Multispecific heavy chain antibodies with modified heavy chain constant regions

Abstract

This invention provides multispecific human heavy chain antibodies (e.g., UniAbs) TM This invention relates to methods of manufacturing such antibodies, compositions comprising such antibodies, including pharmaceutical compositions, and their use in treating conditions characterized by expression of one or more binding targets described herein.

Description

[0001] This application is a divisional application of CN202180030976.1.

[0002] Cross-references to related applications

[0003] This application claims priority to the filing dates of U.S. Provisional Patent Application Serial No. 63 / 017,589, filed April 29, 2020, and U.S. Provisional Patent Application Serial No. 63 / 108,796, filed November 2, 2020, the entire disclosure of which is incorporated herein by reference. Technical Field

[0004] This invention relates to multispecific human heavy chain antibodies (e.g., UniAbs) TM This invention relates to methods of manufacturing such antibodies, compositions comprising such antibodies, including pharmaceutical compositions, and their use in treating conditions characterized by expression of one or more binding targets described herein. Background Technology

[0005] Modified Fc region

[0006] Advances in protein engineering have led to the successful fabrication and clinical use of multispecific antibodies with binding affinity for two or more targets. However, due to their heterodimeric nature, appropriate measures must be employed to facilitate the correct pairing of the desired binding sequence combinations within multispecific antibodies, and thus the correct pairing of peptide subunits. (Wang et al., mAbs 10:8, 1226-1235 (2018)).

[0007] One approach to circumventing the problem of mismatched polypeptide subunits is called “knobs-into-holes” (KiH), which aims to modify the contact interface by introducing mutations in the CH2 and / or CH3 domains to force two distinct antibody heavy chains to pair. On one chain, a large amino acid is replaced by an amino acid with a short side chain to create a “hole.” Conversely, an amino acid with a large side chain is introduced into the other heavy chain to create a “knob.” By co-expressing these two heavy chains, the yield of heterodimer formation (“knob-hole”) was observed to be higher than that of homodimer formation (“hole-hole” or “knob-knob”), which is attributed to the more favorable stability of the knob-hole pair (Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and WO 96 / 027011).

[0008] While this strategy appears attractive for achieving desired heterodimers, other properties of the resulting multispecific antibodies are highly dependent on specific amino acid sequences in the Fc region, namely effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC). Furthermore, effector activity can induce cytokine production, which can lead to an undesirable inflammatory response known as a "cytokine storm." (Gupta et al., Journal of Interferon & Cytokine Research 40:1,19-23 (2019)). Therefore, in some cases, it is necessary to reduce or completely eliminate effector functions, for example, to avoid damaging or killing immune cells (e.g., T cells) that bind to multispecific antibodies, and / or to avoid the production of undesirable cytokines and the resulting undesirable inflammatory response.

[0009] Furthermore, introducing amino acid modifications into proteins can have serious drawbacks, namely, inducing an immune response against the protein based on the presence of non-natural sequences. Therefore, the development of multispecific antibodies requires identifying sequences that are very similar to the general structure of natural antibodies (such as IgA, IgD, IgE, IgG, or IgM) and have minimal deviation from the natural sequence, but which can be successfully incorporated to simultaneously achieve the goal of promoting desired heterodimerization while also reducing or eliminating one or more effector functions.

[0010] To balance these competing needs, the inventors focused on IgG4 Fc, whose native sequence is known to have relatively low levels of effector activity. (Crescioli et al., Curr Allergy Asthma Rep 16:7 (2016)). However, despite this apparent advantage, IgG4 is known to undergo in vivo chain exchange reactions due to its specific hinge region sequence, presenting additional complexity in achieving the desired heterodimerization. (Labrijn et al., Nature Biotechnology 27, 767-71 (2009)). Therefore, modified heavy chain constant region sequences are needed to achieve the desired heterodimerization, incorporating modifications that reduce or eliminate effector activity, while also incorporating modifications that reduce or eliminate chain exchange reactions in IgG4. The molecule described herein addresses these and other challenges.

[0011] Heavy chain antibodies

[0012] In conventional IgG antibodies, the association between the heavy and light chains is attributed to hydrophobic interactions between the light chain constant region and the heavy chain CH1 constant domain. Additional residues in the heavy chain frame region 2 (FR2) and frame region 4 (FR4) further contribute to these hydrophobic interactions between the heavy and light chains.

