of treatment, i.e., the amount of heterodimeric antibody used can be reduced as treatment progresses.

[0314] The present invention also provides a method comprising administering multiple (i.e., two or more) doses of a trispecific antibody during a treatment period. Each dose can be administered at any interval, for example, once a week, twice a week, three times a week, four times a week, or five times a week. Each dose can be administered every two weeks, every three weeks, or every four weeks. In other words, in certain aspects, a two-week waiting period occurs between two administrations of the heterodimeric antibody to the subject. The waiting period between administrations need not be consistent throughout the treatment course. In other words, the interval between doses can be adjusted throughout the treatment course. In certain aspects, the method comprises administering two doses of the heterodimeric antibody to the subject weekly during the first and second weeks of treatment (twice a week during the first and second weeks), administering one dose of the heterodimeric antibody to the subject weekly during the third and fourth weeks of treatment (i.e., once a week during the third and fourth weeks), and administering one dose every two weeks from the fifth week of treatment until the end of treatment (i.e., a two-week waiting period between doses starting in the fifth week and ending at the end of treatment).

[0315] Alternatively, in various aspects, the method comprises administering a dose of the trispecific antibody weekly from the first to the fourth week of treatment, and then administering a dose of the antibody every two weeks starting from the fifth week 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 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, with administration 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 with respect 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 neoplastic cells; (2) an increase in neoplastic cell death; (3) an inhibition of neoplastic cell survival; (4) a decrease in paraprotein production by tumor cells; (5) an inhibition (i.e., slowing to a certain extent, preferably stopping) of tumor growth; (6) an increase in patient survival; and (7) amelioration of one or more symptoms associated with the disease or condition. Tumor response can be assessed by identifying changes in tumor morphology (i.e., overall tumor burden, tumor size, and the like) using screening techniques such as magnetic resonance imaging (MRI) scans, X-ray imaging, computed tomography (CT) scans, bone marrow scans, endoscopy, and tumor biopsy samples, including bone marrow aspiration (BMA) and circulating tumor cell counts. Any degree of improvement is contemplated within the scope of the present invention, without requiring a complete therapeutic response (ie, absence of clinically detectable disease and normalization of any previously abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF) or abnormal monoclonal proteins).

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

[0319] In some instances, the subject has previously received cancer treatment, such as multiple myeloma. For example, the subject may have previously received immunomodulatory drugs (thalidomide, lenalidomide, platinum), proteasome inhibitors (such as platinum, bortezomib, or carfilzomib), dexamethasone, doxorubicin, or a combination thereof.

[0320] Optionally, the subject has previously received treatment with an anti-CD38 monospecific antibody, such as daratumumab (DARZALEX®). In various embodiments, the subject has relapsed or been refractory to the previous anti-CD38 monospecific antibody treatment. The method may include a waiting period between the administration of the previous anti-CD38 monospecific antibody and the administration of the heterodimeric antibody.

[0321] Collaborative treatment

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

[0323] Optionally, the methods of the present invention further comprise administering dexamethasone to the subject. Dexamethasone can be administered by any route, such as those described herein. Preferably, dexamethasone is administered intravenously or orally. If dexamethasone is administered intravenously, it can be optionally administered within one hour prior to administration of the antibody of the present invention. Optionally, the amount of dexamethasone administered is about 8 mg or about 4 mg.

[0324] In various embodiments, the methods of the present invention further comprise administering a chemotherapeutic agent. Non-limiting examples of DNA damaging chemotherapeutic agents include type I topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin and analogs or metabolites, and doxorubicin); type II topoisomerase inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, methotrexate, mitomycin C); C) and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators (e.g., bleomycin); and nucleoside mimetics (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

[0325] Chemotherapeutic agents that disrupt cell replication include: paclitaxel, Europaclitaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib, CEP-18770, MG132, peptide vinyl sulfones), peptide epoxyketones (e.g., ebotamycin and carfilzomib), β-propiolactone inhibitors (e.g., lactacystin, MLN 519, NPI-0052, salinosporamide); A), compounds that form dithiocarbamate complexes with metals (e.g., disulfiram), and certain antioxidants (e.g., epigallocatechin-3-gallate, catechin-3-gallate, and salinosporamide A); NF-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 that are upregulated, overexpressed, or activated in cancer and whose inhibition downregulates cell replication.

