Multispecific heavy chain antibodies with modified heavy chain constant regions

Abstract

To provide multispecific, human heavy chain antibodies that have modified heavy chain constant regions that impart advantageous properties.SOLUTION: The invention provides isolated multispecific antibodies comprising: a first heavy chain polypeptide subunit comprising a mutated human IgG4 constant region comprising mutations S228P, F234A, L235A, and T366W; and a second heavy chain polypeptide subunit comprising a mutated human IgG4 constant region comprising mutations S228P, F234A, L235A, T366S, L368A, and Y407V. The invention further provides methods of making the antibodies, compositions such as pharmaceutical compositions, and their use to treat disorders that are characterized by expression of one or more of the binding targets described herein.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of the filing dates of U.S. Provisional Patent Application No. 63 / 017,589, filed on April 29, 2020, and U.S. Provisional Patent Application No. 63 / 108,796, filed on November 2, 2020, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] The present invention relates to multispecific human heavy - chain antibodies (e.g., UniAbs™) having modified heavy - chain constant regions that confer advantageous properties. The present invention further relates to methods of making such antibodies, compositions such as pharmaceutical compositions comprising such antibodies, and their use for treating disorders characterized by the expression of one or more of the binding targets described herein.

Background Art

[0003] Modified Fc Region Advances in protein engineering have led to the successful production and clinical use of multispecific antibodies that have binding affinity for two or more targets. However, due to their heterodimeric nature, appropriate means must be utilized in multispecific antibodies to facilitate the proper pairing of the desired combination of binding sequences and thus the polypeptide subunits. Wang et al., mAbs 10:8, 1226 - 1235 (2018).

[0004] One approach to avoid the problem of mispaired polypeptide subunits is known as "knob-into-hole" (KiH), which aims to force the pairing of two different antibody heavy chains by introducing mutations into the CH2 and / or CH3 domains to modify the contact interface. On one chain, the bulky amino acids are replaced with amino acids having short side chains to create a "hole". Conversely, amino acids with large side chains are introduced into the other heavy chain to create a "knob". By co-expressing these two heavy chains, the more favorable stability of knob-hole pairing results in the observation of heterodimer formation ("knob-hole") at a higher yield than homodimer formation ("hole-hole" or "knob-knob") (Ridgway, J.B., et al, Protein Eng. 9(1996)617-621; and WO96 / 027011).

[0005] This strategy appears to be attractive for achieving the desired heterodimer, but other properties of the resulting multispecific antibody strongly depend on the specific amino acid sequence of the Fc region, i.e., effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, effector function activity can induce the production of cytokines, which can lead to an unwanted "cytokine storm" of inflammatory responses. Gupta et al., Journal of Interferon & Cytokine Research 40:1, 19-23(2019). Thus, in certain situations, it is necessary to reduce or completely eliminate effector functions, for example, to avoid damage or death of immune cells (e.g., T cells) to which the multispecific antibody binds and / or to avoid unwanted cytokine production and the resulting unwanted inflammatory responses.

[0006] Furthermore, the introduction of amino acid modifications into proteins can have significant drawbacks, namely, based on the presence of non-natural sequences, it may induce an immune response in patients against the protein. Therefore, in the development of multispecific antibodies, there is a need to identify sequences that are very similar in general structure to natural antibodies (such as IgA, IgD, IgE, IgG, IgM, etc.), with minimal deviation from the natural sequence, while at the same time successfully incorporating modifications that promote the desired heterodimerization and achieve the reduction or elimination of one or more effector functions.

[0007] To balance these competing requirements, the inventors focused on IgG4 Fc, which is known to have relatively low levels of effector function activity in its natural sequence. Crescioli et al., Curr Allergy Asthma Rep 16:7 (2016). However, despite such apparent advantages, IgG4 is known to undergo a chain exchange reaction in vivo due to its specific hinge region sequence, presenting further complexity in achieving the desired heterodimerization. Labrijn et al., Nature Biotechnology 27, 767 - 71 (2009). Therefore, there is a need for a modified heavy chain constant region sequence that incorporates modifications to achieve the desired heterodimerization and reduce or eliminate effector functions, while at the same time reducing or eliminating the IgG4 chain exchange reaction. The molecules described herein address these and other issues.

[0008] Heavy chain antibody In conventional IgG antibodies, the association of the heavy and light chains is partially due to hydrophobic interactions between the light chain constant region and the CH1 constant domain of the heavy chain. There are also additional residues in the heavy chain framework 2 (FR2) and framework 4 (FR4) regions that contribute to this hydrophobic interaction between the heavy and light chains.

[0009] However, sera of animals of the family Camelidae (suborder Tylopoda, including camels, dromedaries, and llamas) are known to contain a major type of antibody consisting only of paired H chains (heavy-chain-only antibodies or UniAbs™). The UniAbs™ of Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca, and Lama vicugna) have a unique structure consisting of a single variable domain (VHH), a hinge region, and two constant domains (CH2 and CH3), which are highly homologous to the CH2 and CH3 domains of classical antibodies. These UniAbs™ lack the first domain of the constant region (CH1), which is present in the genome but spliced out during mRNA processing. The absence of the CH1 domain explains the absence of a light chain in UniAbs™, since this domain is the anchoring site for the constant domain of the light chain. Such UniAbs™ have evolved naturally to confer their antigen-binding specificity and high affinity by three CDRs derived from conventional antibodies or fragments thereof (Muyldermans, 2001; J Biotechnol 74:277-302, Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish such as sharks have also evolved a unique type of immunoglobulin called IgNAR, which lacks a light-chain polypeptide and consists entirely of heavy chains. The IgNAR molecule can be engineered by molecular engineering to generate the variable domain of a single heavy-chain polypeptide (vNAR) (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003), Nuttall et al. Function and Bioinformatics 55, 187-197 (2004), Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0010] The ability of antibodies consisting only of heavy chains lacking light chains to bind antigens was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185 - 4195). Heavy chain immunoglobulins physically separated from light chains retained 80% of the antigen - binding activity compared to tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781 - 790 showed that antibodies consisting only of heavy chains lacking light chains were produced in mammalian cell cultures by removing the CH1 domain from the recombinant mouse μ gene. The antibodies produced retained VH - binding specificity and had effector functions.

[0011] By immunization, heavy - chain antibodies with high specificity and affinity can be generated against various antigens (van der Linden, R.H., et al. Biochim. Biophys. Acta. 1431, 37 - 46 (1999)), and the VHH portion can be easily cloned and expressed in yeast (Frenken, L.G.J., et al. J. Biotechnol. 78, 11 - 21 (2000)). Their levels of expression, solubility, and stability are significantly higher compared to classical F(ab) or Fv fragments (Ghahroudi, M.A. et al. FEBS Lett. 414, 521 - 526 (1997)).

[0012] Mice in which the λ (lambda) light (L) chain locus and / or the λ and κ (kappa) L chain loci are functionally silenced, as well as antibodies produced by such mice, are described in U.S. Patent Nos. 7,541,513 and 8,367,888. The recombinant production of antibodies of only the heavy chain in mice and rats is reported, for example, in WO2006008548, U.S. Application Publication No. 20100122358, Nguyen et al., 2003, Immunology; 109(1), 93-101, Bruggemann et al., Crit. Rev. Immunol.; 2006, 26(5):377-90, and Zou et al., 2007, J Exp Med; 204(13):3271-3283. The generation of knockout rats by embryonic microinjection of zinc finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Transgenic rodents having soluble heavy chain-only antibodies and heterologous heavy chain loci that produce such antibodies are described in U.S. Patent Nos. 8,883,150 and 9,365,655. CAR-T constructs containing single domain antibodies as binding (targeting) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.

[0013] B cell maturation antigen (BCMA) BCMA, also known as tumor necrosis factor superfamily member 17 (TNFRSF17) (UniProt Q02223), is a cell surface receptor that is exclusively expressed on plasma cells and plasmablasts. BCMA is a receptor for two ligands of the tumor necrosis factor (TNF) superfamily: APRIL (TNFSF13; proliferation-inducing ligand, also known as TALL-2 and TRDL-1; the high-affinity ligand for BCMA) and B cell activating factor (BAFF) (BLyS; TALL-1; THANK; zTNF4; TNFSF20; and also known as D8Ertd387e; the low-affinity ligand for BCMA). APRIL and BAFF are growth factors that bind to BCMA and promote the survival of plasma cells. BCMA is also highly expressed on malignant plasma cells in human multiple myeloma (MM). Antibodies that bind to BCMA are described, for example, in Gras et al., 1995, Int. Immunol. 7:1093-1106, WO200124811, and WO200124812. Anti-BCMA antibodies that cross-react with TACI are described in WO2002 / 066516. Bispecific antibodies against BCMA and CD3 are described, for example, in US2013 / 0156769 A1 and US2015 / 0376287 A1. Anti-BCMA antibody-MMAE or anti-BCMA antibody-MMAF conjugates have been reported to selectively induce the death of multiple myeloma cells (Tai et al., Blood 2014, 123(20):3128-38). Ali et al., Blood 2016, 128(13):1688-700 reported that in a clinical trial (#NCT02215967), chimeric antigen receptor (CAR) T cells targeting BCMA brought about remission of multiple myeloma in human patients.

[0014] PSMA PSMA, also known as prostate-specific membrane antigen and glutamate carboxypeptidase II (UniProt Q04609), is a type II transmembrane protein with N-acetylated-α-linked-acidic-dipeptidase, folate hydrolase, and dipeptidyl peptidase activities. It is encoded by the human FOLH1 gene and consists of a 19-amino acid cytoplasmic domain, a 24-amino acid transmembrane portion, and a 707-amino acid extracellular portion. This protein is enzymatically active as a non-covalent homodimer. PSMA is expressed in prostate epithelial tissue and is upregulated in the neovasculature of prostate cancer and solid tumors. It is also expressed at low levels in healthy tissues such as the brain, kidney, and salivary gland, but its overexpression in malignant prostate tissue makes it an attractive target for the therapeutic treatment of prostate cancer. Additionally, due to its high expression in malignant neovasculature, it may also be suitable for the treatment or imaging diagnosis of solid tumors. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells targeting PSMA have been described for the treatment of metastatic prostate cancer (Hernandez-Hoyos et al 2016, PMID:27406985, DiPippo et al 2014, PMID:25327986, Serganova et al 2016, PMID:28345023). Furthermore, radionuclide complexes specific for PSMA are being investigated for the imaging and treatment of prostate cancer (e.g., Hofman et al., 2018 PMID:29752180).

[0015] CD19 CD19, also known as B lymphocyte surface antigen B4 (UniProt P15391), is a cell surface receptor that is expressed on all human B cells but not on plasma cells. CD19 is a transmembrane protein that recruits cytoplasmic signaling proteins to the membrane and acts within the CD19 / CD21 complex to lower the threshold of the B cell receptor signaling pathway. CD19 has a relatively large cytoplasmic tail of 240 amino acids. The extracellular Ig-like domain is divided into a potential disulfide bond non-Ig-like domain and an N-linked carbohydrate addition site. The cytoplasmic tail contains at least nine tyrosine residues near the C-terminus, some of which have been shown to be phosphorylated. In addition to CD20 and CD22, CD19 is an attractive target for the treatment of B cell malignancies because its expression is restricted to the B cell lineage. Many monoclonal antibodies and antibody-drug conjugates specific for CD19 have been described (e.g., Naddafi et al. 2015, PMC4644525). Furthermore, anti-CD19 chimeric antigen receptor T cells have been approved for the treatment of leukemia (e.g., Sadelain et al. 2017, PMID: 29245005).

Summary of the Invention

[0016] Aspects of the invention include a first heavy chain polypeptide subunit comprising a mutant human IgG4 constant region comprising the mutations S228P, F234A, L235A, and T366W; and a second heavy chain polypeptide subunit comprising a mutant human IgG4 constant region comprising the mutations S228P, F234A, L235A, T366S, L368A, and Y407V, wherein the isolated multispecific antibody comprises the same. In some embodiments, the mutant human IgG4 constant region of the first heavy chain polypeptide subunit or the mutant human IgG4 constant region of the second heavy chain polypeptide subunit lacks the CH1 domain. In some embodiments, the mutant human IgG4 constant region of the first heavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 73 or 55, and the mutant human IgG4 constant region of the second heavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 72 or 54.

[0017] In some embodiments, the multispecific antibody according to the embodiments of the present invention comprises a heavy chain variable domain comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 36, a CDR2 sequence comprising the sequence of SEQ ID NO: 37, and a CDR3 sequence comprising the sequence of SEQ ID NO: 38; and a light chain variable domain comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 39, a CDR2 sequence comprising the sequence of SEQ ID NO: 40, and a CDR3 sequence comprising the sequence of SEQ ID NO: 41, and further comprises a first binding portion having binding specificity for CD3.

[0018] In some embodiments, the CDR1, CDR2, and CDR3 sequences within the heavy chain variable domain of the first binding portion are present in a human VH framework; the CDR1, CDR2, and CDR3 sequences within the light chain variable domain of the first binding portion are present in a human Vκ framework. In some embodiments, the heavy chain variable domain of the first binding portion comprises a sequence having at least 95% identity with SEQ ID NO: 42, and the light chain variable domain of the first binding portion comprises a sequence having at least 95% identity with SEQ ID NO: 43. In some embodiments, the heavy chain variable domain of the first binding portion comprises the sequence of SEQ ID NO: 42; and the light chain variable domain of the first binding portion comprises the sequence of SEQ ID NO: 43.

[0019] In some embodiments, the multispecific antibody according to the embodiments of the present invention further comprises a second binding portion having binding specificity for a protein other than CD3. In some embodiments, the second binding portion comprises a single heavy chain variable region in a monovalent or bivalent configuration. In some embodiments, the first binding portion comprises a light chain polypeptide subunit and a heavy chain polypeptide subunit, and the second binding portion comprises a heavy chain polypeptide subunit. In some embodiments, the light chain polypeptide subunit of the first binding portion comprises a light chain constant domain. In some embodiments, the protein other than CD3 is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, the TAA is B cell maturation antigen (BCMA). In some embodiments, the TAA is CD19. In some embodiments, the TAA is prostate-specific membrane antigen (PSMA).

[0020] Aspects of the invention include pharmaceutical compositions comprising the multispecific antibodies described herein, polynucleotides encoding the multispecific antibodies described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0021] Aspects of the invention include methods for producing the multispecific antibodies described herein, which include growing the cells described herein under conditions that permit expression of the multispecific antibody and isolating the multispecific antibody from the cells.

[0022] Aspects of the invention include methods of treatment comprising administering to a subject in need thereof an effective dose of the multispecific antibody, or pharmaceutical composition, described herein.

[0023] Aspects of the invention include use of the multispecific antibodies described herein in the preparation of a medicament for the treatment of a disease or disorder in a subject in need thereof.

[0024] Aspects of the invention include methods of treatment of a disease or condition characterized by expression of BCMA, comprising administering to a subject in need thereof an effective dose of the multispecific antibody or pharmaceutical composition described herein. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is myeloma. In some embodiments, the myeloma is multiple myeloma.

[0025] Aspects of the invention include methods of treatment of a disease or condition characterized by expression of PSMA, comprising administering to a subject in need thereof an effective dose of the multispecific antibody or pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the cancer is prostate cancer.