[0013] However, the sera of camelids (Camelidae, suborder Camelidae, which includes camels, dromedary camels, and vicuñas) are known to contain a large class of antibodies consisting only of paired H chains (heavy chain antibodies or UniAbs). TM UniAbs, a family of camels (dromedary camel (Camelus dromedarius), Bactrian camel (Camelus bactrianus), llama (Lama glama), guanaco (Lama guanaco), alpaca (Lama alpaca), and vicugna (Lama vicugna)). TM These UniAbs possess 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. TM The absence of the first domain (CH1) of the constant region, which is present in the genome but is spliced ​​out during mRNA processing, explains UniAbs's... TM The absence of the medium-light chain is because this domain is the anchoring site for the constant structural domains of the light chain. This type of UniAbs TM Natural evolution has endowed conventional antibodies or fragments thereof with antigen-binding specificity and high affinity for the three CDRs (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish (e.g., sharks) have also evolved different types of immunoglobulins called IgNARs, which lack light polypeptide chains and are composed entirely of heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce variable domains (vNARs) of single heavy-chain polypeptides (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)).

[0014] The ability of heavy-chain-only antibodies, lacking the light chain, to bind antigens was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Physically separated heavy-chain immunoglobulins retain 80% of their antigen-binding activity relative to tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that removing the CH1 domain from the rearranged mouse μ gene in mammalian cell cultures produces heavy-chain-only antibodies lacking the light chain. The resulting antibodies retain VH binding specificity and effector function.

[0015] Heavy chain antibodies with high specificity and affinity for a variety of antigens can be generated through immunization (van der Linden, RH et al., Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH moiety can be easily cloned and expressed in yeast (Frenken, LGJ et al., J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of the classic F(ab) or Fv fragments (Ghahroudi, MA et al., FEBS Lett. 414, 521-526 (1997)).

[0016] The λ (lambda) light chain (L) locus and / or the λ and κ (kappa) L chain locus have been described in functionally silenced mice and antibodies produced by such mice in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain-only antibodies in mice and rats has been reported, for example, in WO2006008548; U.S. Application Publication No. 20100122358; Nguyen et al., 2003, Immunology; 109(1), 93-101; Brüggemann et al., Crit. Rev. Immunol.; 2006, 26(5): 377-90; and Zou et al., 2007, J Exp Med; 204(13): 3271-3283. Gene knockout rats generated by embryonic microinjection of zinc finger nucleases are described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy-chain-only antibodies and transgenic rodents containing heterologous heavy-chain loci that produce such antibodies are described in U.S. Patent Nos. 8,883,150 and 9,365,655. CAR-T structures 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.

[0017] B-cell maturation antigen (BCMA)

[0018] BCMA (also known as tumor necrosis factor superfamily member 17, TNFRSF17) (UniProt Q02223) is a cell surface receptor expressed exclusively on plasma cells and plasmablasts. BCMA is the receptor for two ligands in the tumor necrosis factor (TNF) superfamily: APRIL (the proliferation-inducing ligand, also known as TNFSF13; TALL-2 and TRDL-1; a high-affinity ligand of BCMA) and B cell activating factor (BAFF) (also known as BLyS; TALL-1; THANK; zTNF4; TNFSF20; and D8Ertd387e; a low-affinity ligand of BCMA). APRIL and BAFF are growth factors that bind to BCMA and promote plasma cell survival. BCMA is also highly expressed on malignant plasma cells in human multiple myeloma (MM). Antibodies binding 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 US 2013 / 0156769 A1 and US 2015 / 0376287A1. Anti-BCMA antibody-MMAE or anti-BCMA antibody-MMAF conjugates have been reported to selectively induce the killing of multiple myeloma cells (Tai et al., Blood 2014, 123(20):3128-38). Ali et al., Blood 2016, 128(13):1688-700, reported that chimeric antigen receptor (CAR) T cells targeting BCMA could alleviate multiple myeloma in human patients in a clinical trial (#NCT02215967).