[0326] Such treatment regimens may include the administration of other antibody therapies, such as elotuzumab (a humanized monoclonal antibody against SLAMF7; see Tai et al., Blood, 2008; 112: 1329-37); daratumumab, MOR202, and isatuximab, which target CD38; nBT062-SMCC-DM1, nBT062-SPDB-DM4, and nBT062-SPP-DM1, which target CD138; rukatumomab (also known as HCD122) and daclizumab (also known as SGN-40), which target CD40; and lovotozumab, which targets CD56. For studies on antibody therapy for 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 of which are incorporated herein by reference.

[0327] In certain embodiments, the heterodimeric antibody is administered prior to, concurrently with, or after the following treatments: proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), immunomodulatory drugs (IMiDs), such as the thalidomide derivatives platinum and lenalidomide, the histone deacetylase inhibitor panobinostat, antibodies, such as daratumumab, teixumab, ixatuximab, elotuzumab, taquituzumab, onukumab, belantamab, enatuzumab, or any other compound and / or drug and / or biological therapy disclosed above, also including fenacilex, mofortin selinexor, dexamethasone, doxorubicin, melphalan, CAR-T cell therapy, and combinations thereof.

[0328] The specific embodiments described in the present invention are illustrative in nature, and those skilled in the art will appreciate that various changes can be made to the details without departing from the scope of the patent application.

[0329] [example] [1] [:anti] [CD3] [Production of antibodies]

[0330] Materials and Methods

[0331] Recombinant human CD3γε protein (SEQ ID NO: 606) []

[0332] The CD3γ-CD3ε complex (comprising CD3γ residues 23-103, a linker, and CD3ε residues 23-118, hereinafter referred to as recombinant human CD3γε protein) was expressed as a fusion protein in an Escherichia coli expression system according to published information (see Arnett et al., 2004, PNAS, 101 (46): 16268-16273). The protein was renatured from inclusion bodies and purified by affinity chromatography (using an affinity resin prepared with the OKT3 monoclonal antibody) and size exclusion chromatography.

[0333] Recombinant human CD3γε protein was biotinylated using the EZ-link™ Sulfo-NHS-LC-Biotin No-Weigh™ Format Kit (ThermoFisher Scientific, Catalog No. A39257). Briefly, recombinant human CD3γε protein was dialyzed against acetate buffer (pH 5.0) at 4°C and then incubated with a 50-fold molar excess of biotin for 2 hours. The biotinylated recombinant human CD3γε protein was then dialyzed against PBS at 4°C.

[0334] Transient expression of full-length human CD3εδ, human CD3εγ, and cynomolgus monkey CD3εδ in CHO-S cell lines

[0335] The human gene sequence-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 a partial extracellular domain, the full transmembrane and cytoplasmic domains of mouse CD80 (Uniprot ID Q00609 residues 237-306) to generate constructs comprising the sequences of SEQ IDs: 610-614. To achieve protein expression, suspension-adapted CHO-S cells (cGMP cryopreserved, Invitrogen, catalog number A1136401) were co-transfected with 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) using polyethyleneimine (PEI; Polysciences). Briefly, cells were plated at a density of 2 million cells / mL in CD CHO (Gibco) medium. Cells were then transfected with the DNA-PEI mixture at 37°C. Four hours after transfection, the cell culture was diluted 1:1 in PowerCHO 2 (Lonza) supplemented with 4 mM L-glutamine and cultured with orbital shaking at 37°C, 5% CO2, and 80% humidity. The expression of the target antigen was assessed by monitoring the expression of the eGFP reporter protein using a fluorescence microscope.