[0026] Aspects of the invention include methods of treating a disease or condition characterized by the expression of CD19, which include administering to a subject in need thereof an effective dose of a multispecific antibody or pharmaceutical composition described herein. In some embodiments, the disorder is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the disorder is acute lymphoblastic leukemia (ALL). In some embodiments, the disorder is non-Hodgkin lymphoma (NHL). In some embodiments, the disorder is systemic lupus erythematosus (SLE). In some embodiments, the disorder is rheumatoid arthritis (RA). In some embodiments, the disorder is multiple sclerosis (MS).

[0027] Aspects of the invention include kits for treating a disease or disorder of a subject in need thereof, which include a multispecific antibody or pharmaceutical composition described herein, and instructions for use. In some embodiments, the kit further includes at least one additional reagent. In some embodiments, the at least one additional reagent includes a chemotherapeutic agent.

[0028] Aspects of the invention include a bispecific triple-stranded antibody-like molecule comprising a first polypeptide subunit comprising a light chain variable domain (VL) comprising the sequence of SEQ ID NO: 43; and a light chain constant domain (CL); a second polypeptide subunit comprising a heavy chain variable domain (VH) comprising the sequence of SEQ ID NO: 42; and a heavy chain constant domain (CH) comprising the sequence of SEQ ID NO: 72 or 73; wherein the light chain variable domain and the heavy chain variable domain together form a first binding moiety having binding specificity for CD3; and further comprising a third polypeptide subunit comprising a monovalent or bivalent variable region of only the heavy chain having binding specificity for a protein other than CD3, and a heavy chain constant domain (CH) comprising the sequence of SEQ ID NO: 54 or 55. In some embodiments, the third polypeptide subunit comprises a bivalent variable region of only the heavy chain having binding specificity for BCMA.

[0029] Aspects of the present invention include a first polypeptide subunit comprising the sequence of SEQ ID NO: 49; a second polypeptide subunit comprising the sequence of SEQ ID NO: 56; and a third polypeptide subunit comprising the sequence of SEQ ID NO: 58, and a bispecific triple-stranded antibody-like molecule.

[0030] Aspects of the present invention include a pharmaceutical composition comprising the bispecific triple-stranded antibody-like molecule described herein, a polynucleotide encoding the bispecific triple-stranded antibody-like molecule described herein, a vector comprising such a polynucleotide, and a cell comprising such a vector.

[0031] Aspects of the present invention include a method for producing the bispecific triple-stranded antibody-like molecule described herein, which comprises growing the cells described herein under conditions that permit the expression of the bispecific triple-stranded antibody-like molecule and isolating the bispecific triple-stranded antibody-like molecule from the cells.

[0032] Aspects of the present invention include a method of treatment comprising administering to a subject in need thereof an effective dose of the bispecific triple-stranded antibody-like molecule or pharmaceutical composition described herein.

[0033] Aspects of the present invention include the use of the bispecific triple-stranded antibody-like molecule described herein in the preparation of a medicament for the treatment of a disease or disorder in a subject in need thereof.

[0034] Aspects of the present invention include a method of treatment of a disease or condition characterized by the expression of BCMA, which comprises administering to a subject in need thereof an effective dose of the bispecific triple-stranded antibody-like molecule or pharmaceutical composition described herein. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is myeloma. In some embodiments, the myeloma is multiple myeloma.

[0035] Aspects of the invention include kits for treating a disease or disorder of an individual in need thereof, comprising a bispecific triple-stranded antibody-like molecule or pharmaceutical composition described herein, and instructions for use. In some embodiments, the kit further comprises at least one additional reagent. In some embodiments, the at least one additional reagent comprises a chemotherapeutic agent.

[0036] These aspects, and further aspects, are further described in the remainder of the disclosure, including the examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

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Mode for Carrying Out the Invention

[0038] The practice of the present invention, unless otherwise specified, uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology within the skill of the art. Such techniques are fully described in the literature such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989), “Oligonucleotide Synthesis” (M.J. Gait, ed., 1984), “Animal Cell Culture” (R.I. Freshney, ed., 1987), “Methods in Enzymology” (Academic Press, Inc.), “Current Protocols in Molecular Biology” (F.M. Ausubel et al., eds., 1987, and periodic updates), “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994), “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988), “Phage Display: A Laboratory Manual” (Barbas et al., 2001), Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0039] When a range of values is provided, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, as well as any other stated value or intervening value within the stated range, is understood to be encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges, and are also included herein in accordance with any specific exclusion limits within the stated range. When the stated range includes one or both of its upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the present invention.

[0040] Unless otherwise specified, the antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0041] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0042] All references cited through this disclosure, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0043] I. Definitions "Comprising" means that the recited element is essential to the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claims.

[0044] "Consisting essentially of" means limiting the scope of the described composition or method to certain substances or steps that do not substantially affect the basic and novel characteristics (s) of the present invention.

[0045] "Consisting of" means that any element, step, or component not specified in the claims is excluded from the composition, method, or kit.

[0046] The antibody residues in this specification are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues within the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al. (supra)). The "EU index described in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody mean residue numbering according to the Kabat numbering system. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0047] "Antibody" or "immunoglobulin" refers to a molecule containing at least one heavy chain and one light chain. Since the amino-terminal domains of the heavy and light chains have variable sequences, they are generally called variable region domains or variable heavy chain (VH) domains or variable light chain (VL) domains. The two domains have conventionally associated to form a specific binding region. However, as discussed herein, specific binding can also be obtained with variable sequences of only the heavy chain. Various non-natural configurations of antibodies are known and are used in the art.

[0048] A "functional" or "biologically active" antibody or antigen-binding molecule (including heavy-chain-only antibodies and multispecific (e.g., bispecific) triple-stranded antibody-like molecules (TCAs described herein)) is a molecule that can exhibit one or more of its natural activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule, such as a TCA, can have the ability to specifically bind to an antigen, and that binding can then induce or alter cellular or molecular events such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as a TCA, can also block receptor ligand activation or act as an agonist or antagonist. The ability of an antibody or other binding molecule, such as a TCA, to exhibit one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

[0049] The term "antibody" can refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that contains an antigen-binding site that immunospecifically binds to an antigen of a target of interest or a portion thereof, such targets including, but not limited to, cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass (including subclasses engineered with modified Fc portions that attenuate or enhance effector cell activity). The light chain of the antibody of interest can be a κ light chain (Vκ) or a λ light chain (Vλ). The immunoglobulin can be derived from any species. In one aspect, the immunoglobulin is predominantly of human origin.

[0050] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site and are highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The monoclonal antibodies according to the invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495 and can also be produced, for example, via recombinant protein production methods (see, e.g., U.S. Patent No. 4,816,567).

[0051] The term "variable," when used in connection with antibodies, refers to the fact that the sequences of certain portions of the antibody variable domains vary widely between antibodies and that these sequences are used for the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed throughout the entire antibody variable domain. Instead, it is concentrated in three segments called hypervariable regions in both the light-chain variable domain and the heavy-chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, which mainly adopt a β-sheet conformation and are linked by three hypervariable regions, which form loops that connect to the β-sheet structure and, in some cases, form part of it. The hypervariable regions in each chain are held in close proximity to the hypervariable regions of the other chain by the FRs and contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as the antibody's involvement in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0052] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include amino acid residues derived from "complementary determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues derived from "hypervariable loop" residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain, Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, "CDR" means the complementary determining regions of an antibody as defined in Lefranc, M.P. et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). "Framework region" or "FR" residues are variable domain residues other than the hypervariable region / CDR residues defined herein.

[0053] Exemplary CDR designations are shown herein, and one of ordinary skill in the art will understand that several definitions of CDRs are commonly used, including the most commonly used Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694-700). The Chothia definition is based on the location of structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877-883).Alternative CDR definitions for purposes include, but are not limited to, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657-670, Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes.” J Immunol. 2008;181:6230-6235, Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143, and Padlan et al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139., each of which is specifically incorporated herein by reference.

[0054] The terms "heavy-chain only antibody" and "heavy-chain antibody" are used interchangeably herein and, in the broadest sense, refer to an antibody, or one or more portions of an antibody, for example, one or more arms of an antibody lacking the light chain of a conventional antibody. These terms specifically include, but are not limited to, homodimeric antibodies that include a VH antigen-binding domain and CH2 and CH3 constant domains but lack a CH1 domain, functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs that include a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR), and functional fragments thereof, as well as soluble single-domain antibodies (sUniDabs™). In one embodiment, the heavy-chain only antibody consists of a variable region antigen-binding domain consisting of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In another embodiment, the heavy-chain only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In another embodiment, the heavy-chain only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and a CH2 domain. In a further embodiment, the heavy-chain only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and a CH3 domain. Also included herein are heavy-chain only antibodies in which the CH2 and / or CH3 domains are truncated. In a further embodiment, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain but does not include a hinge region. The heavy-chain only antibody can be in the form of a dimer in which the two heavy chains are disulfide-bonded or, alternatively, covalently or non-covalently bound to each other. The heavy-chain only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as IgM, IgA, IgD, and IgE subclasses are also included herein. In certain embodiments, the heavy-chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype. In one embodiment, the heavy-chain only antibody herein is used as the binding (targeting) domain of a chimeric antigen receptor (CAR).This definition specifically includes only human heavy chain-only antibodies produced by a human immunoglobulin transgenic rat (UniRat™) called UniAbs™. The variable region (VH) of UniAbs™ is called UniDabs™ and is a variable building block that can bind to the Fc region or serum albumin in order to develop novel therapeutic agents that have multispecificity, increased potency, and an extended half-life. Homodimeric UniAbs™ lack a light chain and thus lack a VL domain, so that the antigen is recognized by a single domain, namely the variable domain of the heavy chain of the heavy chain antibody (VH or VHH).

[0055] As used herein, an "intact antibody chain" is an antibody chain that includes a full-length variable region and a full-length constant region (Fc). An intact "conventional" antibody includes an intact light chain and an intact heavy chain, as well as the light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2, and CH3 for secreted IgG. Other isotypes such as IgM or IgA may have different CH domains. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody can have one or more "effector functions," which refers to biological activities attributable to the Fc constant region of the antibody (the native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors. Constant region variants include variants that alter effector properties, binding to Fc receptors, and the like.

[0056] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be provided as different classes. There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and some of these can be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Fc constant domains corresponding to different classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The forms of Ig include forms with or without hinge modifications (Roux et al (1998) J. Immunol. 161:4083-4090, Lund et al (2000) Eur. J. Biochem. 267:7246-7256, US2005 / 0048572, US2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types called κ (kappa) and λ (lambda) based on the amino acid sequence of their constant domains. Antibodies according to embodiments of the present invention can include a κ light chain sequence or a λ light chain sequence.

[0057] A "functional Fc region" possesses the "effector functions" of the native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC, Fc receptor binding, ADCC, ADCP, downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with receptors such as FcγRI, FCγRIIA, FcγRIIB1, FCγRIIB2, FCγRIIIA, FCγRIIIB receptors, and the low affinity FcRn receptor, and can be evaluated using various assays known in the art. A "dead" or "silenced" Fc is an Fc that has been mutated, for example, to retain activity with respect to an extended serum half-life, but does not activate high affinity Fc receptors or has a reduced affinity for Fc receptors.

[0058] The "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Native sequence human Fc regions include, for example, the native sequence human IgG1 Fc region (non-A and A allotypes), the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region, and the native sequence human IgG4 Fc region, as well as their natural variants.

[0059] The "variant Fc region" includes an amino acid sequence different from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions (multiple possible). Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, and preferably about 1 to about 5 amino acid substitutions. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology with them, more preferably at least about 95% homology with them.

[0060] The human IgG4 Fc amino acid sequence (UniProtKB accession number P01861) is shown herein as SEQ ID NO: 45. Silenced IgG1 is described, for example, in Boesch, A.W., et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4): p. 915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0061] Other Fc variants are possible and include, but are not limited to, variants lacking regions capable of forming disulfide bonds, variants with specific amino acid residues removed at the N-terminus of native Fc, or variants with methionine residues added. Thus, in some embodiments, one or more Fc portions of the antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the Fc hinge region can be completely removed. In yet another embodiment, the antibody may contain an Fc variant.

[0062] Furthermore, the Fc variant can be engineered to remove or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to effect complement binding or Fc receptor binding. For example, but not limited to, deletions may be made at complement binding sites such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO97 / 34631 and WO96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0063] In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence containing the T366W mutation, which may optionally be referred to herein as the IgG4 CH3 knob sequence. In some embodiments, the antibody comprises a variant human IgG4 CH3 domain sequence containing the T366S, L368A, and Y407V mutations, which may optionally be referred to herein as the IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner to place a "knob" in the first heavy chain constant region of the first monomer of the antibody dimer and a "hole" in the second heavy chain constant region of the second monomer of the antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pairs of heavy chain polypeptide subunits in the antibody.

[0064] In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising the S228P mutation, F234A mutation, L235A mutation, and T366W mutation (the knob). In some embodiments, the antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising the S228P mutation, F234A mutation, L235A mutation, and T366S mutation, L368A mutation, and Y407V mutation (the hole).

[0065] The term "antibody comprising an Fc region" refers to an antibody that comprises an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering scheme) may be removed, for example, during purification of the antibody or by recombinant manipulation of the nucleic acid encoding the antibody. Thus, antibodies having an Fc region according to the present invention can include antibodies that contain or do not contain K447.

[0066] Aspects of the invention include antibodies that contain variable regions of only heavy chains in a monovalent or bivalent configuration. As used herein, the term "monovalent configuration" with respect to a variable region domain of only a heavy chain means that there is only one variable region domain of only a heavy chain having a single binding site (see the right arm of the antibody in FIG. 1, panel A). In contrast, the term "bivalent configuration" with respect to a variable region domain of only a heavy chain means that there are two variable region domains of only heavy chains (each having a single binding site) that are connected by a linker sequence (see the right arm of the antibody in FIGS. 1, panels B and C). Non-limiting examples of linker sequences are further discussed herein and include, but are not limited to, GS linker sequences of various lengths. When the variable region of only the heavy chain is in a bivalent configuration, each of the two variable region domains of only the heavy chain has a binding affinity for the same antigen or different antigens (e.g., for different epitopes on the same protein, for two different proteins, etc.). However, unless otherwise specified, a variable region of only a heavy chain indicated to be in a "bivalent configuration" is understood to include two identical variable region domains of only heavy chains connected by a linker sequence, in which case each of the two identical variable region domains of only heavy chains has a binding affinity for the same target antigen.

[0067] Aspects of the invention include antibodies having a multispecific configuration, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein constructs are known and used in bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0068] Various methods for producing multivalent artificial antibodies have been developed by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, a first and a second antigen-binding domain on a polypeptide are connected by a polypeptide linker. One non-limiting example of such a polypeptide linker is a GS linker having an amino acid sequence of four glycine residues followed by one serine residue, and this sequence is repeated n times in the GS linker, where n is an integer in the range of 1 to about 10, for example, 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 70) (n = 1) and GGGGSGGGGS (SEQ ID NO: 71) (n = 2). Other suitable linkers can also be used, for example, as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is hereby incorporated by reference in its entirety.

[0069] The term "triple-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which are one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain, comprising an antigen-binding region and at least one CH domain, and having binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion comprising a CH2 and / or CH3 and / or CH4 domain and no CH1 domain, and a heavy-chain-only antibody comprising, consisting essentially of, or consisting of one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, such binding domains being derived from or having sequence identity to the variable region of the heavy or light chain of the antibody. A portion of such a variable region is V H and / or V L gene segments, D and J HA gene segment, or J L It may be encoded by a gene segment. The variable region is a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It may be encoded by a gene segment.