[0019] PSMA

[0020] 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, folic acid hydrolase, and dipeptidyl peptidase activities. In humans, it is encoded by the FOLH1 gene and consists of a 19-amino acid cytoplasmic domain, a 24-amino acid transmembrane portion, and a 707-amino acid extracellular portion. The protein functions as a non-covalent homodimer. PSMA is expressed in prostate epithelial tissue and is upregulated in the neovascularization system of prostate cancer and solid tumors. It is also expressed at low levels in healthy tissues (e.g., brain, kidneys, and salivary glands), but its overexpression in malignant prostate tissue makes it an attractive target for therapeutic treatment of prostate cancer. Given its high expression in the malignant neovascularization system, it may also be associated with therapies or imaging 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). Additionally, radionuclide conjugates specifically targeting PSMA are being investigated for imaging and treatment of prostate cancer (e.g., Hofman et al., 2018, PMID:29752180).

[0021] CD19

[0022] CD19 (also known as B-lymphocyte surface antigen B4, UniProt P15391) is a cell surface receptor expressed on all human B cells but not found on plasma cells. CD19 is a transmembrane protein that recruits cytoplasmic signaling proteins to the membrane and functions in the CD19 / CD21 complex to lower the threshold of B-cell receptor signaling pathways. CD19 has a relatively large 240-amino acid cytoplasmic tail. The extracellular Ig-like domain is separated by a potentially disulfide-linked 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. Like CD20 and CD22, the restricted expression of CD19 in the B-cell lineage makes it an attractive target for therapeutic treatment of B-cell malignancies. Numerous monoclonal antibodies and antibody-drug conjugates specifically targeting CD19 have been described (e.g., Naddafi et al., 2015, PMC4644525). In addition, 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

[0023] Aspects of the present invention include isolated multispecific antibodies comprising: a first heavy chain polypeptide subunit containing a mutated human IgG4 constant region containing mutations of S228P, F234A, L235A, and T366W; and a second heavy chain polypeptide subunit containing a mutated human IgG4 constant region containing mutations of S228P, F234A, L235A, T366S, L368A, and Y407V. In some embodiments, 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 lacks a CH1 domain. In some embodiments, the mutated human IgG4 constant region of the first heavy chain polypeptide subunit contains the sequence of SEQ ID NO: 73 or 55, and the mutated human IgG4 constant region of the second heavy chain polypeptide subunit contains the sequence of SEQ ID NO: 72 or 54.

[0024] In some embodiments, the multispecific antibody according to embodiments of the present invention further comprises a first binding portion having binding specificity to CD3, the first binding portion comprising: a heavy chain variable domain comprising a CDR1 sequence containing the sequence of SEQ ID NO:36, a CDR2 sequence containing the sequence of SEQ ID NO:37, and a CDR3 sequence containing the sequence of SEQ ID NO:38; and a light chain variable domain comprising a CDR1 sequence containing the sequence of SEQ ID NO:39, a CDR2 sequence containing the sequence of SEQ ID NO:40, and a CDR3 sequence containing the sequence of SEQ ID NO:41.

[0025] In some embodiments, the CDR1, CDR2, and CDR3 sequences in the heavy chain variable domain of the first binding portion are present in the human VH framework; and the CDR1, CDR2, and CDR3 sequences in the light chain variable domain of the first binding portion are present in the human Vκ framework. In some embodiments, the heavy chain variable domain of the first binding portion contains a sequence having at least 95% identity with SEQ ID NO:42; and the light chain variable domain of the first binding portion contains 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 contains the sequence of SEQ ID NO:42; and the light chain variable domain of the first binding portion contains the sequence of SEQ ID NO:43.

[0026] In some embodiments, the multispecific antibody according to embodiments of the present invention further comprises a second binding moiety having binding specificity to proteins other than CD3. In some embodiments, the second binding moiety comprises a single heavy chain variable region in a monovalent or divalent configuration. In some embodiments, the first binding moiety comprises a light chain polypeptide subunit and a heavy chain polypeptide subunit, wherein the second binding moiety comprises a heavy chain polypeptide subunit. In some embodiments, the light chain polypeptide subunit of the first binding moiety 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 a B-cell maturation antigen (BCMA). In some embodiments, the TAA is CD19. In some embodiments, the TAA is a prostate-specific membrane antigen (PSMA).