[0336] Generation of CD3ε knockout (KO) HPB-ALL cell pools

[0337] HPB-ALL CD3-deficient cell lines were generated from primary HPB-ALL cells (DSMZ, catalog number: ACC483, Lot 15) using Clustered Regularly Interspaced Palindromic Repeats (CRISPR) / Cas9 technology targeting the first exon of the CD3 gene. See Ran et al. (2013, Cell, 154: 1380-1389). HPB-ALL cell lines were transfected using a Neon electroporator (Invitrogen Life Technologies) according to the manufacturer's instructions. After transfection, cells were stained with an anti-CD3 antibody (OKT3 clone, ThermoFisher, catalog number: 14-0037-85) and sorted using a Melody FACS sorter (BD) to generate a CD3-deficient cell pool. The lack of CD3 expression was further confirmed by flow cytometry using Qifikit (DAKO, catalog number: K007811-8).

[0338] Gene bank generation

[0339] The gene library used here was of synthetic origin, with diversity limited to the heavy chain (CDR-H1, CDR-H2, and CDR-H3) and a fixed Vκ3-15 / Jκ1 light chain (SEQ ID NO: 1). The gene library comprises four sub-gene libraries based on the VH1-69, VH3-23, VH3-15, and VH3-53 antibody strains. CDRs were randomized using trimer oligonucleotides. Primers were designed for diversification of CDR-H1 and CDR-H2 for each sub-gene library, encoding strain-specific naturally occurring diversity at Kabat residues 27-35 and 50-58, respectively. CDR-H3 was randomized using a pool of oligonucleotides encoding 15 CDR-H3 lengths (6-20) and length-specific naturally occurring diversity at Kabat residues 95-102. The diversified scFv fragments were pooled to mimic the natural CDR-H3 length distribution and cloned into pNGLEN (a custom-modified pUC119 phage vector). The resulting ligation reaction was electroporated into E. coli TG1 cells. The diversity of each sub-library ranged from 1.2 × 10⁻¹⁰ to 1.7 × 10⁻¹⁰, for a total diversity of 5.6 × 10⁻¹⁰ across the four sub-libraries.

[0340] Gene pool screening

[0341] The panning strategy used involved four rounds of selection using either the constitutive CD3 protein or two rounds of selection using the recombinant CD3 protein, followed by three rounds of selection using CD-3 expressing cells.

[0342] Purified phage particles (10 12 plaque-forming units) from individual or pooled libraries were blocked for one hour at room temperature in 1 ml of 3% (w / v) nonfat milk phosphate buffered saline (3% MPBS). Two (100) μl of Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Invitrogen, catalog number 65002) were blocked under the same conditions. Phage were subtracted with the pre-blocked magnetic beads for one hour at room temperature. The remaining pre-blocked magnetic beads were incubated with 100 nM biotinylated recombinant human CD3εδ protein (Creative Biomart, catalog number CD3E & CD3D-377H) or in-house biotinylated recombinant human CD3γε protein (SEQ ID NO: 606) for 30 minutes at room temperature. The subtracted phage were then incubated with pre-coated magnetic beads for two hours at room temperature. The magnetic beads were washed five times with PBS containing 0.1% (v / v) Tween (PBS-Tween 0.1%) and then twice with PBS. Phage were eluted with 100 mM triethylamine for 10 minutes at room temperature and then neutralized with 1 M Tris-HCl (pH 8). The eluted phage were used to infect 10 ml of exponentially growing Escherichia coli TG1 cells. The infected cells were grown in 2YT medium at 37°C and 100 revolutions per minute (RPM) for one hour, then spread onto 2YTAG (2YT medium supplemented with 100 μg / ml ampicillin and 2% glucose) agar plates and incubated at 30°C overnight (ON). The selected colonies were scraped from the plates into 10 ml of 2YT, supplemented with 15% v / v glycerol, and stored at -80°C. Glycerol-derived TG1 cells were grown in 2YTAG medium at 37°C and 240 rpm until the optical density at 600 nm reached 0.5. The cells were then superinfected with M13K07 helper phage at an MOI of 10 at 37°C and 100 rpm for one hour. The medium was then switched to 2YTAK (2YT medium supplemented with 100 μg / ml ampicillin and 50 μg / ml kanamycin), and the cells were further incubated overnight at 30°C and 280 rpm. Alternatively, the eluted phage was used to infect 2 ml of exponentially growing E. coli TG1 cells. The infected cells were grown in 2YT medium at 37°C and 100 rpm for one hour, followed by growth in 2YT medium supplemented with 2% glucose at 37°C and 240 rpm for another hour. The cells were then superinfected with M13K07 helper phage and the phage produced as described above. The following day, 10 μl of phage containing cell-free supernatant was used for subsequent selection rounds.