[0070] The TCA-binding compound utilizes a "heavy-chain-only antibody" or "heavy-chain antibody" or "heavy-chain polypeptide", which, as used herein, means a single-chain antibody that contains the heavy-chain constant regions CH2 and / or CH3 and / or CH4 but does not contain the CH1 domain. In one embodiment, the heavy-chain antibody consists of an antigen-binding domain, at least a part of the hinge region, and CH2 and CH3 domains. In another embodiment, the heavy-chain antibody consists of an antigen-binding domain, at least a part of the hinge region, and the CH2 domain. In a further embodiment, the heavy-chain antibody consists of an antigen-binding domain, at least a part of the hinge region, and the CH3 domain. Heavy-chain antibodies with shortened CH2 and / or CH3 domains are also included herein. In a further embodiment, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain but does not contain the hinge region. The heavy-chain-only antibody can be in the form of a dimer in which two heavy chains are disulfide-bonded or covalently or non-covalently bound to each other, and optionally, it can contain an asymmetric interface between one or more CH domains to facilitate proper pairing between polypeptide chains. The heavy-chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses such as IgM, IgA, IgD, and IgE subclasses are also included herein. In certain embodiments, the heavy-chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype or the IgG4 subtype. Non-limiting examples of the TCA-binding compound are described, for example, in WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entirety.

[0071] Heavy-chain antibodies constitute approximately one-fourth of the IgG antibodies produced by camels, such as dromedaries and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but do not have light chains. As a result, the variable antigen-binding part is called the VHH domain, which represents the smallest natural intact antigen-binding site, with a length of only about 120 amino acids (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). By immunization, heavy-chain antibodies with high specificity and affinity can be generated against various antigens (van der Linden, R.H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH part can be easily cloned and expressed in yeast (Frenken, L.G.J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility, and stability are significantly higher compared to classical F(ab) or Fv fragments (Ghahroudi, M.A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in antibodies called VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003), Nuttall et al. Function and Bioinformatics 55, 187-197 (2004), Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0072] The term "CD3" refers to the human CD3 protein multi-subunit complex. The multi-subunit complex of CD3 protein is composed of six characteristic polypeptide chains. These include the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and one CD3ζ chain homodimer (SwissProt 20963), which is associated with the T cell receptor α chain and β chain. The term "CD3" includes, unless otherwise specified, any CD3 variant, isoform, and species homolog that can be expressed on cells (including T cells) that are naturally expressed or transfected with genes or cDNAs encoding their polypeptides.

[0073] The "BCMA×CD3 antibody" is a multispecific heavy-chain-only antibody, for example, a bispecific heavy-chain-only antibody containing two different antigen-binding regions, one of which specifically binds to the antigen BCMA and the other specifically binds to CD3. The "PSMA×CD3 antibody" is a multispecific heavy-chain-only antibody, for example, a bispecific heavy-chain-only antibody containing two different antigen-binding regions, one of which specifically binds to the antigen PSMA and the other specifically binds to CD3. The "CD19×CD3 antibody" is a multispecific heavy-chain-only antibody, for example, a bispecific heavy-chain-only antibody containing two different antigen-binding regions, one of which specifically binds to the antigen CD19 and the other specifically binds to CD3.

[0074] The term "BCMA" as used herein refers to the human B cell maturation antigen, also known as BCMA, CD269, and TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of human BCMA consists of amino acids 1-54 (or 5-51) according to UniProt.

[0075] The terms "anti-BCMA heavy chain only antibody" and "BCMA heavy chain only antibody" are used herein to refer to the heavy chain only antibody as defined above that immunospecifically binds to BCMA.

[0076] As used herein, the term "PSMA" refers to a type II transmembrane protein having N-acetylated-α-linked acidic dipeptidase, folate hydrolase, and dipeptidyl peptidase activities. The term "PSMA" includes PSMA proteins of any human and non-human animal species, specifically including human PSMA and PSMA of non-human mammals.

[0077] As used herein, the term "human PSMA" includes any variant, isoform, and species homolog of human PSMA (UniProt Q04609), regardless of its source or mode of preparation. Thus, "human PSMA" includes human PSMA naturally expressed by cells and PSMA expressed on cells transfected with the human PSMA gene.

[0078] As defined above, the terms "anti-PSMA heavy chain only antibody", "PSMA heavy chain only antibody", "anti-PSMA heavy chain antibody", and "PSMA heavy chain antibody" are used herein interchangeably to refer to the heavy chain only antibody that immunospecifically binds to PSMA, including human PSMA as defined above. This definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals such as transgenic rats or transgenic mice expressing human immunoglobulins, including UniRats (trademark) that produce human anti-PSMA UniAb (trademark) antibodies as defined above.

[0079] As used herein, the terms "CD19" and "cluster of differentiation 19" refer to molecules expressed at all stages of B cell development up to the final differentiation into plasma cells. The term "CD19" includes CD19 proteins of any human and non-human animal species, specifically including human CD19 and CD19 of non-human mammals.

[0080] As used herein, the term "human CD19" includes any variant, isoform and species homolog of human CD19 (UniProt P15391), regardless of its source or mode of preparation. Thus, "human CD19" includes human CD19 as naturally expressed by cells, and CD19 expressed on cells transfected with the human CD19 gene.

[0081] The terms "anti-CD19 heavy chain only antibody", "CD19 heavy chain only antibody", "anti-CD19 heavy chain antibody" and "CD19 heavy chain antibody" are used interchangeably herein and refer to an antibody consisting of only the heavy chain as defined above that immunospecifically binds to CD19, including human CD19 as defined above. This definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals such as transgenic rats or transgenic mice that express human immunoglobulins, including UniRats (TM) that produce human anti-CD19 UniAb (TM) antibodies as defined above.

[0082] "Percent amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity (any conservative substitutions are not considered part of the sequence identity). Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms required to obtain the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the sequence comparison computer program ALIGN-2 is used to generate the amino acid sequence identity values (%).

[0083] An "isolated" antibody is one that has been identified, separated, and / or recovered from the components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified to homogeneity by (1) greater than 95% by weight, most preferably greater than 99% by weight of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain at least a 15-residue N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver stain. Isolated antibodies include in situ antibodies in recombinant cells since at least one component of the antibody's natural environment will not be present. However, typically, isolated antibodies will be prepared by at least one purification step.

[0084] The antibodies of the invention include multispecific antibodies. Multispecific antibodies have multiple binding specificities. The term "multispecific" specifically includes "bispecific" and "trispecific", as well as higher order independent specific binding affinities such as higher order polyepitope specificities, and also includes tetravalent antibodies and antibody fragments. The terms "multispecific antibody", "multispecific heavy chain only antibody", "multispecific heavy chain antibody", and "multispecific UniAb™" are used herein in the broadest sense and encompass all antibodies having multiple binding specificities.

[0085] The multispecific antibodies of the present invention specifically include antibodies that immunospecifically bind to two or more non-overlapping epitopes on BCMA proteins, PSMA proteins, or CD19 proteins such as human BCMA protein, human PSMA protein, or human CD19 protein (i.e., bivalent and bivalent paratopic). The multispecific heavy chain antibodies of the present invention also specifically include antibodies that immunospecifically bind to epitopes on BCMA proteins, PSMA proteins, or CD19 proteins such as human BCMA protein, human PSMA protein, or human CD19 protein, and epitopes on different proteins such as CD3 protein, such as human CD3 (i.e., bivalent and bivalent paratopic). The multispecific heavy chain antibodies of the present invention also specifically include antibodies that immunospecifically bind to two or more non-overlapping or partially overlapping epitopes on BCMA proteins, PSMA proteins, or CD19 proteins such as human BCMA protein, human PSMA protein, or human CD19 protein, and epitopes on different proteins such as CD3 protein, such as human CD3 protein (i.e., bivalent and bivalent paratopic).

[0086] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. This term specifically includes linear epitopes and conformational epitopes.

[0087] "Epitope mapping" is the process of identifying the binding site or epitope of an antibody on a target antigen. An antibody epitope can be a linear epitope or a conformational epitope. A linear epitope is formed by a continuous sequence of amino acids in a protein. A conformational epitope is formed by amino acids whose protein sequences are discontinuous but come together when the protein folds into its three-dimensional structure.

[0088] "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As described above, the present invention specifically includes heavy-chain antibodies having polyepitope specificity, i.e., heavy-chain antibodies that bind to one or more non-overlapping epitopes on BCMA proteins such as human BCMA protein, human PSMA protein, or human CD19 protein, PSMA proteins, or CD19 proteins, as well as heavy-chain antibodies that bind to one or more epitopes on BCMA proteins, PSMA proteins, or CD19 proteins and epitopes on different proteins such as, for example, CD3 protein. The term "non-overlapping epitope(s)" or "non-competing epitope(s)" of an antigen is defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies but not by the other member. Antibody pairs that recognize non-overlapping epitopes, or antigen-binding regions on a multispecific antibody that target the same antigen, do not compete for binding to that antigen and can bind to that antigen simultaneously.

[0089] When two antibodies recognize the same or sterically overlapping epitopes, the antibody binds to the "essentially the same epitope" as the reference antibody. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is the competition assay, which can be configured in various formats using either a labeled antigen or a labeled antibody. Typically, the antigen is immobilized on a 96-well plate, and the ability of the unlabeled antibody to block the binding of the labeled antibody is measured using a radioisotope label or an enzyme label.

[0090] The term "valence" as used herein refers to a specific number of binding sites within an antibody molecule.

[0091] A "monovalent" antibody has one binding site. Thus, a monovalent antibody is also monospecific.

[0092] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent", "trivalent", and "tetravalent" refer to the presence of two, three, and four binding sites, respectively. Thus, the bispecific antibodies according to the invention are at least bivalent and can be trivalent, tetravalent, or multivalent. The bivalent antibodies according to embodiments of the invention can have two binding sites for the same epitope (i.e., bivalent, monospecific), or two different epitopes (i.e., bivalent, bispecific).

[0093] A variety of methods and protein constructs are known and used for preparing bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0094] The term "triple-chain antibody-like molecule" or "TCA" is used herein to refer to an antibody-like molecule that comprises, consists essentially of, or consists of three polypeptide subunits, two of which comprise one heavy chain and one light chain of a monoclonal antibody, or a functional antigen-binding fragment of such an antibody chain, that contain an antigen-binding region and at least one CH domain, and that have binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion that does not contain a CH1 domain and contains CH2 and / or CH3 and / or CH4 domains, and a heavy-chain-only antibody that contains an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen, and such a binding domain is derived from or has sequence identity to the variable region of an antibody heavy or light chain. A portion of such a variable region can be encoded by V H and / or V L gene segments, D and J H gene segments, or J L gene segments. The variable region can be rearranged V H DJ H 、V L DJ H 、VH J L or V L J L may be encoded by a gene segment. The TCA protein utilizes only the heavy chain antibodies as defined above.

[0095] The term "chimeric antigen receptor" or "CAR" is used herein in the broadest sense and refers to a modified receptor that grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) onto a transmembrane domain and an intracellular signaling domain. Typically, the receptor is used to graft the specificity of a monoclonal antibody onto a T cell to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015;108(7):dvj439, and Jackson et al., Nature Reviews Clinical Oncology, 2016;13:370 - 383). CAR-T cells are T cells that have been genetically modified to produce an artificial T cell receptor for use in immunotherapy. In one embodiment, "CAR-T cells" means therapeutic T cells that express a transgene encoding one or more chimeric antigen receptors minimally composed of an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0096] The term "human antibody" is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. The term "human antibody" specifically includes, as defined above, heavy chain only antibodies having human heavy chain variable region sequences produced by transgenic animals such as transgenic rats or mice, particularly UniAbs™ produced by UniRats™.

[0097] The term "chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule containing amino acid sequences derived from at least two different Ig loci, for example, a transgenic antibody containing a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies having a non-human Fc region or an artificial Fc region, and human idiotypes. Such immunoglobulins can be isolated from the animals of the present invention modified to produce such chimeric antibodies.

[0098] As used herein, the term "effector cell" refers to an immune cell involved in the effector phase of an immune response, as contrasted with the recognition and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells such as natural killer cells can induce antibody-dependent cell cytotoxicity (ADCC). For example, monocytes and macrophages expressing FcR are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system, or the binding to antigen-presenting cells. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0099] "Human effector cells" are leukocytes that express receptors such as T cell receptors and FcRs and perform effector functions. Preferably, these cells express at least FcγRIII and perform an ADCC effector function. Examples of human leukocytes mediating ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with NK cells being preferred. Effector cells can be isolated from their natural source, for example, from blood or PBMC, as described herein.

[0100] The term "immune cell" is used herein in the broadest sense and includes, without limitation, cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., T cells including B cells and cytotoxic T cells (CTL)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells, and basophils.

[0101] The "effector function" of an antibody refers to the biological activities attributed to the Fc region of the antibody (either the native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptor, BCR).

[0102] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. NK cells, which are the main cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay as described in U.S. Patent No. 5,500,362 or 5,821,337 may be performed. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells. Alternatively, or in addition, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model, such as the model disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0103] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a homologous antigen. To evaluate complement activation, for example, a CDC assay as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) may be performed.

[0104] "Binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies typically bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind to antigens more quickly and tend to remain bound.

[0105] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in the reaction rate mode. For example, a mouse Fc fusion antigen is loaded onto an anti-mouse Fc sensor and immersed in a well containing an antibody to measure the concentration-dependent association rate (kon). In the final step of immersing the sensor in a well containing only buffer, the dissociation rate (koff) of the antibody is measured. Kd is the ratio of koff / kon. (See Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009 for details).

[0106] As used herein, the terms "treatment", "treating", etc. generally mean obtaining a desired pharmacological and / or physiological effect. Such an effect can be prophylactic in terms of completely or partially preventing the disease or condition, and / or therapeutic in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, including (a) preventing the onset in a subject who may be susceptible to but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., preventing its expression, or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. In the treatment of an ongoing disease, of particular interest are those treatments that stabilize or alleviate the undesirable clinical symptoms of the patient. Such treatment is desirably carried out before the function of the affected tissue is completely lost. Therapeutic agents for a subject may be administered during the symptomatic period of the disease and, in some cases, after the symptomatic period of the disease.

[0107] A "therapeutically effective amount" is intended to mean the amount of an active agent necessary to provide a therapeutic effect in a subject. For example, a "therapeutically effective amount" is an amount that induces, alleviates, or causes improvement in the pathological symptoms associated with a disease, the progression of the disease, or the physiological state, or improves resistance to a disorder.

[0108] As used herein, the term "prostate cancer" refers to a malignant tumor of glandular origin in the prostate.

[0109] The term "characterized by the expression of PSMA" broadly refers to any disease or disorder in which PSMA expression is associated with or involved in one or more pathological processes characteristic of the disease or disorder. Such disorders include, but are not limited to, prostate cancer.

[0110] With respect to the present invention, the terms "B cell neoplasm" or "mature B cell neoplasm" include, but are not limited to, all lymphocytic leukemias and lymphomas, chronic lymphocytic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, precursor B cell lymphoblastic leukemia, hairy cell leukemia, small lymphocytic lymphoma, B cell prolymphocytic lymphoma, B cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B cell lymphoma (DLBCL), multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma tumors such as plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, non-Hodgkin lymphoma, Hodgkin lymphoma, hairy cell leukemia, primary pleural effusion lymphoma, and AIDS-related non-Hodgkin lymphoma.