[0027] Aspects of the present invention include pharmaceutical compositions comprising multispecific antibodies as described herein, polynucleotides encoding multispecific antibodies as described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0028] Aspects of the present invention include a method for generating multispecific antibodies as described herein, comprising growing cells as described herein under conditions that allow for the expression of multispecific antibodies and isolating multispecific antibodies from the cells.

[0029] Aspects of the invention include treatment methods comprising administering an effective dose of the multispecific antibody or pharmaceutical composition described herein to an individual in need.

[0030] Aspects of the invention include the use of the multispecific antibodies described herein for the preparation of medicaments for treating diseases or conditions in individuals in need.

[0031] Aspects of the invention include methods for treating diseases or ailments characterized by BCMA expression, comprising administering an effective dose of the multispecific antibody or pharmaceutical composition described herein to an individual in need. 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.

[0032] Aspects of the invention include methods for treating diseases or ailments characterized by PSMA expression, comprising administering an effective dose of the multispecific antibody or pharmaceutical composition described herein to an individual in need. In some embodiments, the disease is cancer. In some embodiments, the cancer is prostate cancer.

[0033] Aspects of the present invention include methods of treating diseases or ailments characterized by CD19 expression, comprising administering an effective dose of the multispecific antibody or pharmaceutical composition described herein to an individual in need. In some embodiments, the ailment is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the ailment is acute lymphoblastic leukemia (ALL). In some embodiments, the ailment is non-Hodgkin's lymphoma (NHL). In some embodiments, the ailment is systemic lupus erythematosus (SLE). In some embodiments, the ailment is rheumatoid arthritis (RA). In some embodiments, the ailment is multiple sclerosis (MS).

[0034] Aspects of the present invention include a kit for treating a disease or condition in an individual in need, comprising a multispecific antibody or pharmaceutical composition as described herein and instructions for use. In some embodiments, the kit further comprises at least one other agent. In some embodiments, at least one other agent comprises a chemotherapeutic agent.

[0035] Aspects of the present 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 specific for binding to CD3; and a third polypeptide subunit comprising: a heavy chain-only variable region in a monovalent or divalent configuration specific for binding to proteins 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 heavy chain-only variable region in a divalent configuration specific for binding to BCMA.

[0036] Aspects of the present invention include a bispecific triple-stranded antibody-like molecule comprising: 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.

[0037] Aspects of the present invention include pharmaceutical compositions comprising the bispecific triple-stranded antibody-like molecules described herein, polynucleotides encoding the bispecific triple-stranded antibody-like molecules described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0038] Aspects of the invention include a method for generating the bispecific triple-stranded antibody-like molecule described herein, comprising growing the cells described herein under conditions that allow the expression of the bispecific triple-stranded antibody-like molecule and isolating the bispecific triple-stranded antibody-like molecule from the cells.

[0039] Aspects of the invention include treatment methods comprising administering an effective dose of the bispecific triple-stranded antibody-like molecule or pharmaceutical composition described herein to an individual in need.

[0040] Aspects of the invention include the use of the bispecific triple-stranded antibody-like molecule described herein for the preparation of medicaments for the treatment of diseases or conditions in individuals in need.

[0041] Aspects of the invention include methods for treating diseases or ailments characterized by BCMA expression, comprising administering an effective dose of the bispecific triple-stranded antibody-like molecule or pharmaceutical composition described herein to an individual in need. 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.

[0042] Aspects of the present invention include a kit for treating a disease or condition in an individual in need, comprising a bispecific triple-stranded antibody-like molecule or pharmaceutical composition as described herein and instructions for use. In some embodiments, the kit further comprises at least one other reagent. In some embodiments, at least one other reagent comprises a chemotherapeutic agent.

[0043] These and other aspects will be further explained in the remainder of this disclosure, including the embodiments. Attached Figure Description

[0044] Figures A-C of Figure 1 provide a graphical illustration of various multispecific antibodies according to embodiments of the present invention.

[0045] Figure 2 Figures A and B show images of non-reducing SDS-PAGE analysis of multiple antibody species purified by protein A chromatography.

[0046] Figure 3 Figure A shows images of non-reducing SDS-PAGE analysis of multiple antibody species purified by protein A chromatography. Figure B shows images of reduced SDS-PAGE analysis of the same antibody species shown in Figure A.

[0047] Figure 4 Figures A-D are diagrams illustrating the Fcγ receptor-antibody interactions of various IgG1 antibody species according to embodiments of the present invention.