[0343] For cell-based panning, 2-10 × 107 non-expressing cells (CHO, HEK, or CD3ε-deleted HPB-ALL) and 2-10 × 107 CD3ε-expressing cells (HPB-ALL or CHO cells transiently expressing the membrane-binding extracellular domain of cynomolgus monkey CD3ε (SEQ ID NO: 613) and CD3δ (SEQ ID NO: 614)) were blocked for one hour at room temperature with PBS / BSA 3% supplemented with 0.1% azide to prevent receptor endocytosis. Phage were back-selected using the non-expressing cells for one hour at room temperature. The back-selected phage were then incubated with CD3ε-expressing cells for two hours at room temperature. To remove nonspecific phage, cells were washed four times with PBS-Tween 0.1% and twice with PBS. Phage were eluted with 76 mM citric acid, pH 2.0, for 10 minutes at room temperature and then with 1% Tris-HCl, pH 8. The eluted phages were used to infect exponentially growing E. coli TG1 cells. Phage amplification was performed as described above.

[0344] scFv screening by flow cytometry

[0345] Flow cytometry was used to evaluate the binding of selected scFv clones 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 clones from the fourth or fifth round of selection were selected and plated in 96-well plates and grown in 2YT medium supplemented with 100 μg / ml ampicillin and 0.1% glucose. scFv expression was induced by the addition of 0.02 mM IPTG and incubation at 30°C and 260 rpm overnight. Cells were centrifuged, and the pellets were resuspended in TES buffer (50 mM Tris-HCl, pH 8; 1 mM EDTA, pH 8; 20% sucrose) and incubated on ice for 30 minutes. Periplasmic extracts were obtained. Cell debris was removed by centrifugation, and the scFv-containing supernatant was used for flow cytometry. CD3-expressing and non-transfected CHO cells were seeded in microtiter plates at a density of 105 cells / well. The supernatant was then removed by centrifugation, and 100 µl of periplasmic extract, previously diluted 1:1 in PBS containing 3% (w / v) bovine serum albumin (PBS-BSA 3%), was added to each well and incubated at 4°C for 30 minutes. The cells were then washed with PBS-BSA 3% and incubated with biotinylated chicken anti-c-Myc antibody (Gallus Immunotech, catalog number: ACMYC-B) diluted 1:200 in PBS-BSA 3% for 30 minutes at 4°C. The cells were then washed with 3% PBS-BSA and incubated with streptavidin-APC (eBioscience, catalog number 17-4317) diluted 1:100 in 3% PBS-BSA for 30 minutes at 4°C. Finally, cell fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences).