[0111] The term "characterized by expression of CD19" broadly refers to any disease or disorder in which CD19 expression is associated or involved in one or more pathological processes that are characteristic of the disease or disorder. Such disorders include, but are not limited to, B cell neoplasms.

[0112] The term "characterized by expression of BCMA" broadly refers to any disease or disorder in which BCMA expression is associated or involved in one or more pathological processes that are characteristic of the disease or disorder. Such disorders include, but are not limited to, B cell neoplasms.

[0113] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal being evaluated for and / or being treated for a condition. In one embodiment, the mammal is a human. The terms "subject", "individual", and "patient" include, but are not limited to, an individual having cancer, an individual having an autoimmune disease, an individual having a pathogen infection, etc. The subject can be a human, but also includes other mammals, particularly mammals useful as laboratory models of human diseases, such as mice, rats, etc.

[0114] The term "pharmaceutical preparation" refers to a preparation in a form in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are toxic to an unacceptable degree to the subject to which the preparation is administered. Such a preparation is sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is an excipient that can be administered to a subject mammal in an appropriate amount to provide the active ingredient in an effective dose used.

[0115] A "sterile" preparation is sterile or does not contain or essentially does not contain all viable microorganisms and their spores. A "frozen" preparation is a preparation having a temperature below 0°C.

[0116] A "stable" formulation is a formulation in which the internal protein essentially retains its physical stability and / or chemical stability and / or biological activity during storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity, during storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are outlined, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10:29-90)(1993). Stability can be measured over a selected period at a selected temperature. Stability can be qualitatively and / or quantitatively evaluated in a variety of different ways, including assessment of aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or by visual inspection), cation exchange chromatography, charge heterogeneity assessment using imaging capillary isoelectric focusing (icIEF) or capillary zone electrophoresis, amino-terminal or carboxy-terminal sequence analysis, mass spectrometry, SDS-PAGE analysis comparing reduced intact antibodies, peptide mapping (e.g., trypsin or LYS-C) analysis, evaluation of the biological activity or antigen-binding function of the antibody, etc. Instability can include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., fragmentation of the hinge region), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differences in glycosylation, etc.

[0117] II. Detailed Description

[0118] Anti-BCMA antibody The present invention relates to several families of related antibodies that bind to human BCMA. The variable regions of the antibodies of these families are described in US Patent Publications Nos. US20190352412, US20200157232, and US20200048348, as well as PCT Publications Nos. WO2018237037 and WO2019006072, the disclosures of which are hereby incorporated by reference in their entireties. A non-limiting selection of representative anti-BCMA heavy chain antibody variable domain sequences is shown in Table 1 below. [Table 1]

[0119] The anti-BCMA antibody sequences can be selected from the antibodies provided herein for development and therapeutic methods or other uses including, but not limited to, use as multispecific, e.g., bispecific, antibodies. In some embodiments, bispecific or multispecific antibodies are provided, which can have any of the configurations described herein including, but not limited to, those containing TCA. The bispecific antibody includes at least the heavy chain variable region of an antibody specific for a protein other than BCMA.

[0120] When the protein of the present invention is a bispecific antibody, one binding moiety is specific for human BCMA and the other arm can be specific for a target cell, tumor-associated antigen, target antigen such as an integrin, a pathogen antigen, a checkpoint protein, etc. The target cells specifically include cancer cells of hematological tumors such as B cell tumors, as described below.

[0121] Various formats of bispecific antibodies, including but not limited to single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and multiples thereof, are within the scope of the present invention. The bispecific antibodies herein specifically include T cell bispecific antibodies (anti-BCMA × anti-CD3 antibodies) that bind to BCMA, which is selectively expressed on plasma cells (PC) and multiple myeloma (MM), and CD3. Such antibodies induce potent T cell-mediated cell death of cells carrying BCMA and can be used to treat tumors, particularly hematological tumors such as B cell tumors, as further described herein.

[0122] In a preferred embodiment, the bispecific antibody is a TCA comprising an anti-CD3 VH domain paired with a light chain variable domain (VL) (the VH domain and the VL domain together have a binding affinity for CD3); a heavy chain variable domain of an antibody having a binding affinity for BCMA in a monovalent or bivalent configuration; and a first heavy chain constant region sequence comprising S228P mutation, F234A mutation, L235A mutation, and T366W mutation (knob), and a second heavy chain constant region sequence comprising S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (hole), and a variant human IgG4 Fc domain. This variant, i.e., the modified IgG4 Fc domain, prevents unwanted Fab exchange, reduces the effector function of the antibody, and further promotes the heterodimerization of the heavy chain polypeptide subunits to form a bispecific antibody.

[0123] In some embodiments, the present invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 58.

[0124] In some embodiments, the present invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59.

[0125] In some embodiments, the invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 58, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0126] In some embodiments, the invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0127] In some embodiments, the invention includes a human monoclonal IgG4 bispecific antibody comprising an anti-CD3 heavy chain comprising SEQ ID NO: 56, an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 58.

[0128] In some embodiments, the invention includes a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59.

[0129] In some embodiments, the invention includes a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 58, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0130] In some embodiments, the invention includes a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0131] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 58.

[0132] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 59.

[0133] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the second binding arm does not include a light chain.

[0134] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 59, and the second binding arm does not comprise a light chain.

[0135] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 58.

[0136] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59.

[0137] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 58, and the bispecific antibody does not comprise an anti-BCMA light chain.

[0138] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59, and the bispecific antibody does not comprise an anti-BCMA light chain.

[0139] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising an anti-CD3 heavy chain comprising SEQ ID NO: 56, an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 58.

[0140] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59.

[0141] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 58, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0142] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 56, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 59, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0143] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm includes a first heavy chain and a light chain, the second binding arm includes a bivalent second heavy chain, the first heavy chain includes the amino acid sequence of SEQ ID NO: 56, the light chain includes the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain includes the amino acid sequence of SEQ ID NO: 58.

[0144] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 59.

[0145] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the second binding arm does not include a light chain.

[0146] In some embodiments, the invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 56, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 59, and the second binding arm does not include a light chain.

[0147] In some embodiments, the invention includes a bispecific antibody comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 76.

[0148] In some embodiments, the present invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77.

[0149] In some embodiments, the present invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 76, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0150] In some embodiments, the present invention includes a bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0151] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising an anti-CD3 heavy chain comprising SEQ ID NO: 75, an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 76.

[0152] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77.

[0153] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 76, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0154] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77, wherein the bispecific antibody does not include an anti-BCMA light chain.

[0155] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 76.

[0156] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 77.

[0157] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 76, and the second binding arm does not comprise a light chain.

[0158] In some embodiments, the invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 77, and the second binding arm does not comprise a light chain.

[0159] In some embodiments, the invention comprises a bispecific triple-chain antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 76.

[0160] In some embodiments, the invention comprises a bispecific triple-chain antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77.

[0161] In some embodiments, the invention comprises a bispecific triple-chain antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 76, and the bispecific antibody does not comprise an anti-BCMA light chain.

[0162] In some embodiments, the invention comprises a bispecific triple-chain antibody-like molecule (TCA) comprising (i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, (ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and (iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77, and the bispecific antibody does not comprise an anti-BCMA light chain.

[0163] In some embodiments, the invention comprises a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising an anti-CD3 heavy chain comprising SEQ ID NO: 75, an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 76.

[0164] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising: i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77.

[0165] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising: i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and an anti-BCMA heavy chain comprising SEQ ID NO: 76, wherein the bispecific antibody does not comprise an anti-BCMA light chain.

[0166] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising: i) an anti-CD3 heavy chain comprising SEQ ID NO: 75, ii) an anti-CD3 light chain comprising SEQ ID NO: 49, and iii) an anti-BCMA heavy chain comprising SEQ ID NO: 77, wherein the bispecific antibody does not comprise an anti-BCMA light chain.

[0167] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising: (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 76.

[0168] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, and the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 77.

[0169] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 76, and the second binding arm does not include a light chain.

[0170] In some embodiments, the present invention includes a human monoclonal IgG4 bispecific triple-chain antibody-like molecule (TCA) comprising (i) a first binding arm that binds to human CD3 and (ii) a second binding arm that binds to human BCMA, wherein the first binding arm comprises a first heavy chain and a light chain, the second binding arm comprises a bivalent second heavy chain, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 75, the light chain comprises the amino acid sequence of SEQ ID NO: 49, the bivalent second heavy chain comprises the amino acid sequence of SEQ ID NO: 77, and the second binding arm does not include a light chain.

[0171] Anti-CD19 antibody The present invention provides a family of related antibodies that bind to human CD19. The variable regions of the antibodies of this family are described in PCT Publication No. WO2020018922, the disclosure of which is hereby incorporated by reference in its entirety. The anti-CD19 antibody sequences can be selected from the sequences provided herein for development and therapeutic or other uses, including but not limited to use as multispecific, e.g., bispecific, antibodies. In some embodiments, bispecific or multispecific antibodies are provided, which can have any of the configurations described herein, including but not limited to those containing a TCA. The bispecific antibody includes at least the heavy chain variable region of an antibody specific for a protein other than CD19.

[0172] When the protein of the present invention is a bispecific antibody, one binding moiety is specific for human CD19, while the other arm can be specific for a target cell, tumor-associated antigen, target antigen such as integrin, pathogen antigen, checkpoint protein, etc. Target cells specifically include cancer cells, such as hematological tumors, e.g., B cell tumors, as described below.

[0173] Various forms of bispecific antibodies, including but not limited to single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and multiples thereof, are within the scope of the present invention. Specifically included in the bispecific antibodies herein are T cell bispecific antibodies (anti-CD19 × anti-CD3 antibodies) that bind to CD19 selectively expressed on mature B cells and CD3. Such antibodies can induce potent T cell-mediated cell death of cells expressing CD19 and can be used to treat tumors, particularly hematological tumors such as B cell tumors, as further described herein.

[0174] In a preferred embodiment, the bispecific antibody comprises an anti-CD3 VH domain paired with a light chain variable domain (VL) (the VH domain and the VL domain together have a binding affinity for CD3); a heavy chain variable domain of a monovalent or bivalent antibody having a binding affinity for CD19; and a first heavy chain constant region sequence comprising S228P, F234A, L235A, and T366W mutations (knobs), and a second heavy chain constant region sequence comprising S228P, F234A, L235A, T366S, L368A, and Y407V mutations (holes), and is a TCA comprising a variant human IgG4 Fc domain. This variant, i.e., the modified IgG4 Fc domain, prevents unwanted Fab exchange, reduces the effector function of the antibody, and also promotes heterodimerization of the heavy chain polypeptide subunits to form a bispecific antibody.

[0175] Anti-PSMA antibody The present invention provides a family of related antibodies that bind to human PSMA. Antibodies of this family are exemplified by the heavy chain variable region (VH) sequences provided in SEQ ID NOs: 24-54 shown in Table 2. This family of antibodies provides many advantages that contribute to their utility as clinical therapeutic agents (plural). Since the antibodies include members with various binding affinities, a specific sequence with the desired binding affinity can be selected.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0176] In a preferred embodiment, the bispecific antibody comprises an anti-CD3 VH domain paired with a light chain variable domain (VL) (the VH domain and the VL domain together have a binding affinity for CD3); a heavy chain variable domain of a monovalent or bivalent only-for-PSMA-binding affinity antibody; and a first heavy chain constant region sequence containing S228P, F234A, L235A, and T366W mutations (knob), and a second heavy chain constant region sequence containing S228P, F234A, L235A, T366S, L368A, and Y407V mutations (hole), and is a TCA comprising a variant human IgG4 Fc domain. This variant, i.e., the modified IgG4 Fc domain, prevents unwanted Fab exchange, reduces the effector function of the antibody, and also promotes the heterodimerization of the heavy chain polypeptide subunits to form the bispecific antibody.

[0177] CD3×target protein triple-chain antibody-like molecule (TCA) In some embodiments, bispecific or multispecific antibodies are provided, which can have any of the configurations described herein, including but not limited to bispecific triple-chain antibody-like molecules. In some embodiments, the multispecific antibody can comprise a heavy chain / light chain pair having binding specificity for a first antigen (e.g., CD3), and a heavy chain derived from a heavy-chain-only antibody. In certain embodiments, the heavy chain derived from the heavy-chain-only antibody does not contain a CH1 domain and contains an Fc portion containing CH2 and / or CH3 and / or CH4 domains. In one particular embodiment, the bispecific antibody comprises a heavy chain / light chain pair having binding specificity for an antigen on an effector cell (e.g., the CD3 protein on a T cell), and a heavy chain derived from a heavy-chain-only antibody containing an antigen-binding domain having binding specificity for BCMA, PSMA, or CD19.

[0178] In a preferred embodiment, the bispecific antibody comprises an anti-CD3 VH domain paired with a light chain variable domain (VL) (the VH domain and the VL domain together have a binding affinity for CD3); a heavy chain variable domain of a heavy chain-only antibody having a binding affinity for BCMA, PSMA, or CD19; a first heavy chain constant region sequence comprising S228P, F234A, L235A, and T366W mutations (knob), and a second heavy chain constant region sequence comprising S228P, F234A, L235A, T366S, L368A, and Y407V mutations (hole), and is a TCA comprising a variant human IgG4 Fc domain. This variant, i.e., the modified IgG4 Fc domain, prevents unwanted Fab exchange, reduces the effector function of the antibody, and further promotes heterodimerization of the heavy chain polypeptide subunits to form the bispecific antibody.

[0179] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In certain embodiments, the light chain is a fixed light chain. In some embodiments, the CD3-binding VH domain comprises the CDR1 sequence of SEQ ID NO: 36, the CDR2 sequence of SEQ ID NO: 37, and the CDR3 sequence of SEQ ID NO: 38 in a human VH framework. In some embodiments, the fixed light chain comprises the CDR1 sequence of SEQ ID NO: 39, the CDR2 sequence of SEQ ID NO: 40, and the CDR3 sequence of SEQ ID NO: 41 in a human VL framework. Together, the CD3-binding VH domain and the light chain variable domain have binding affinity for CD3. In some embodiments, the CD3-binding VH domain comprises the heavy chain variable region sequence of SEQ ID NO: 42. In some embodiments, the CD3-binding VH domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 42. In some embodiments, the fixed light chain comprises the light chain variable region sequence of SEQ ID NO: 43. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to the heavy chain variable region sequence of SEQ ID NO: 43.

[0180] The multispecific antibody comprising the CD3-binding VH domain and the light chain variable domain described above has advantageous properties, for example, as described in PCT Publication No. WO2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the multispecific antibodies and antigen-binding domains described herein having binding affinity for BCMA, PSMA, or CD19, in combination with the CD3-binding domain and the fixed light chain domain described herein, can generate a multispecific antibody having binding affinity for one or more BCMA epitopes, PSMA epitopes, or CD19 epitopes and CD3. [Table 3] [Table 4]

Table 5

Table 6-1

Table 6-2

Table 6-3

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

[0181] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the configurations described herein, including but not limited to bispecific triple-stranded antibody-like molecules. In some embodiments, the bispecific antibody may comprise at least one heavy chain variable region having binding specificity for BCMA, PSMA, or CD19, and at least one heavy chain variable region having binding specificity for a different protein, such as CD3. In some embodiments, the bispecific antibody is in a monovalent or bivalent configuration and comprises a heavy chain / light chain pair having binding specificity for a first antigen, and a heavy chain-only antibody-derived heavy chain comprising an Fc portion that does not include a CH1 domain but includes a CH2 and / or CH3 and / or CH4 domain, and an antigen-binding domain that binds to an epitope of a second antigen or a different epitope of the first antigen. In one particular embodiment, the bispecific antibody is in a monovalent or bivalent configuration and comprises a heavy chain / light chain pair having binding specificity for an antigen on an effector cell (e.g., the CD3 protein on a T cell), and a heavy chain-only antibody-derived heavy chain comprising an antigen-binding domain having binding specificity for BCMA, PSMA, or CD19.