[0048] Figure 5Figures A-E are diagrams illustrating the Fcγ receptor-antibody interactions of various IgG4 antibody species according to embodiments of the present invention.

[0049] Figure 6 Figures A-D are diagrams illustrating the Fcγ receptor-antibody interactions of various IgG1 antibody species according to embodiments of the present invention.

[0050] Figure 7 Figures A-E are diagrams illustrating the Fcγ receptor-antibody interactions of various IgG4 antibody species according to embodiments of the present invention.

[0051] Figure 8 Figure A shows the binding of cells to human PSMA. Figure 8 Figure B shows the binding of cells to PSMA in cynomolgus monkeys.

[0052] Figure 9 This is a diagram illustrating T cell-mediated lysis of PSMA-positive cells using unstimulated T cells.

[0053] Figure 10 This is a diagram illustrating T cell-mediated lysis of PSMA-positive cells using pre-activated T cells.

[0054] Figure 11 This is a graph showing the percentage of specific lysis of PSMA-negative DU145 cells as a function of multispecific antibody concentration in the presence of pre-activated T cells.

[0055] Figure 12 This is a diagram showing the binding of PSMA×CD3 bispecific antibody to PSMA-positive and negative cells.

[0056] Figure 13 This is a diagram showing T cell-mediated lysis of PSMA-positive cells.

[0057] Figure 14 Figure A shows the change in T cell-mediated PSMA-positive cell lysis with antibody concentration. Figure B shows the change in cytokine (IFNγ) release with antibody concentration. Figure C shows the change in cytokine (IL-2) release with antibody concentration.

[0058] Figure 15 Figure A shows the change in T cell proliferation with antibody concentration. Figure B shows the change in T cell proliferation with antibody concentration. Figure C shows the CD8 to CD4 ratio of proliferating T cells. Figure D shows the CD8 to CD4 ratio of proliferating T cells.

[0059] Figure 16 This is a diagram illustrating the inhibition of 22Rv1 tumor growth in a tumor xenograft model.

[0060] Figure 17 This is a graph illustrating the change in the percentage of CD4+CD69+ T cells with bispecific antibody concentration, as shown in the legend.

[0061] Figure 18 This is a graph illustrating the change in the percentage of CD8+CD69+ T cells with bispecific antibody concentration, as shown in the legend.

[0062] Figure 19 This is a graph illustrating the change in the percentage of CD8+CD69+ T cells with bispecific antibody concentration, as shown in the legend.

[0063] Figure 20 This is a graph illustrating the change in the percentage of CD8+CD69+ T cells with bispecific antibody concentration, as shown in the legend.

[0064] Figure 21 Figures A-D provide several diagrams illustrating bispecific antibody-mediated tumor cell lysis. The ability of anti-CD3× anti-BCMA bispecific antibodies to kill three different BCMA+ tumor cell lines and one BCMA-negative cell line by redirecting activated primary T cells was analyzed. In this experiment, tumor cells were mixed with activated pan-T cells at a 10:1 E:T ratio, and bispecific antibodies were added simultaneously. Figure A shows the killing of RPMI-8226 cells, Figure B shows the killing of NCI-H929 cells, Figure C shows the killing of U-266 cells, and Figure D shows the killing of K562 cells (i.e., the negative control). The x-axis shows the concentration of the antibody used, and the y-axis shows the percentage of tumor cell lysis 6 hours after antibody addition.

[0065] Figure 22 Figures A-D provide several diagrams illustrating bispecific antibody-mediated IL-2 release. The levels of IL-2 cytokine release were measured after culturing resting human T cells with various tumor cell lines and escalating doses of anti-CD3×anti-BCMA bispecific antibodies. Figure A shows IL-2 release stimulated by RPMI-8226 cells, Figure B shows IL-2 release stimulated by NCI-H929 cells, Figure C shows IL-2 release stimulated by U-266 cells, and Figure D shows IL-2 release stimulated by K562 cells (i.e., the negative control).