[0346] IgG1 LALA expression

[0347] cDNAs encoding the constant regions of various antibodies were synthesized by GENEART AG (Regensburg, Germany) and modified using standard molecular biology techniques. PCR products were digested with appropriate DNA restriction enzymes, purified, and ligated into a modified pcDNA3.1 plasmid (Invitrogen) containing a CMV promoter and a bovine hormone polyadenylation (poly(A)) construct. The expression vector also contained oriP, a plasmid replication source for human herpesvirus type 4, and a mouse VJ2C leader peptide for secretion of the encoded polypeptide. To reformat the scFv library clones into the human IgG1 LALA (human IgG1 with L234A and L235A substitutions, EU numbering) format, PCR amplified the individual VH cDNAs of each scFv in its phage library vector. The VH cDNAs were then PCR cloned into the modified pcDNA 3.1 vector described above, upstream of the cDNA encoding the human IgG1 heavy chain CH1 domain. Furthermore, the fixed Vκ3-15 / Jκ1 light chain (SEQ ID NO: 1) was cloned into the modified pcDNA 3.1 vector described above, upstream of the cDNA encoding the human kappa constant light chain domain.

[0348] To achieve 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 cultured at a concentration of 8 million cells / mL (Biowest, catalog number L0501) in RPMI 1640 supplemented with 0.1% Pluronic F-68 (ThermoScientific, catalog number 24040032). The cells were then transfected with the DNA-PEI mixture. Four hours after transfection, the cell culture was diluted 1:1 into BalanceCD HEK293 cells (Irvine Scientific, catalog number 91165) supplemented with 4 mM L-glutamine and incubated with rotational shaking at 37°C, 5% CO2, and 80% humidity for 5 days. The cell-free culture supernatant containing the recombinant protein was prepared by centrifugation, filtered, and used for further purification. The IgG1 LALA protein was purified using CaptivA® Protein A affinity resin (Repligen, catalog number CA-PRI-0100). The affinity resin was added to the filtered culture supernatant and incubated overnight at 4°C with gentle agitation. The next day, the beads were collected onto a Poly-Prep® chromatography column (Bio-Rad Laboratories), washed with PBS, and the recombinant protein was eluted with an acidic buffer (typically 0.1 M glycine at pH 3). After neutralization with 1 / 10 volume of Tris-HCl, pH 8, the preparation was buffer exchanged into PBS.

[0349] Screening of anti-CD3 IgG1 LALA clones on CD3 expressing cells

[0350] Screening for the generation of human IgG1-LALA as an anti-CD3 antibody included binding studies in selected HPB-ALL wild-type and CD3 knockout (KO) HPB-ALL cell lines, as well as binding studies in primary isolated PBMCs using flow cytometry.

[0351] Binding to HPB-ALL and CD3ε-deficient HPB-ALL cells

[0352] Cells were plated at 1 × 10 5 cells per well in a 96-well round-bottom plate (TPP, catalog number 92097). The cells were centrifuged at 350 g for 3 minutes at 4°C and resuspended in 100 µl of cold FACS buffer (PBS 1X supplemented with 2.5% heat-inactivated fetal bovine serum, 2 mM EDTA, and 0.05% sodium azide) containing increasing concentrations of anti-CD3 IgG1-LALA antibody. The stained cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer, centrifuged at 350 g for 3 minutes, and resuspended in 100 µl of a cocktail of goat polyclonal anti-human IgG PE secondary antibodies (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 SYTOX Green Dead Cell Stain (ThermoFisher Scientific, catalog number S34860) at a 1 / 2000 dilution. Samples were acquired on a CytoFlex instrument (Beckman Coulter). Cells were gated based on size and debris using FSC and SSC scalars, excluding doublets and dead cells. The PE geometric mean fluorescence intensity of the cells was analyzed using FlowJo software. Only samples showing at least 200 cells in the final gate were analyzed. Finally, the geometric mean fluorescence intensity values ​​were plotted using PE Prism software (GraphPad). Binding status is shown in Table 1.