[0182] In some embodiments where the antibody of the invention is a bispecific antibody, one arm (one binding portion or one binding unit) of the antibody is specific for human BCMA, human PSMA, or human CD19, and the other arm may be specific for a target cell, a tumor-associated antigen, a targeted antigen, such as an integrin, a pathogen antigen, a checkpoint protein, etc. Target cells specifically include, but are not limited to, cancer cells including cells derived from solid tumors, such as prostate tumors, as described below. In some embodiments, one arm (one binding portion, or one binding unit) of the antibody is specific for human BCMA, human PSMA, or human CD19, and the other arm is specific for CD3.

[0183] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 43 linked to the sequence of SEQ ID NO: 48, an anti-CD3 heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 44, 45, 46, 47, 50, 51, 52, 53, 56 or 57, and an anti-BCMA heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 58, 59 or 60 in a monovalent or divalent configuration linked to any one of the sequences of SEQ ID NO: 75, 76, 77, 78, 84 or 85. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 58, 59 or 60. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 58. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 58. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 59. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 59. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 60. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 60.

[0184] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 43 linked to the sequence of SEQ ID NO: 48, an anti-CD3 heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 44, 45, 46, 47, 50, 51, 52, 53, 56 or 57, and an anti-PSMA heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 61, 62, 63, 64, 65 or 66. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 61. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 61. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 62. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 62. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 63. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 63. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 64. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 64. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 65.In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 65. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 66. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 66.

[0185] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide comprising the sequence of SEQ ID NO: 43 linked to the sequence of SEQ ID NO: 48, an anti-CD3 heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 44, 45, 46, 47, 50, 51, 52, 53, 56 or 57, and an anti-CD19 heavy chain polypeptide comprising any one of the sequences of SEQ ID NO: 67, 68 or 69. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 67. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 67. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 68. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 68. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide comprising SEQ ID NO: 49, a second polypeptide comprising SEQ ID NO: 56, and a third polypeptide comprising SEQ ID NO: 69. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide consisting of SEQ ID NO: 49, a second polypeptide consisting of SEQ ID NO: 56, and a third polypeptide consisting of SEQ ID NO: 69.

[0186] Although not limited thereto, various forms of multispecific antibodies including single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and multiples thereof are within the scope of the present invention. The multispecific antibodies herein specifically include T cell multispecific (e.g., bispecific) antibodies that bind to BCMA, PSMA, or 19, and CD3 (anti-BCMA × anti-CD3 antibody, anti-PSMA × anti-CD3 antibody, anti-CD19 × anti-CD3 antibody), and a first heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, and T366W mutation (knob), and a second heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (hole), and a variant human IgG4 Fc domain. This variant, i.e., the modified IgG4 Fc domain, prevents unwanted Fab exchange, reduces the effector function of the antibody, and promotes heterodimerization of the heavy chain polypeptide subunits to form a bispecific antibody. Such antibodies each induce potent T cell-mediated cell death of cells expressing BCMA, PSMA, or CD19.

[0187] Preparation of Antibodies The multispecific antibodies of the present invention can be prepared by methods known in the art. In a preferred embodiment, the heavy chain antibodies herein are produced by transgenic animals, preferably rats, in which the endogenous immunoglobulin genes have been knocked out or inactivated. In a preferred embodiment, the heavy chain antibodies herein are produced by UniRat™. UniRat™ has the endogenous immunoglobulin genes silenced and uses the human immunoglobulin heavy chain locus to express a diverse and naturally optimized repertoire of fully human HCAb. The endogenous immunoglobulin loci of rats can be knocked out or silenced using various techniques, but in UniRat™, zinc finger (endo) nuclease (ZNF) technology is used to inactivate the endogenous rat heavy chain J locus, the light chain Cκ locus, and the light chain Cλ locus. ZNF constructs for microinjection into oocytes can generate IgH and IgL knockout (KO) lines. For details, see, for example, Geurts et al., 2009, Science 325:433. The characterization of Ig heavy chain knockout rats has been reported by Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. The advantage of ZNF technology is that non-homologous end joining for silencing genes or loci via deletions of up to several kb can also provide target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). Human heavy chain antibodies produced by UniRat™ are called UniAbs™ and can bind to epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity, and small size are ideal for single and multispecific applications.

[0188] In addition to UniAbs (trademark), specifically, heavy-chain only antibodies lacking the VHH frameworks and mutations of camels, as well as their functional VH regions, are included herein. Such heavy-chain only antibodies can be produced, for example, in transgenic rats or mice containing a fully human heavy-chain only locus, as described in WO2006 / 008548, but other transgenic mammals such as rabbits, guinea pigs, rats, etc. can also be used, with rats and mice being preferred. Heavy-chain only antibodies containing VHH or VH functional fragments can also be produced by recombinant DNA techniques, for example, by expressing the encoded nucleic acid in a suitable eukaryotic or prokaryotic host including mammalian cells (e.g., CHO cells), E. coli or yeast.

[0189] The domains of heavy-chain only antibodies combine the advantages of antibodies and small molecule drugs, can be monovalent or multivalent, have low toxicity, and are cost-effective for production. Due to their small size, these domains are easy to administer, including oral or topical administration, are characterized by high stability including gastrointestinal stability, and their half-lives can be adjusted according to the desired use or efficacy. Furthermore, the VH and VHH domains of HCAb can be produced in a cost-effective manner.

[0190] In certain embodiments, the heavy chain antibodies of the invention, including UniAbs™, have the natural amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) replaced with a different amino acid residue, whereby a hydrophobic patch that includes or is associated with the natural amino acid residue at that position and is exposed on the surface can be disrupted. Such hydrophobic patches are normally buried at the interface with the light chain constant region of the antibody, but in HCAbs are exposed on the surface and are used, at least in part, for undesirable aggregation of the HCAb and association with the light chain. The substituted amino acid residue is preferably charged, more preferably positively charged such as lysine (Lys, K), arginine (Arg, R) or histidine (His, H), preferably arginine (R). In a preferred embodiment, the heavy chain only antibodies derived from transgenic animals contain a mutation from Trp to Arg at position 101. The resulting HCAb preferably has high antigen binding affinity and solubility under physiological conditions without aggregation.

[0191] As part of the invention, human heavy chain antibodies (UniAbs™) having unique sequences derived from UniRat™ animals that bind to human CD3, BCMA, PSMA, or CD19 in ELISA protein and cell binding assays were identified. The identified heavy chain variable region (VH) sequences (see, e.g., Tables 1 and 2) were positive for protein binding and / or binding to cells expressing the target protein (e.g., CD3, BCMA, PSMA, or CD19) and negative for binding to cells that do not express the target protein.

[0192] Heavy chain antibodies that bind to non-overlapping epitopes on a target protein, such as UniAbs™, can be identified by competitive binding assays such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between a known antibody that binds to a target antigen and the antibody of interest can be used. By using this approach, a set of antibodies can be classified into those that compete and those that do not compete with a reference antibody. Non-competing antibodies are identified as those that bind to a distinct epitope that does not overlap with the epitope to which the reference antibody binds. In many cases, one antibody is immobilized, an antigen is bound, and a second labeled (e.g., biotinylated) antibody is tested in an ELISA assay for its ability to bind to the captured antigen. This can also be performed using surface plasmon resonance (SPR) platforms such as the ProteOn XPR36 (BioRad, Inc.), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and the MX96 SPR Imager (Ibis Technologies B.V.), as well as biolayer interferometry platforms such as the Octet Red384 and Octet HTX (ForteBio, Pall Inc.). For details, see the examples in this specification.

[0193] Generally, an antibody "competes" with a reference antibody if it causes a decrease of about 15-100% in the binding of the reference antibody to the target antigen, as measured by standard techniques such as the competitive binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0194] Pharmaceutical Compositions, Uses, and Methods of Treatment Providing a pharmaceutical composition comprising one or more multispecific binding compounds of the present invention admixed with a suitable pharmaceutically acceptable carrier is another aspect of the present invention. Pharmaceutically acceptable carriers as used herein are exemplified by, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0195] In one embodiment, the pharmaceutical composition comprises a heavy chain antibody (e.g., UniAb™) that binds to a target protein (e.g., CD3, BCMA, PSMA, or CD19). In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) having binding specificity for two or more non-overlapping epitopes on a target protein (e.g., CD3, BCMA, PSMA, or CD19). In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb™) having binding specificity for a target protein (e.g., CD3, BCMA, PSMA, or CD19) and binding specificity for a binding target on an effector cell (e.g., a binding target on a T cell, e.g., the CD3 protein on a T cell).

[0196] The pharmaceutical compositions of the antibodies used according to the present invention are prepared for storage, for example, in the form of lyophilized formulations or aqueous solutions, by mixing a protein having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (see, e.g., Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosages and concentrations employed, and includes buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0197] The pharmaceutical composition for oral administration is preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., the dosage for a single administration). The formulation depends on the selected route of administration. The antibodies herein can be administered by intravenous injection or infusion, or by subcutaneous administration. In the case of administration by injection, the antibodies herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can contain a carrier, excipient, or stabilizer as described above. Alternatively, the antibodies can be in lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, prior to use.

[0198] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy-chain antibodies including the UniAbs™ of the present invention. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689.

[0199] Method of Use The heavy-chain only antibodies, bispecific antibodies, and pharmaceutical compositions described herein can be used for the treatment of diseases and conditions characterized by the expression of a target protein (e.g., CD3, BCMA, PSMA, or CD19), including but not limited to the diseases and conditions further described herein.

[0200] The pharmaceutical compositions herein containing anti-BCMA antibodies can be used for the treatment of B-cell related diseases including B-cell and plasma cell malignancies and autoimmune diseases characterized by the expression or overexpression of BCMA.

[0201] Such B cell-related disorders include, but are not limited to, plasmacytoma, Hodgkin lymphoma, follicular lymphoma, small non-cleaved cell lymphoma, endemic Burkitt lymphoma, sporadic Burkitt lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodular monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell type lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, potentially malignant B cell proliferation, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immune regulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves disease, Wegener granulomatosis, polyarteritis nodosa, Sjogren syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-related amyloidosis, or monoclonal immunoglobulinemia, including B cell and plasma cell malignancies and autoimmune diseases.

[0202] Plasma cell disorders characterized by the expression of BCMA include multiple myeloma (MM). MM is a B cell malignancy characterized by the monoclonal proliferation and accumulation of abnormal plasma cells in the bone marrow compartment. Current treatments for MM often induce remission, but almost all patients ultimately relapse and die. There is substantial evidence that myeloma cells are removed via the immune system in the setting of allogeneic hematopoietic stem cell transplantation; however, the toxicity of this approach is high and few patients are cured. Some monoclonal antibodies have shown promise in the treatment of MM in preclinical studies and early clinical trials, but the consistent clinical efficacy of monoclonal antibody therapy for MM has not been ultimately proven. Thus, novel therapies, including immunotherapies for MM, are greatly needed (see, e.g., Carpenter et al., Clin Cancer Res 2013, 19(8):2048-2060).

[0203] Overexpression or activation of BCMA by APRIL, a proliferation-inducing ligand, is known to promote the progression of human multiple myeloma (MM) in vivo. BCMA has also been shown to promote the in vivo growth of xenograft MM cells with p53 mutations in mice. Since the activity of the APRIL / BCMA pathway plays a central role in the pathogenesis and drug resistance of MM through bidirectional interactions between tumor cells and the bone marrow microenvironment that supports them, BCMA has been identified as a therapeutic target for MM. For further details, see, for example, Yu-Tsu Tai et al., Blood 2016;127(25):3225-3236.

[0204] Another B cell disorder in which plasma cells, i.e., those expressing BCMA, are involved is systemic lupus erythematosus (SLE), also known as lupus. SLE is a systemic autoimmune disease that can affect all parts of the body and typically involves the immune system attacking the body's own cells and tissues, causing chronic inflammation and tissue damage. This is a type III hypersensitivity reaction in which antibody-immune complexes precipitate and trigger further immune responses (Inaki & Lee, Nat Rev Rheumatol 2010;6:326-337).

[0205] The anti-BCMA heavy chain-only antibody (UniAb) of the present invention can be used to develop therapeutic agents for the treatment of MM, SLE, and other B cell or plasma cell disorders characterized by BCMA expression, such as those listed above. In particular, the anti-BCMA heavy chain-only antibody (UniAb) of the present invention is a candidate for treating MM alone or in combination with other MM treatments.

[0206] PSMA is a type II transmembrane protein that is expressed in prostate epithelial tissue and upregulated in the neovasculature of prostate cancer and solid tumors. It is also expressed at low levels in healthy tissues such as the brain, kidney, and salivary gland, but its overexpression in malignant prostate tissue makes it an attractive target for therapeutic treatment of prostate cancer. Moreover, due to its high expression in malignant neovasculature, it may also be suitable for the treatment or imaging diagnosis of solid tumors. Monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells targeting PSMA have been described for the treatment of metastatic prostate cancer (Hernandez-Hoyos et al 2016, PMID:27406985, DiPippo et al 2014, PMID:25327986, Serganova et al 2016, PMID:28345023). Furthermore, radionuclide complexes specific for PSMA are being investigated for prostate cancer imaging and treatment (e.g., Hofman et al., 2018 PMID:29752180).

[0207] In one aspect, the PSMA heavy chain antibodies (e.g., UniAbs™) and pharmaceutical compositions herein can be used to treat disorders characterized by the expression of PSMA, including but not limited to, prostate cancer and solid tumors.

[0208] CD19 is a cell surface receptor expressed on all human B cells but not on plasma cells. CD19 has a relatively large cytoplasmic tail of 240 amino acids. The extracellular Ig-like domain is divided by a potential disulfide bond non-Ig-like domain and an N-linked carbohydrate addition site. The cytoplasmic tail contains at least nine tyrosine residues near the C-terminus, some of which have been shown to be phosphorylated. In addition to CD20 and CD22, CD19 is an attractive target for therapeutic treatment of B cell malignancies because its expression is restricted to the B cell lineage. CD19 is a promising target for antibody-based therapies because its expression has been observed in many hematological malignancies.

[0209] In one aspect, the CD19 heavy chain antibodies (e.g., UniAbs™) and pharmaceutical compositions of the present specification are used to treat hematological malignancies characterized by the expression of CD19, including but not limited to diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia (CLL), and B-cell acute lymphoblastic leukemia (ALL).

[0210] Diffuse large B-cell lymphoma (DLBCL or DLBL) is the most common form of non-Hodgkin lymphoma in adults (Blood 1997 89(11):3909-18), with an estimated annual incidence of 7-8 cases per 100,000 people in the United States and the United Kingdom. It is characterized as a progressive cancer that can occur in virtually any part of the body. The cause of DLBCL is not fully understood and can arise from the malignant transformation of not only normal B cells but also other types of lymphoma or leukemia cells. Treatment approaches generally include chemotherapy and radiotherapy, and the average overall 5-year survival rate in adults is approximately 58%. Although some monoclonal antibodies have been shown to be promising for the treatment of DLBCL, consistent clinical efficacy has not yet been ultimately proven. Therefore, novel treatment methods for DLBCL, including immunotherapy, are highly needed.