[0066] Figure 23Figures A-D provide several diagrams illustrating bispecific antibody-mediated IFN-γ release. The levels of IFN-γ cytokine release were measured after culturing resting human T cells with various tumor cell lines and escalating doses of anti-CD3×anti-BCMA bispecific antibodies. Figure A shows IFN-γ release stimulated by RPMI-8226 cells, Figure B shows IFN-γ release stimulated by NCI-H929 cells, Figure C shows IFN-γ release stimulated by U-266 cells, and Figure D shows IFN-γ release stimulated by K562 cells (i.e., the negative control).

[0067] Figure 24 These are images obtained from non-reducing SDS-PAGE analysis of multiple antibody species purified by protein A chromatography.

[0068] Figure 25 This is a table showing the percentage of HMW types, monomers, and LMW types of the sample after purification of the indicated construct.

[0069] Figure 26 Figures A-D are diagrams illustrating the Fcγ receptor-antibody interactions of various IgG4 antibody species according to embodiments of the present invention.

[0070] Figure 27 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0071] Figure 28 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0072] Figure 29 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0073] Figure 30 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0074] Figure 31Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0075] Figure 32 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0076] Figure 33 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0077] Figure 34 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend.

[0078] Figure 35 Figures A and B are plots illustrating the changes in CD4+CD69+ T cell percentage (Figure A) and CD8+CD69+ T cell percentage (Figure B) with the concentration of the bispecific antibody as shown in the legend. Detailed Implementation

[0079] Unless otherwise indicated, the practice of this invention will employ conventional molecular biology techniques (including recombinant techniques), microbiological techniques, cell biology techniques, biochemical techniques, and immunological techniques, which are well known to those skilled in the art. These techniques are fully explained in references such as: "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Animal Cell Culture" (edited by R.R. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by F.M. Usubel et al., 1987 and periodically updated); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 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).

[0080] When providing ranges of values, it should be understood that, unless the context explicitly states otherwise, every intermediate value between the upper and lower limits of the range and any other value or intermediate value in the range, up to the tenths of the lower limit, is included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the scope of this invention, subject to any explicitly excluded limits in the range. When the range includes one or two limits, the range excluding any or both of the included limits is also included in this invention.

[0081] Unless otherwise indicated, antibody residues in this article 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)).

[0082] In the following description, numerous specific details are set forth to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures familiar to those skilled in the art have not been set forth to avoid obscuring the invention.

[0083] All references cited throughout this disclosure (including patent applications and publications) are incorporated herein by reference in their entirety.

[0084] I. definition

[0085] "Comprising" means that the listed elements are essential to the composition / method / kit, but may include other elements within the scope of the claims to form the composition / method / kit, etc.

[0086] "consisting essentially of..." means to limit the scope of the composition or method to the specified materials or steps that do not substantially affect the essential and novel features of the invention.

[0087] "Composed of" means excluding from the composition, method, or kit any element, step, or component not specified in the claims.

[0088] The antibody residues in this article are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in 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” (e.g., the EU index reported above by Kabat et al.) is generally used when referring to residues in the constant region of the immunoglobulin heavy chain. “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to residue numbers using the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbers using the EU numbering system.

[0089] An "antibody" or "immunoglobulin" is a molecule containing at least one heavy chain and one light chain, wherein the sequences of the amino-terminal domains of the heavy and light chains are variable, and are therefore often referred to as variable region domains or variable heavy chain (VH) or variable light chain (VH) domains. The two domains typically associate to form a specific binding region; however, as will be discussed here, specific binding can also be achieved using only the variable sequence of the heavy chain, and various non-natural conformations of antibodies are known and used in this technique.

[0090] "Functional" or "bioactive" antibodies or antigen-binding molecules (including heavy-chain-only antibodies and multispecific (e.g., bispecific) triple-chain antibody-like molecules (TCAs) as described herein) are antibodies or antigen-binding molecules capable of exerting one or more of their native activities in structural, regulatory, biochemical, or biophysical events. For example, functional antibodies or other binding molecules (e.g., TCAs) may have the ability to specifically bind antigens, and this binding may initiate or alter cellular or molecular events, such as signal transduction or enzyme activity. Functional antibodies or other binding molecules (e.g., TCAs) may also block ligand activation of receptors or act as agonists or antagonists. The ability of antibodies or other binding molecules (e.g., TCAs) to exert one or more of their native activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0091] The term antibody can refer to a full-length heavy chain, full-length light chain, or intact immunoglobulin molecule; or the immunoactive portion of any of these polypeptides, i.e., a polypeptide containing an antigen-binding site that specifically binds to a target antigen or a portion thereof, including (but not limited to) cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including modified subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. The light chain of the subject antibody can be a κ light chain (Vκ) or a λ light chain (Vλ). Immunoglobulins can be derived from any species. In one respect, immunoglobulins are primarily derived from humans.