[0353] Binding to CD4+ human T cells in PBMCs

[0354] Human PBMCs were obtained from buffy coat / apheresis filters at the Blood Transfusion Center of La Chaux-de-Fonds / Bern (Switzerland) using Ficoll density gradient separation and frozen on the day of retrieval. 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% nonessential amino acids, 1% sodium pyruvate, and 1% penicillin / streptomycin) and incubated overnight at a concentration of 1 × 10⁶ cells per ml. On the day of the assay, cells were plated at a density of 1 × 10⁵ cells per well in a 96-well round-bottom plate. After centrifugation at 350 g for 3 minutes at 4°C, cells were resuspended in 100 µl of cold FACS buffer containing serial dilutions of anti-CD3 IgG1-LALA antibody, starting at 800 nM and increasing to 3- or 10-fold. Cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer, centrifuged at 350 g for 3 minutes, and resuspended in 100 µl of a mixture of a goat polyclonal anti-human IgG PE secondary antibody (ThermoFisher Scientific, Catalog No. 12-4998-82, 1 / 200) and a co-staining antibody for human CD4-AlexaFluor700 (ThermoFisher Scientific, Catalog No. 56-0048-82, 1 / 100) diluted in FACS buffer for 30 minutes. Cells were then washed twice and resuspended in 100 µl of FACS buffer containing SYTOX Green Dead Cell Stain (ThermoFisher Scientific, Catalog No. S34860) diluted 1 / 2000. Samples were acquired on a CytoFlex instrument (Beckman Coulter). Cells were gated based on size and debris using FSC and SSC sizing, excluding doublets and dead cells. Finally, the geometric mean fluorescence intensity of PE in the CD4+ T cell population was analyzed using FlowJo software. Finally, Prism software (GraphPad) was used to plot the geometric mean fluorescence intensity of PE, as shown in Figure 1A. The binding status is shown in Table 1.

[0355] Screening of anti-CD3 IgG1 LALA clones with T cell activation ability

[0356] To evaluate the potential of humanized anti-CD3 IgG1-LALA to induce T cell activation, additional screening was performed using a T cell activation assay. Serial dilutions of anti-CD3 IgG1-LALA, starting at 200 nM and ranging up to 3 or 10 times, were plated in 96-well plates (TPP, catalog number 92096) with PBS. The plates were then incubated overnight at 4°C. Frozen isolated human T cells were thawed in pre-warmed complete RPMI medium and incubated overnight at a concentration of 1 × 10⁶ cells per ml. On the day of the assay, the plates were washed twice with PBS, and primary T cells were harvested, counted, and plated at a density of 1 × 10⁵ cells per well (100 µl). After incubation at 37°C, 5% CO2 for 48 h, the plates were centrifuged (350 g, 5 min), and the cells were transferred to 96-well round-bottom plates, washed, and resuspended in 100 μl of a mixture of human CD4-PE-eFluor610 (ThermoFisher Scientific, catalog no. 61-0049-42, 1 / 200), CD8-AlexaFluor700 (BioLegend, catalog no. 344724, 1 / 100), CD25-PE, and CD69-PE-Cy7 (ThermoFisher Scientific, catalog nos. 12-0259-42 and 25-0699-42, respectively, 1 / 100) antibodies diluted in FACS buffer and incubated at 4°C for 30 min. The cells were then washed twice and resuspended in 100 µl of FACS buffer containing SYTOX Green Dead Cell Stain (ThermoFisher Scientific, catalog number S34860) at a 1 / 2000 dilution. Samples were acquired on a CytoFlex instrument (Beckman Coulter). Cells were gated based on size and debris based on FSC and SSC scalars, and doublets were excluded. Finally, cells negative for viability staining were gated, and the PE geometric mean fluorescence intensity of the CD4+ population was analyzed using FlowJo software. T cell activation was assessed by gated CD4+ T cells expressing CD69. Finally, the percentage of activated CD69+ cells within the CD4+ T cell count was plotted using Prism software (GraphPad) as shown in Figure 1B. The binding status is shown in Table 1. A total of eight individual experiments were performed.

[0357] Results and Conclusions

[0358] Phage display panning and scFv screening using flow cytometry []

[0359] 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 exhibited specific binding to CD3ε-expressing CHO cells were sequenced, an...