[0211] In another aspect, the CD19 heavy chain antibodies (e.g., UniAbs™) and pharmaceutical compositions of the present specification are used to treat autoimmune disorders characterized by pathogenic B cells expressing CD19, including but not limited to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).

[0212] The effective dosage of the composition of the present invention for treating a disease varies widely depending on many different factors including the means of administration, the target site, the physiological state of the patient, whether the patient is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is human, but non-human mammals such as companion animals like dogs, cats, horses, etc., and experimental mammals such as rabbits, mice, rats, etc. can also be treated. The treatment dosage can be gradually increased to optimize safety and effectiveness.

[0213] Dosage levels can be readily determined by those skilled in the art and can be varied as needed, for example, to vary the response of the subject to the treatment. The amount of active ingredient that can be combined with a carrier substance to produce a single dosage form varies depending on the host to be treated and the particular mode of administration. Generally, a unit dosage form contains from about 1 mg to about 500 mg of the active ingredient.

[0214] In some embodiments, the therapeutic dosage of the drug can range from about 0.0001 to 100 mg / kg of the host's body weight, more generally from 0.01 to 5 mg / kg. For example, the dosage can be within the range of 1 mg / kg body weight or 10 mg / kg body weight or 1 - 10 mg / kg. Exemplary treatment regimens involve administration once every two weeks or once a month or once every 3 - 6 months. The therapeutic substance of the present invention is usually administered in multiple doses. The interval between single doses can be weekly, monthly or annually. The interval can also be irregular as specified by measuring the blood level of the therapeutic substance in the patient. Alternatively, the therapeutic substance of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency vary widely depending on the half-life of the polypeptide in the patient.

[0215] Generally, the compositions are prepared as either liquid solutions or suspensions for injection, and solid forms suitable for dissolving or suspending in a liquid vehicle prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, either directly or after reconstitution of solid (e.g., lyophilized) compositions. The formulations can also, as described above, be emulsified or encapsulated in liposomes, or microparticles such as polylactide, polyglycolide, or copolymers, to enhance the adjuvant effect. Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injections or implant formulations that can be formulated in such a way as to permit sustained or pulsed release of the active ingredient. Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with the regulations of all Good Manufacturing Practice (GMP) standards of the United States Food and Drug Administration.

[0216] The toxicity of the antibodies and antibody constructs described herein can be determined by standard pharmacological procedures in cell culture or experimental animals, for example, by measuring the LD50 (lethal dose for 50% of the population) or LD100 (lethal dose for 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal experiments can be used to formulate a dosage range that is not toxic for use in humans. The dosage of the antibodies described herein preferably lies within a range of circulating concentrations that includes an effective dosage with little or no toxicity. The dosage can vary widely within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition.

[0217] The composition for administration will generally comprise an antibody or other agent (e.g., another abrasive agent) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional well-known sterilization techniques. The compositions can contain pharmaceutically acceptable adjunct substances (pH adjusters and buffers, toxicity adjusters, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.) as required to approximate physiological conditions. The concentration of the active agent in these formulations can vary widely and is selected mainly based on, for example, the volume of liquid, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the patient (e.g., Remington’s Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0218] Kits containing the active agent of the present invention, its formulations, and instructions for use are also within the scope of the present invention. The kit can further include at least one additional reagent, such as a chemotherapeutic agent, etc. The kit usually includes a label indicating the purpose of use of the contents of the kit. As used herein, the term “label” includes any written or recorded matter supplied on or with the kit or otherwise associated with the kit.

[0219] It will be apparent to those skilled in the art that the present invention, fully described herein, can be subjected to various changes and modifications without departing from the spirit or scope of the present invention.

Examples

[0220] Example 1: Formation of Heterodimers Heterodimer formation was analyzed by non-reducing and reducing SDS-PAGE analysis to determine whether antibodies according to embodiments of the invention, including various mutations in the hinge and Fc regions, and knob-in-hole mutations, could be successfully expressed and assembled into the desired heterodimer combinations. To test this, antibody constructs were expressed in recombinant CHO cell culture. The harvested cell culture was then purified by protein A affinity chromatography and the various antibody fragments produced were analyzed. The protein A elution pool was then analyzed on reducing and non-reducing gels to visualize the different species.

[0221] The results of these analyses are shown in panels A and B of FIG. 2, panels A and B of FIG. 3, and FIG. 24, showing that the percentage of heterodimer formation of antibody species containing knob-in-hole mutations is excellent even in the presence of effector function silencing mutations (F234A, L235A) and Fab arm exchange prevention mutations (S228P) in the heavy chain sequence. As shown in FIG. 25, the purified CD19 constructs were evaluated to analyze the percentage of high molecular weight (HMW) and low molecular weight (LMW) species, as well as the percentage of monomers.

[0222] Example 2: Fcγ Receptor Binding by Biolayer Interferometry (BLI) Fcγ receptor-IgG interactions were analyzed on the Octet platform using a Ni-NTA biosensor (ForteBio). The Ni-NTA biosensor has QIAGEN's Tris-NTA filled with nickel (Ni2+) immobilized at the tip. Ni-NTA binds to the HIS tag attached to the recombinant protein. In this format, the Fcγ receptor protein is loaded as a ligand onto the biosensor, followed by binding to IgG. Antibodies according to embodiments of the invention were investigated to analyze the degree of interaction between their Fc regions and the Fcγ receptor protein immobilized on the biosensor.

[0223] Here, the Fcγ receptor was human Fcγ receptor I / CD64 (Acro Biosystems). The antibody concentrations tested included a two-fold serial dilution series from 100 nM to 1.6 nM. The results of these studies are shown in Panels A - D of Figure 4, Panels A - E of Figure 5, Panels A - D of Figure 6, Panels A - E of Figure 7, and Panels A - D of Figure 26, and the results indicate that the binding of the silenced Fc receptor antibody to human FcγR1 is significantly inhibited even when the knob-in-hole mutation and Fab arm exchange mutation are present in the Fc region.

[0224] Specifically, Panel A of Figure 4 shows the results of a bispecific CD3×BCMA (monovalent) IgG1 antibody that does not contain the KiH mutation or the silencing mutation. The data indicate that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Panel B of Figure 4 shows the results of the same bispecific antibody used in Panel A, but here the CH2 domain contains the silencing mutation. These results indicate that the presence of the silencing mutation significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor. Panel C of Figure 4 shows the results of a bispecific CD3×BCMA (monovalent) IgG1 antibody that contains the KiH mutation but not the silencing mutation. These data indicate that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a very similar manner to that observed for the antibody in Panel A. Panel D of Figure 4 shows the results of the same bispecific antibody used in Panel C, but here the CH2 domain contains the silencing mutation. These results indicate that even when the KiH mutation is present, the presence of the silencing mutation significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor.

[0225] Panel A of Figure 5 shows the results of a bispecific CD3×BCMA (monovalent) IgG4 antibody that does not contain a KiH mutation or a silencing mutation but contains an S228P mutation to prevent Fab arm exchange. The data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Panel B of Figure 5 shows the results of the same bispecific antibody used in Panel A, but here the CH2 domain contains silencing mutations (F234A, L235A). These results show that the presence of the silencing mutations significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even in the presence of the S228P mutation. Panel C of Figure 5 shows the results of a bispecific CD3×BCMA (monovalent) IgG4 antibody that contains a KiH mutation and an S228P mutation but no silencing mutations. These data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a very similar manner to that observed for the antibody in Panel A. Panel D of Figure 5 shows the results of the same bispecific antibody used in Panel C, but here, in addition to the S228P and KiH mutations, the CH2 domain contains silencing mutations (F234A, L235A). These results show that the presence of the silencing mutations significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even when the S228P and KiH mutations are included. Panel E shows the results of a bispecific CD3×BCMA (bivalent) IgG4 antibody that contains an S228P mutation, silencing mutations F234A and L235A, and a KiH mutation in the CH3 domain. These data show that the interaction between this antibody and the Fcγ receptor immobilized on the biosensor was significantly reduced, even in the presence of the S228P and KiH mutations.

[0226] Panel A of Figure 6 shows the results of a bispecific CD3×PSMA (monovalent) IgG1 antibody that does not contain a KiH mutation or a silencing mutation. The data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Panel B of Figure 6 shows the results of the same bispecific antibody used in Panel A, but here the CH2 domain contains a silencing mutation. These results show that the presence of the silencing mutation significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor. Panel C of Figure 6 shows the results of a bispecific CD3×PSMA (monovalent) IgG1 antibody that contains a KiH mutation but does not contain a silencing mutation. These data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a very similar manner to that observed for the antibody in Panel A. Panel D of Figure 6 shows the results of the same bispecific antibody used in Panel C, but here the CH2 domain contains a silencing mutation. These results show that the presence of the silencing mutation significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even when the KiH mutation is present.

[0227] Panel A of Figure 7 shows the results of a bispecific CD3×PSMA (monovalent) IgG4 antibody that does not contain a KiH mutation or a silencing mutation but contains an S228P mutation to prevent Fab arm exchange. The data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Panel B of Figure 7 shows the results of the same bispecific antibody used in Panel A, but here the CH2 domain contains silencing mutations (F234A, L235A). These results show that the presence of the silencing mutations significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even in the presence of the S228P mutation. Panel C of Figure 7 shows the results of a bispecific CD3×PSMA (monovalent) IgG4 antibody that contains the KiH mutation and the S228P mutation but does not contain a silencing mutation. These data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a very similar manner to that observed with the antibody in Panel A. Panel D of Figure 7 shows the results of the same bispecific antibody used in Panel C, but here, in addition to the S228P and KiH mutations, the CH2 domain contains silencing mutations (F234A, L235A). These results show that the presence of the silencing mutations significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even in the presence of the S228P and KiH mutations. Panel E shows the results of a bispecific CD3×PSMA (bivalent) IgG4 antibody that contains the S228P mutation, the silencing mutations F234A and L235A, and a KiH mutation in the CH3 domain. These data show that the interaction between this antibody and the Fcγ receptor immobilized on the biosensor was significantly reduced, even in the presence of the S228P and KiH mutations.

[0228] Panel A of FIG. 26 shows the results of a bispecific CD3×CD19 (monovalent) IgG4 antibody that does not contain a KiH mutation or a silencing mutation but contains an S228P mutation to prevent Fab arm exchange. The data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Panel B of FIG. 26 shows the results of the same bispecific antibody used in Panel A, but here the CH2 domain contains silencing mutations (F234A, L235A). These results show that the presence of the silencing mutation significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even in the presence of the S228P mutation. Panel C of FIG. 26 shows the results of a bispecific CD3×CD19 (monovalent) IgG4 antibody that contains the KiH mutation and the S228P mutation but does not contain a silencing mutation. These data show that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a manner very similar to that observed with the antibody in Panel A. Panel D of FIG. 26 shows the results of the same bispecific antibody used in Panel C, but here, in addition to the S228P and KiH mutations, the CH2 domain contains silencing mutations (F234A, L235A). These results show that the presence of the silencing mutation significantly reduces the interaction between the antibody and the Fcγ receptor on the biosensor, even in the presence of the S228P and KiH mutations.

[0229] Collectively, the data shown in FIGS. 4, 5, 6, 7, and 26 show that the VH region sequences of the bispecific antibodies do not affect the functional properties of the IgG4 Fc mutations described herein. Thus, the IgG4 Fc modifications described herein (S228P; F234A, L235A; T366W, T366S, L368A, and Y407V) can be implemented in antibodies with different VH sequences (i.e., different binding targets) to achieve a reduction in Fab arm exchange (S228P), a decrease in effector function activity (F234A, L235A), and proper heterodimerization (T366W; T366S, L368A, and Y407V).

[0230] Example 3: Flow Cytometry Analysis of Binding to PSMA-Positive and -Negative Cells by Anti-PSMA UniAbs™ Flow cytometry (Guava easyCyte 8HT, EMD Millipore) was used to evaluate binding to PSMA-positive cells using LNCaP cell line (ATCC: CRL-1740), 22Rv1 cell line (ATCC CRL-2505), and PC3 cell line (ATCC CRL-1435) that were stably transfected to express human PSMA or DU-145 cell line (ATCC HTB-81). Briefly, 50,000 target cells were stained with a dilution series of purified UniAbs™ for 30 minutes at 4°C. After incubation, the cells were washed twice with flow cytometry buffer (1×PBS, 1% BSA, 0.1% NaN 3 ) and stained with goat F(ab’) 2 anti-human IgG conjugated to R-phycoerythrin (PE) (Southern Biotech, catalog number 2042-09) to detect the antibody bound to the cells. After incubation at 4°C for 20 minutes, the cells were washed twice with flow cytometry buffer, and the mean fluorescence intensity (MFI) was measured by flow cytometry. The MFI of cells stained with secondary antibody only was used to measure the background signal, and the binding of each antibody was converted to multiples of the background. Binding to cynomolgus monkey PSMA-positive cells was measured using the same protocol with the following modifications: The target cells were cells derived from Freestyle 293-F cells (ThermoFisher R79007) transiently transfected to express the extracellular domain of cynomolgus monkey PSMA. In some experiments, the EC50 value was calculated using GraphPad Prism 7.

[0231] Table 8 summarizes the target binding activity of the anti-PSMA heavy chain antibodies (HCAbs) described herein. Column 1 shows the clone ID of the HCAbs. Column 2 shows the binding to LNCaP cells measured as multiples of the background MFI signal. [Table 8]

[0232] As shown in Panels A and B of Figure 8, the differences in the binding of cynomolgus monkey PSMA support the differences in the human PSMA epitopes recognized by HCAb346181 and 345497.

[0233] Example 4: Composition of Biparatopic and Bivalent Anti-PSMA Antibodies As shown in Table 9, anti-PSMA clone ID350123 consists of the ID346181 sequence linked to the ID345497 sequence with the crosslinking sequence GGGGSGGGGS (SEQ ID NO: 71). Clone ID350122 consists of two repeats of ID346181 linked by the same linker sequence. Clone ID350123 is biparatopic because it consists of two anti-PSMA domains that recognize different epitopes on PSMA. Clone ID350122 is bivalent but not biparatopic because it consists of a tandem of the same anti-PSMA domain. Schematic diagrams of various triple-stranded antibody-like molecules (TCAs) are shown in Panels A - C of Figure 1. [Table 9]

[0234] Example 5: Multispecific Antibody-Mediated Cell Death of PSMA-Positive Prostate Tumor Cells by T Cell Redirecting Assay Using Resting T Cells Target cells were seeded at 15,000 cells per well in a 96-well plate and grown overnight at 37°C. After incubation, a large amount of multi-specific antibody was added at an effector cell to target cell ratio of 10:1 together with resting human T cells and incubated for an additional 48 hours or 72 hours at 37°C (48 hours for assays with LNCaP, MDA-PCa-2b and PC3-PSMA cells, 72 hours for assays with 22Rv1 cells). Cell death was measured using either the cell proliferation reagent WST-1 (Sigma catalog number: 11644807001) or flow cytometry. In some experiments, a small sample of each supernatant was collected and stored for cytokine production analysis after incubation but before analysis of target cell viability. When analyzing cell viability with the WST-1 reagent, the reagent stock was added to each well at a 1:10 dilution and incubated at 37°C for 90 minutes. Absorbance was then measured at 450 nm (reference 690 nm) and the specific lysis rate (%) was calculated.