[0092] As used herein, the term "monoclonal antibody" refers to an antibody derived from a largely homologous population of antibodies, meaning that the individual antibodies constituting the population are identical, except for the possibility of small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The monoclonal antibodies of the present invention can be manufactured by a hybridoma method first described by Kohler et al., (1975) Nature 256:495, and can also be manufactured by, for example, recombinant protein production methods (see, for example, U.S. Patent No. 4,816,567).

[0093] The term "variable" as used in the context of antibodies refers to the fact that certain portions of the antibody variable domain differ significantly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. It is concentrated in three segments of both the light and heavy chain variable domains, called hypervariable regions. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, predominantly in a β-sheet configuration, linked by three hypervariable regions that form loops that link the β-sheet structure and, in some cases, form a portion of the β-sheet structure. The hypervariable regions of each chain are tightly bound together by the FRs and, together with the hypervariable regions of the other chain, help form 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)). Constant domains do not directly participate in the binding of antibodies to antigens, but they exhibit a variety of effector functions, such as antibody-dependent cytotoxicity (ADCC).

[0094] The term “hypervariant region” as used herein refers to the amino acid residues of an antibody responsible for antigen binding. Hypervariant regions typically contain amino acid residues from the “complementarity-determining region” or “CDR” (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in 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 from the “hypervariant loop” (residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some implementations, "CDR" refers to the complementarity-determining region of an antibody as defined in Lefranc, MP et al., IMGT, International ImMunoGeneTics Database, Nucleic Acids Res., 27:209-212 (1999). "Frame region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0095] This article shows exemplary CDR names; however, those skilled in the art will understand that multiple definitions of CDR are commonly used, including the Kabat definition (see Zhao et al., “A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010; 47:694-700), which is based on sequence variability and is the most commonly used. 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).The alternative CDR definitions of interest include (but are not limited to) those published in the following literature: 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 explicitly incorporated herein by reference.

[0096] 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, such as one or more arms of an antibody, lacking the light chain of a conventional antibody. The term specifically includes (but is not limited to) homodimeric antibodies comprising a VH antigen-binding domain and constant CH2 and CH3 domains in the absence of the CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, homodimeric Ig-NARs comprising one variable domain (V-NAR) and five C-like constant domains (C-NAR), and their functional fragments; and soluble single-domain antibodies (sUniDabs). TMIn one embodiment, the heavy-chain-only antibody comprises a variable region antigen-binding domain, said variable region antigen-binding domain comprising frame 1, CDR1, frame 2, CDR2, frame 3, CDR3, and frame 4. In another embodiment, the heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In another embodiment, the heavy-chain-only antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy-chain-only antibodies with truncated CH2 and / or CH3 domains are also included herein. In another embodiment, the heavy chain comprises an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but without a hinge region. The heavy-chain-only antibody may be in dimer form, wherein the two heavy chains are covalently or non-covalently linked to each other by disulfide bonds or otherwise. Heavy chain antibodies may belong to the IgG subclass, but antibodies belonging to other subclasses (e.g., IgM, IgA, IgD, and IgE subclasses) are also included herein. In a particular embodiment, the heavy chain antibody is an IgG1, IgG2, IgG3, or IgG4 subtype, specifically the IgG1 subtype. In one embodiment, the heavy chain antibody is used herein as a binding (targeting) domain of a chimeric antigen receptor (CAR). The definition specifically includes human heavy chain antibodies (UniRat) generated from human immunoglobulin transgenic rats. TM ), called UniAbs TM UniAbs TM The variable region (VH) is called UniDabs TM It is also a universal structural unit that can be linked to the Fc region or serum albumin for the development of novel therapeutic agents with multispecificity, increased potency, and prolonged half-life. Due to the homodimer UniAbs TM Lacking a light chain and therefore a VL domain, the antigen is recognized by a single domain, namely the variable domain (VH or VHH) of the heavy chain of the heavy chain antibody.