Claims

1. A trispecific antibody binding to human CD3, human BCMA, and human CD38, comprising: a first heavy chain polypeptide comprising: a first heavy chain variable region including CDR1 having the amino acid sequence of SEQ ID NO: 181, CDR2 having the amino acid sequence of SEQ ID NO: 307, and CDR3 having the amino acid sequence of SEQ ID NO: 433; and a second heavy chain variable region including CDR1 having the amino acid sequence of SEQ ID NO: 234, CDR2 having the amino acid sequence of SEQ ID NO: 360, and CDR3 having the amino acid sequence of SEQ ID NO: 486; and a second heavy chain polypeptide comprising: a heavy chain variable region including CDR1 having the amino acid sequence of SEQ ID NO: 239, CDR2 having the amino acid sequence of SEQ ID NO: 365, and CDR3 having the amino acid sequence of SEQ ID NO:

486. The amino acid sequence CDR3 of SEQ ID NO: 721; and three light chain polypeptides, each light chain polypeptide comprising: a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 721, CDR2 having the amino acid sequence of SEQ ID NO: 722, and CDR3 having the amino acid sequence of SEQ ID NO:

723.

2. The three-specific antibody as described in claim 1, wherein, Two of the three light chain polypeptides are covalently linked to the first heavy chain polypeptide via disulfide bonds, and the third light chain polypeptide is covalently linked to the second heavy chain polypeptide via disulfide bonds.

3. The three-specific antibody as described in claim 1, wherein, The first heavy chain polypeptide includes a first heavy chain variable region containing an amino acid sequence having SEQ ID NO:

592.

4. The three-specific antibody as described in claim 1, wherein, The first heavy chain polypeptide includes a second heavy chain variable region containing an amino acid sequence having SEQ ID NO:

591.

5. The three-specific antibody as described in claim 1, wherein, The second heavy chain polypeptide contains a heavy chain variable region which contains an amino acid sequence having SEQ ID NO:

594.

6. The three-specific antibody as described in claim 1, wherein, The first heavy chain polypeptide includes a first heavy chain variable region containing an amino acid sequence having SEQ ID NO: 592, the first heavy chain polypeptide includes a second heavy chain variable region containing an amino acid sequence having SEQ ID NO: 591, and the second heavy chain polypeptide includes a heavy chain variable region containing an amino acid sequence having SEQ ID NO:

594.

7. The three-specific antibody as described in claim 1, wherein, The second heavy chain variable region of the first heavy chain polypeptide is fused to the C-terminus of the first heavy chain variable region of the first heavy chain polypeptide via a peptide linker at the N-terminus.

8. The trispecific antibody as claimed in claim 1, wherein the trispecific antibody comprises a modified Fc domain comprising a first modified IgG1 CH3 domain and a second modified IgG1 CH3 domain.

9. A trispecific antibody binding to human CD3, human BCMA, and human CD38, comprising: a first polypeptide comprising an amino acid sequence having SEQ ID NO: 546; a second polypeptide comprising an amino acid sequence having SEQ ID NO: 547; and a third polypeptide, a fourth polypeptide, and a fifth polypeptide, wherein the third polypeptide, the fourth polypeptide, and the fifth polypeptide each comprise an amino acid sequence having SEQ ID NO:

1.

10. A trispecific antibody as claimed in claim 1, comprising: one heavy chain polypeptide comprising a first modified IgG1 CH3 domain and another heavy chain polypeptide comprising a second modified IgG1 CH3 domain; the first modified IgG1 CH3 domain comprising all substitutions selected from the group consisting of: Q347A, S364K, T366V, K370T, K392Y, F405S, Y407V, K409W, T411N (according to EU designations); and the second modified IgG1 CH3 domain comprising all substitutions selected from the group consisting of: Q347E, Y349A, L351F, S364T, T366V, K370T, T394D, V397L, D399E, F405A, Y407S, K409R, T411R (according to EU designations).

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