[0235] When analyzing target cell viability by flow cytometry, the assay was then initiated with the membrane dye DiR (ThermoFisher D12731) after labeling the target cells. After incubation with T cells and antibody, the supernatant was either stored for cytokine analysis or discarded. The wells were then washed once to recover dead tumor cells and T cells and transferred to a flow cytometry plate. The remaining adherent tumor cells were trypsinized and then added to the corresponding wells of the flow cytometry plate. Dead cells were stained using Annexin V reagent and flow cytometry was performed (BD FACSCelesta) to quantify the percentage of dead tumor cells in each sample gated by DiR staining. Wells containing untreated target cells were used to normalize for spontaneous cell death. In some experiments, a negative control antibody consisting of the same CD3 targeting arm as the PSMA×CD3 multi-specific molecule but with the tumor targeting arm replaced by a VH specific for the HIV protein gp120 was used.

[0236] Figure 9 shows T cell-mediated lysis of PSMA-positive cells using unstimulated T cells. Unstimulated human T cells were incubated with PSMA-expressing cells (LNCaP) and various concentrations of the multispecific antibody. The biparatopic anti-PSMA×CD3 antibody (350123×CD3) was superior to the monospecific PSMA×CD3 antibody (346181×CD3).

[0237] Assay using pre-activated T cells Human pan T cells were pre-activated with plate-bound OKT3 and IL-2 for 3 days and then incubated for an additional 1 day in fresh IL-2. Target cells were trypsinized, loaded with Calcein-AM (ThermoFisher C3100MP), mixed with activated T cells at an E:T ratio of 20:1, and added to the wells of a 96-well plate. After adding dilution series of different multispecific antibodies, the plate was incubated at 37 °C for 4 hours. The supernatant was then transferred to a black 96-well plate, and the absorbance was measured at 480 nm / 520 nm ex / em to quantify the release of calcein. The spontaneous release of calcein from intact tumor cells was normalized using target cells incubated in the absence of T cells. The calcein signal corresponding to maximal cell lysis could be calculated by adding 2% Triton-X to the control wells containing target cells. This value was used to report each experimental well as a percentage of maximal cell lysis. Data analysis was performed using GraphPad prism 7.

[0238] Figure 10 shows T cell-mediated lysis of PSMA-positive cells using pre-activated T cells. Pre-activated human T cells were incubated with human PSMA-expressing cells (LNCaP) and various concentrations of the multispecific antibody. Tumor cell death was measured by calcein release and normalized to the spontaneous release of tumor cells in the absence of T cells. The biparatopic anti-PSMA×CD3 antibody (350123×CD3) was superior to both monospecific PSMA×CD3 antibodies.

[0239] Figure 11 shows that the multispecific antibody does not lyse PSMA-negative cells. Pre-activated human T cells were incubated with PSMA-negative prostate cancer cells (DU145) and various concentrations of the multispecific antibody. Lysis of these cells did not occur with any of the antibodies tested.

[0240] Figure 12 shows the binding of the PSMA×CD3 multispecific antibody to PSMA-positive and negative cells. The multispecific anti-PSMA×anti-CD3 antibody shows binding to PSMA-positive prostate tumor cells (22Rv1), but does not show binding to PSMA-negative prostate tumor cells (DU145). The biparatopic molecule (350123) showed the strongest on-target cell binding.

[0241] Figure 13 shows T cell-mediated lysis of PSMA-positive cells. The data in Figure 13 show that binding to PSMA via two different epitopes increases cell killing compared to a version of the antibody that is bivalent but monospecific.

[0242] Example 6: Monoparatopic PSMA×CD3 bispecific antibodies do not induce cytokine production compared to biparatopic PSMA×CD3 multispecific antibodies Cytokine production was analyzed in a tumor cell cytotoxicity assay using resting T cells. The design of these assays is described in detail elsewhere. Supernatants were collected at the end of the assay (after 72 hours of incubation for assays using 22Rv1 cells and after 48 hours for all other cell lines). ELISA kits were used according to the manufacturer's protocol to detect IL-2 (Biolegend 431804) and IFNγ (Biolegend 430104). Test supernatants were diluted prior to ELISA analysis so that cytokine levels fell within the linear portion of the standard curve supplied with each kit. In some cases, cytokines could not be detected in the test wells and values were reported as below the lower limit of quantification of the assay.

[0243] Figure 14 (Panels A, B, and C) shows the comparison of T cell-mediated lysis of PSMA-positive cells and cytokine production. The multispecific PSMA×CD3 antibody induces T cell-mediated lysis of the PSMA-positive prostate cancer cell line LNCaP. The biparatopic molecule (350123) stimulated tumor cell death more potently compared to the monospecific molecule (346181), but also caused higher levels of production of the cytokines interferon γ (IFNγ) and interleukin 2 (IL-2), as shown in Panels B and C of Figure 14.

[0244] Table 10 shows T cell-mediated lysis and cytokine production against four PSMA-positive prostate tumor cell lines. The PSMA×CD3 multispecific antibody was tested in an in vitro cytotoxicity assay of tumor cells using non-stimulated T cells and a series of doses of the antibody against a panel of four PSMA-positive tumor cell lines. After 72 hours (22Rv1) or 48 hours (MDA-PCa-2b, LNCAP, PC3-PSMA), the percentage of tumor cell death was calculated, described as the EC50, and the highest death rate achieved was also described. Supernatants were recovered from these test wells and analyzed by ELISA for the cytokines interferon γ (IFNγ) or interleukin-2 (IL-2). The monospecific molecule (3461881) induced a nearly equivalent level of tumor cell cytotoxicity against all four cell lines tested compared to the biparatopic molecule, but had a higher EC50 for cytokine production and in most cases a lower level of stimulation of maximal cytokine production.

Table 10

[0245] Example 7: The PSMA×CD3 Multispecific Antibody Induces T Cell Proliferation PSMA-positive tumor cells were seeded at 25,000 cells per well in a 96-well plate and grown overnight at 37°C. Human pan T cells (Miltenyi 130-096-535) isolated from resting PBMC were labeled with the lineage-tracing dye CFSE according to the manufacturer's instructions (ThermoFisher C34554). Then, 100,000 labeled pan T cells were added to the wells containing tumor cells, followed by dilution of a series of antibodies and incubation at 37°C, 8% CO 2 2. After 5 days of incubation, the cells were gently mixed and transferred to a flow cytometry plate. After pelleting the cells and removing the supernatant, the cells were stained on ice for 20 minutes with anti-CD8 conjugated to APC (Biolegend 301049) and anti-CD4 conjugated to PE (Biolegend 317410). The cells were then washed and resuspended in flow cytometry buffer (BD FACSCelesta) for analysis. The cells were gated on forward scatter and side scatter, as well as CD4 expression or CD8 expression. The percentage of proliferated T cells, as indicated by CD4 or CD8 positive staining and low or negative CFSE signal, was calculated for the entire T cell population as well as the CD4 and CD8 subsets. Flow cytometry data were analyzed using FlowJo and plotted in GraphPad Prism 7.

[0246] Figure 15 (Panels A, B, C, and D) shows that the PSMA×CD3 multispecific antibody stimulates T cell proliferation in the presence of PSMA-positive tumor cells, and the monovalent PSMA bispecific antibody preferentially activates CD3 T cells. The multispecific antibody was incubated with PSMA-expressing tumor cells and T cells labeled with the cell-tracking dye CFSE. After 5 days of incubation, T cell proliferation and the composition of the proliferated T cells (CD8+ vs. CD4+) were analyzed by flow cytometry. Panels A and B show the total proliferation of T cells, while panels C and D show the ratio of CD8+ to CD4+ T cells in the proliferated wells. The horizontal dashed line indicates the CD8:CD4 ratio of unstimulated T cells, which is approximately 1:2 (actual value = 0.64). The monovalent PSMA×CD3 bispecific antibody (346181) preferentially activates CD8 T cells (the CD8:CD4 ratio after proliferation is approximately 2:1), while the bivalent PSMA×CD3 multispecific antibody (350123) does not preferentially activate CD8+ T cells as much (the CD8:CD4 ratio is about 1:1).

[0247] Example 8: Multispecific antibodies inhibit prostate tumor growth in xenograft models Male immunodeficient CIEA-NOG mice (Taconic) at 5-6 weeks of age were subcutaneously implanted with 10 million 22Rv1 cells in the lower right abdomen, and 10 million human PBMCs were added by tail vein injection 1 day after tumor implantation. These animals were treated with 100 μg of the multispecific antibody or vehicle by tail vein injection starting 1 day after tumor implantation, on days 1, 5, 9, and 13. Tumor volume was quantified using calipers and recorded for 25 days.

[0248] Figure 16 shows the results of the 22Rv1 tumor xenograft model. The biparatopic PSMA×CD3 molecule (350123) showed inhibition of 22Rv1 tumor growth in the tumor xenograft model. Three mice were tested for each treatment group, and the change in tumor volume of each animal was plotted in cubic millimeters. PBMC was administered to the animals on day 1 after tumor transplantation, and the animals were treated with the antibody on days 1, 5, 9, and 13. Two of the three animals treated with the multispecific antibody showed a delay in tumor progression.

[0249] Example 9: Analysis of T cell activation CD69 is a cell surface marker on T cells that is upregulated by stimulation and thus functions as an indicator of T cell activation. In this experiment, the activation of CD69 was evaluated under three different conditions: 1) whole peripheral blood mononuclear cells (PBMC) without BCMA coating; 2) BCMA-coated pan T cells; and 3) pan T cells without BCMA coating. PBMC was separated from the buffy coat using Ficoll (density 1.077 g / ml), and the cryopreserved PBMC was thawed and placed in RPMI 1640 supplemented with 10% FBS at 2 × 10 6 cells / ml and incubated at 37°C for 24 hours. On day 2, pan T cells were separated from the resting PBMC using the Miltenyi negative selection kit, and the separated cells were used in the second and third assay conditions. In the first assay condition, the PBMC was counted and seeded onto the assay plates.

[0250] For the cells evaluated under antigen coating conditions, 96-well plates were coated with recombinant BCMA protein (human BCMA protein, Fc tag, Acro Biosystems, catalog number BC7-H5254) at a concentration of 1 μg / mL, recombinant PSMA protein (recombinant human PSMA / FOLH1 protein, RND systems, catalog number -4234-ZN-0101)) at a concentration of 1 μg / mL, or recombinant CD19 protein (human CD19 protein, His tag, Acro Biosystems, catalog number -CD9-H52H2) at a concentration of 10 μg / mL. Bispecific antibodies were analyzed using a 12-point dose curve of a 3-fold dilution series with a maximum dose of 300 nM. The bispecific antibodies and T cells were resuspended in RPMI1640 supplemented with 10% FBS and incubated for 18 hours. Pan T cells, which are effector cells, were plated at 100,000 cells / well.

[0251] For the cells evaluated under conditions without antigen coating, bispecific antibodies were incubated with the cells using a 12-point dose curve of a 3-fold dilution series. 300 nM bispecific antibody was the highest concentration tested in this assay. Samples were incubated at 37 °C for 18 hours in RPMI1640 supplemented with 10% FBS. Pan T cells isolated from PBMCs are effector cells and were plated at 100,000 cells / well.

[0252] For all experimental conditions, the cells were washed and labeled with cell surface T cell antibodies. Labeling was performed using the following antibodies: (1) CD4-positive T cells (FITC anti-human CD4 antibody), (2) CD8-positive T cells (PE anti-human CD8a antibody), (3) CD69 activation (Alexa Fluor 647 anti-human CD69 antibody) (Biolegend). Cells were then analyzed on a BD Celesta using an appropriate template to measure CD69 activation.

[0253] The results of T cell activation studies are shown in the following figures: Figures 17 - 18, Panels A - B of Figure 27 (without BCMA antigen coating, using PBMC); Panels A - B of Figure 30 (without PSMA coating); and Panels A - B of Figure 33 (without CD19 coating).

[0254] Figure 17 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Generally, bispecific antibodies containing the IgG1 Fc sequence showed CD4+ T cell activation at lower concentrations of bispecific antibody. Bispecific antibodies containing the IgG4 Fc sequence showed CD4+ T cell activation at higher concentrations of bispecific antibody. In particular, the CD4+ T cell activation achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations was low, and it was shown that the introduction of PAA and KiH mutations decreased the BCMA - independent activation of T cells by these bispecific antibodies.

[0255] Figure 18 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Similar to the case of CD4+ T cells, bispecific antibodies containing the IgG1 Fc sequence showed CD8+ T cell activation at lower concentrations of bispecific antibody. Bispecific antibodies containing the IgG4 Fc sequence showed CD8+ T cell activation at higher concentrations of bispecific antibody. In particular, the CD8+ T cell activation achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations was low, and it was shown that the introduction of PAA and KiH mutations decreased the BCMA - independent activation of T cells by these bispecific antibodies.

[0256] Panel A of Figure 27 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. The CD4+ T cell activation achieved by the IgG4 Fc bispecific antibodies containing PAA and KiH mutations is similar to that of the negative control (gp120, CD3 (F2B)), indicating that the introduction of PAA and KiH mutations reduces the BCMA-independent activation of T cells by these bispecific antibodies. Panel B of Figure 27 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Similar to the case of CD4+ T cells, the CD8+ T cell activation achieved by the IgG4 Fc bispecific antibodies containing PAA and KiH mutations is similar to that of the negative control, indicating that the introduction of PAA and KiH mutations reduces the BCMA-independent activation of T cells by these bispecific antibodies.

[0257] Panel A of Figure 30 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. The CD4+ T cell activation achieved by the IgG4 Fc bispecific antibodies containing PAA and KiH mutations is similar to that of the negative control (gp120, CD3 (F2B)), indicating that the introduction of PAA and KiH mutations reduces the PSMA-independent activation of T cells by these bispecific antibodies. Panel B of Figure 30 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Similar to the case of CD4+ T cells, the CD8+ T cell activation achieved by the IgG4 Fc bispecific antibodies containing PAA and KiH mutations is similar to that of the negative control, indicating that the introduction of PAA and KiH mutations reduces the PSMA-independent activation of T cells by these bispecific antibodies.

[0258] Panel A of FIG. 33 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. The activation of CD4+ T cells achieved by the IgG4 Fc bispecific antibody containing the PAA and KiH mutations is much lower than that observed from other antibody constructs without the PAA and KiH mutations, indicating that the introduction of the PAA and KiH mutations reduces the CD19-independent activation of T cells by these bispecific antibodies. Panel B of FIG. 33 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Similar to the case of CD4+ T cells, the activation of CD8+ T cells achieved by the IgG4 Fc bispecific antibody containing the PAA and KiH mutations is much lower than that observed from other antibody constructs without the PAA and KiH mutations, indicating that the introduction of the PAA and KiH mutations reduces the CD19-independent activation of T cells by these bispecific antibodies.

[0259] The results of CD8+ T cell activation with antigen coating using pan T cells isolated from resting PBMCs are shown in FIG. 19, Panel B of FIG. 28, Panel B of FIG. 31, and Panel B of FIG. 34. The results of CD4+ T cell activation with antigen coating using pan T cells isolated from resting PBMCs are shown in Panel A of FIG. 28, Panel A of FIG. 31, and Panel A of FIG. 34. The antigen coating concentrations of BCMA and PSMA were 1 μg / mL, while the antigen coating concentration of CD19 was 10 μg / mL.

[0260] FIG. 19 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without BCMA coating, all bispecific antibodies showed activation of CD8+ T cells at concentrations similar to those of the bispecific antibodies against antigen-coated cells. In particular, activation of CD8+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the CD8+ T cell activation activity of these molecules.

[0261] Panel B of FIG. 28 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without BCMA coating, all bispecific antibodies showed activation of CD8+ T cells at concentrations similar to those of the bispecific antibodies against antigen-coated cells. In particular, activation of CD8+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the activity of CD8+ T cell activation of these molecules.

[0262] Panel B of FIG. 31 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without PSMA coating, all bispecific antibodies showed CD8+ T cell activation at concentrations similar to those of the bispecific antibodies against antigen-coated cells. In particular, activation of CD8+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the activity of CD8+ T cell activation of these molecules.

[0263] Panel B of Figure 34 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without CD19 coating, all bispecific antibodies showed CD8+ T cell activation at concentrations similar to those of the bispecific antibody against antigen-coated cells. In particular, activation of CD8+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the activity of these molecules in activating CD8+ T cells.

[0264] Panel A of Figure 28 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without BCMA coating, all bispecific antibodies showed CD4+ T cell activation at concentrations similar to those of the bispecific antibody against antigen-coated cells. In particular, activation of CD4+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the activity of these molecules in activating CD4+ T cells.

[0265] Panel A of Figure 31 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without PSMA coating, all bispecific antibodies showed CD4+ T cell activation at concentrations similar to those of the bispecific antibody against antigen-coated cells. In particular, activation of CD4+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the activity of these molecules in activating CD4+ T cells.

[0266] Panel A of FIG. 34 is a graph showing CD4+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. Unlike the experiments without CD19 coating, all bispecific antibodies showed CD4+ T cell activation at concentrations similar to those of the bispecific antibodies against antigen-coated cells. In particular, the activation of CD4+ T cells was achieved by IgG4 Fc bispecific antibodies containing PAA and KiH mutations, indicating that the introduction of PAA and KiH mutations did not eliminate the activity of these molecules in activating CD4+ T cells.

[0267] The results of CD8+ T cell activation without antigen coating using pan T cells isolated from resting PBMCs are shown in FIG. 20, Panel B of FIG. 29, Panel B of FIG. 32, and Panel B of FIG. 35. The results of CD4+ T cell activation without antigen coating using pan T cells isolated from resting PBMCs are shown in Panel A of FIG. 29, Panel A of FIG. 32, and Panel A of FIG. 35.

[0268] FIG. 20 is a graph showing CD8+CD69+ T cells (%) as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. These results indicate that CD69 activation in CD8+ T cells is BCMA-dependent for all bispecific antibody molecules tested.

[0269] Panels A and B of FIG. 29 are graphs showing CD4+CD69+ T cells (%) and CD8+CD69+ T cells (%), respectively, as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. These results indicate that CD69 activation in CD4+ T cells and CD8+ T cells is BCMA-dependent for all bispecific antibody molecules tested. CD69 activation increased slightly in both CD4+ T cells and CD8+ T cells in higher concentration bispecific antibody constructs without silencing mutations.

[0270] Panels A and B of FIG. 32 are graphs showing CD4+CD69+ T cells (%) and CD8+CD69+ T cells (%) respectively as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. These results indicate that CD69 activation in CD4+ T cells and CD8+ T cells is PSMA-dependent with all bispecific antibody molecules tested.

[0271] Panels A and B of FIG. 35 are graphs showing CD4+CD69+ T cells (%) and CD8+CD69+ T cells (%) respectively as a function of bispecific antibody concentration for the bispecific antibody constructs shown in the legend. These results indicate that CD69 activation in CD4+ T cells and CD8+ T cells is CD19-dependent with all bispecific antibody molecules tested. CD69 activation increased slightly in both CD4+ T cells and CD8+ T cells in higher concentrations of bispecific antibody constructs without silencing mutations.

[0272] Example 10: Lysis of Tumor Cells An anti-CD3×anti-BCMA bispecific antibody was assayed for its ability to kill three different BCMA+ tumor cells and one BCMA-negative cell line through redirection of activated primary T cells. In this experiment, tumor cells and activated pan T cells were mixed at an E:T ratio of 10:1 with the addition of the bispecific antibody. The results are shown in Panels A-D of FIG. 21. Panel A shows the killing of RPMI-8226 cells, Panel B shows the killing of NCI-H929 cells, Panel C shows the killing of U-266 cells, and Panel D shows the killing of K562 cells as a negative control. The x-axis indicates the concentration of the antibody used, and the y-axis indicates the lysis rate (%) of the tumor cells 6 hours after antibody addition.

[0273] Resting human T cells were cultured with various tumor cell lines and increasing doses of anti-CD3×anti-BCMA bispecific antibody, and then the levels of IL-2 cytokine release were measured. Panel A of FIG. 22 shows IL-2 release stimulated by RPMI-8226 cells. Panel B of FIG. 22 shows IL-2 release stimulated by NCI-H929 cells. Panel C of FIG. 22 shows IL-2 release stimulated by U-266 cells. Panel D of FIG. 22 shows IL-2 release stimulated by K562 cells, which is a negative control.

[0274] Resting human T cells were cultured with various tumor cell lines and increasing doses of anti-CD3×anti-BCMA bispecific antibody, and then the levels of IFN-γ cytokine release were measured. Panel A of FIG. 23 shows IFN-γ release stimulated by RPMI-8226 cells. Panel B of FIG. 23 shows IFN-γ release stimulated by NCI-H929 cells. Panel C of FIG. 23 shows IFN-γ release stimulated by U-266 cells. Panel D of FIG. 23 shows IFN-γ release stimulated by K562 cells, which is a negative control.

[0275] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are merely illustrative. Those skilled in the art will find numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternative means to the embodiments of the present invention described herein may be employed in the practice of the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures included in these claims, as well as their equivalents, be thereby encompassed.

Claims

1. a first heavy chain polypeptide subunit comprising a mutated human IgG4 constant region comprising the mutations S228P, F234A, L235A, and T366W; and A second heavy chain polypeptide subunit comprising a mutated human IgG4 constant region comprising the mutations S228P, F234A, L235A, T366S, L368A, and Y407V.

1. An isolated multispecific antibody comprising:

2. 2. The isolated multispecific antibody of claim 1, wherein the mutated human IgG4 constant region of the first heavy chain polypeptide subunit or the mutated human IgG4 constant region of the second heavy chain polypeptide subunit is deleted for the CH1 domain.

3. 3. The isolated multispecific antibody of claim 1 or 2, wherein the mutated human IgG4 constant region of the first heavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 73 or 55 and the mutated human IgG4 constant region of the second heavy chain polypeptide subunit comprises the sequence of SEQ ID NO: 72 or 54.

4. a heavy chain variable domain comprising a CDR1 sequence comprising the sequence of SEQ ID NO:36, a CDR2 sequence comprising the sequence of SEQ ID NO:37, and a CDR3 sequence comprising the sequence of SEQ ID NO:38; and a light chain variable domain comprising a CDR1 sequence comprising the sequence of SEQ ID NO:39, a CDR2 sequence comprising the sequence of SEQ ID NO:40, and a CDR3 sequence comprising the sequence of SEQ ID NO:

41. and further comprising a first binding moiety having binding specificity for CD3, comprising: An isolated multispecific antibody according to any one of claims 1 to 3.

5. the CDR1, CDR2 and CDR3 sequences in the heavy chain variable domain of the first binding moiety are present in a human VH framework; 5. The isolated multispecific antibody of claim 4, wherein the CDR1, CDR2 and CDR3 sequences in the light chain variable domain of the first binding moiety are present in a human Vκ framework.

6. the heavy chain variable domain of the first binding moiety comprises a sequence having at least 95% identity to SEQ ID NO:42; the light chain variable domain of the first binding moiety comprises a sequence having at least 95% identity to SEQ ID NO: 43; 6. An isolated multispecific antibody according to claim 5.

7. the heavy chain variable domain of the first binding moiety comprises the sequence of SEQ ID NO:42; the light chain variable domain of the first binding moiety comprises the sequence of SEQ ID NO: 43; 7. An isolated multispecific antibody according to claim 6.

8. 8. The isolated multispecific antibody of claim 1, further comprising a second binding moiety having binding specificity for a protein other than CD3.

9. 9. The isolated multispecific antibody of claim 8, wherein the second binding moiety comprises a single heavy chain variable region in a monovalent or bivalent configuration.

10. 10. The isolated multispecific antibody of claim 9, wherein the first binding moiety comprises a light chain polypeptide subunit and a heavy chain polypeptide subunit and the second binding moiety comprises a heavy chain polypeptide subunit.

11. 11. The isolated multispecific antibody of claim 10, wherein the light chain polypeptide subunit of the first binding moiety comprises a light chain constant domain.

12. The isolated multispecific antibody according to any one of claims 8 to 11, wherein said protein other than CD3 is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

13. 13. The isolated multispecific antibody of claim 12, wherein the TAA is B-cell maturation antigen (BCMA).

14. 13. The isolated multispecific antibody of claim 12, wherein the TAA is CD19.

15. 13. The isolated multispecific antibody of claim 12, wherein the TAA is prostate-specific membrane antigen (PSMA).

16. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 15.

17. A polynucleotide encoding a multispecific antibody according to any one of claims 1 to 15.

18. A vector comprising the polynucleotide of claim 17.

19. A cell comprising the vector of claim 18.

20. A method for producing a multispecific antibody according to any one of claims 1 to 15, comprising growing a cell according to claim 19 under conditions permissive for expression of said multispecific antibody and isolating said multispecific antibody from said cell.

21. A method of treatment comprising administering to an individual in need thereof an effective dose of a multispecific antibody according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16.

22. 16. Use of a multispecific antibody according to any one of claims 1 to 15 in the preparation of a medicament for the treatment of a disease or disorder in an individual in need thereof.

23. A multispecific antibody according to any one of claims 1 to 15, or a pharmaceutical composition according to claim 16, for use in therapy in an individual in need thereof.

24. A method for treating a disease or condition characterized by expression of BCMA, comprising administering to an individual in need thereof an effective dose of a multispecific antibody according to claim 13, or a pharmaceutical composition comprising the multispecific antibody according to claim 13.

25. 25. The method of claim 24, wherein the disease is an autoimmune disease.

26. 25. The method of claim 24, wherein the disease is cancer.

27. 27. The method of claim 26, wherein the cancer is myeloma.

28. 28. The method of claim 27, wherein the myeloma is multiple myeloma.

29. A method for treating a disease or condition characterized by expression of PSMA, comprising administering to an individual in need thereof an effective dose of a multispecific antibody according to claim 15, or a pharmaceutical composition comprising the multispecific antibody according to claim 15.

30. 30. The method of claim 29, wherein the disease is cancer.

31. 30. The method of claim 29, wherein the cancer is prostate cancer.

32. 15. A method for treating a disease or condition characterized by expression of CD19, comprising administering to an individual in need thereof an effective dose of a multispecific antibody according to claim 14, or a pharmaceutical composition comprising a multispecific antibody according to claim 14.

33. 33. The method of claim 32, wherein the disorder is diffuse large B-cell lymphoma (DLBCL).

34. 33. The method of claim 32, wherein the disorder is acute lymphoblastic leukemia (ALL).

35. 33. The method of claim 32, wherein the disorder is non-Hodgkin's lymphoma (NHL).

36. 33. The method of claim 32, wherein the disorder is systemic lupus erythematosus (SLE).

37. 33. The method of claim 32, wherein the disorder is rheumatoid arthritis (RA).

38. 33. The method of claim 32, wherein the disorder is multiple sclerosis (MS).

39. A kit for treating a disease or disorder in an individual in need thereof comprising a multispecific antibody according to any one of claims 1 to 15, or a pharmaceutical composition according to claim 16, and instructions for use.

40. 40. The kit of claim 39, further comprising at least one additional reagent.

41. 41. The kit of claim 40, wherein the at least one additional reagent comprises a chemotherapeutic agent.

42. A light chain variable domain (VL) comprising the sequence of SEQ ID NO: 43; and Light chain constant domain (CL) a first polypeptide subunit comprising: A heavy chain variable domain (VH) comprising the sequence of SEQ ID NO: 42; and A heavy chain constant domain (CH) comprising the sequence of SEQ ID NO: 72 or 73 A second polypeptide subunit comprising: the second polypeptide subunit, wherein the light chain variable domain and the heavy chain variable domain together form a first binding moiety that binds to CD3; and A variable region of only the heavy chain, in a monovalent or bivalent configuration, that binds to a protein other than CD3; and A heavy chain constant domain (CH) comprising the sequence of SEQ ID NO: 54 or 55 A third polypeptide subunit comprising A bispecific three-chain antibody-like molecule comprising:

43. 43. The bispecific three-chain antibody-like molecule of claim 42, wherein the third polypeptide subunit comprises the variable region of only the heavy chain in a bivalent configuration that binds BCMA.

44. a first polypeptide subunit comprising the sequence of SEQ ID NO:49; A second polypeptide subunit comprising the sequence of SEQ ID NO:56; and A third polypeptide subunit comprising the sequence of SEQ ID NO:

58.

44. The bispecific three-chain antibody-like molecule of claim 43, comprising:

45. A pharmaceutical composition comprising the bispecific three-chain antibody-like molecule of any one of claims 42 to 44.

46. A polynucleotide encoding the bispecific three-chain antibody-like molecule of any one of claims 42 to 44.

47. A vector comprising the polynucleotide of claim 46.

48. A cell comprising the vector of claim 47.

49. A method for producing a bispecific three-chain antibody-like molecule according to any one of claims 42 to 44, comprising growing a cell according to claim 48 under conditions permissive for expression of said bispecific three-chain antibody-like molecule and isolating said bispecific three-chain antibody-like molecule from said cell.

50. A method of treatment comprising administering to an individual in need thereof an effective dose of the bispecific triple-chain antibody-like molecule of any one of claims 42 to 44, or the pharmaceutical composition of claim 45.

51. Use of a bispecific three-chain antibody-like molecule according to any one of claims 42 to 44 in the preparation of a medicament for the treatment of a disease or disorder in an individual in need thereof.

52. A bispecific three-chain antibody-like molecule according to any one of claims 42 to 44, or a pharmaceutical composition according to claim 45, for use in therapy in an individual in need thereof.

53. A method for treating a disease or condition characterized by expression of BCMA, comprising administering to an individual in need thereof an effective dose of the bispecific triple-chain antibody-like molecule of any one of claims 42 to 44, or the pharmaceutical composition of claim 45.

54. 54. The method of claim 53, wherein the disease is an autoimmune disease.

55. 54. The method of claim 53, wherein the disease is cancer.

56. 56. The method of claim 55, wherein the cancer is myeloma.

57. 57. The method of claim 56, wherein the myeloma is multiple myeloma.

58. A kit for treating a disease or disorder in an individual in need thereof comprising a bispecific triple-chain antibody-like molecule according to any one of claims 42 to 44, or a pharmaceutical composition according to claim 45, and instructions for use.

59. 60. The kit of claim 58, further comprising at least one additional reagent.

60. 60. The kit of claim 59, wherein the at least one additional reagent comprises a chemotherapeutic agent.

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