[0097] As used herein, a “complete antibody chain” is an antibody chain containing a full-length variable region and a full-length constant region (Fc). A complete “standard” antibody contains a complete light chain and a complete heavy chain, as well as the light chain constant domain (CL) and heavy chain constant domain of secreted IgG, CH1, hinge, CH2, and CH3. Other isotypes (e.g., 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 a variant of its amino acid sequence. A complete antibody may have one or more “effective functions,” which refer to those biological activities attributable to the antibody’s Fc constant region (native sequence Fc region or 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 those that alter effector profiles, Fc receptor binding, etc.

[0098] Antibodies and various antigen-binding proteins can be provided in different classes based on the amino acid sequence of their heavy chain Fc (constant domain). There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the Fc corresponding to different classes of antibodies are respectively named α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310). Antibody light chains from any vertebrate species can be assigned to one of two types (referred to as κ (kappa) and λ (lambda)) based on the amino acid sequence of their constant structural domains. Antibodies according to embodiments of the present invention may comprise either a κ light chain sequence or a λ light chain sequence.

[0099] A “functional Fc region” possesses the “effective function” of the native Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions typically require interaction between the Fc region and receptors (e.g., FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and low-affinity FcRn receptors); and can be evaluated using a variety of analyses known in this technique. A “dead” or “silent” Fc is an Fc that has been mutated to retain activity (e.g., prolonged serum half-life) but does not activate high-affinity Fc receptors or has reduced Fc receptor affinity.

[0100] A “natural sequence Fc region” contains an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Natural sequence human Fc regions include, for example, the natural sequence human IgG1 Fc region (non-A and A variants); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their natural variants.

[0101] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native Fc region or the Fc region of the parental polypeptide, for example, about 1 to about 10 amino acid substitutions in the native Fc region or the Fc region of the parental polypeptide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, and more preferably about 1 to about 5 amino acid substitutions. The variant Fc region described herein preferably has at least about 80% homology with the native Fc region and / or the Fc region of the parental polypeptide, and most preferably at least about 90% homology, more preferably at least about 95% homology.

[0102] The human IgG4 Fc amino acid sequence (UniProtKB number P01861) is provided herein as SEQ ID NO:45. Silent IgG1 is described, for example, in Boesch, AW et al., “Highly parallel characterization of IgGFc binding interactions.” MAbs, 2014, 6(4): 915-27, the entire contents of which are incorporated herein by reference.

[0103] Other Fc variants are possible, including (but not limited to) Fc variants with the deletion of regions capable of forming disulfide bonds, or with certain amino acid residues eliminated at the N-terminus of the native Fc, or with methionine residues added thereto. Therefore, 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 another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, the antibody may contain an Fc variant.

[0104] Furthermore, Fc variants can be constructed to remove or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to achieve complement binding or Fc receptor binding. For example, and without limitation, deletion can occur at complement binding sites (e.g., C1q binding sites). Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0105] In some embodiments, the antibody comprises a variant human IgG4CH3 domain sequence containing the T366W mutation, which may optionally be referred to herein as the IgG4CH3 button sequence. In some embodiments, the antibody comprises a variant human IgG4CH3 domain sequence containing the T366S, L368A, and Y407V mutations, which may optionally be referred to herein as the IgG4CH3 pore sequence. The IgG4CH3 mutations described herein may be used in any suitable manner to place a “button” on the first heavy chain constant region of the first monomer in the antibody dimer and a “pore” on the second heavy chain constant region of the second monomer in the antibody dimer, thereby facilitating the correct pairing (heterodimerization) of desired heavy chain polypeptide subunit pairs in the antibody.

[0106] In some embodiments, the antibody comprises a heavy chain polypeptide subunit containing a variant human IgG4 Fc region having S228P mutation, F234A mutation, L235A mutation, and T366W mutation (twist). In some embodiments, the antibody comprises a heavy chain polypeptide subunit containing a variant human IgG4 Fc region having S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (well).

[0107] The term "Fc-region-comprising antibody" refers to an antibody containing an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody purification or by recombinant modification of the nucleic acid encoding the antibody. Therefore, the Fc-region-comprising antibody of the present invention may contain an antibody with or without K447.

[0108] Aspects of the present invention include antibodies comprising heavy chain-only variable regions in a monovalent or bivalent configuration. As used herein, the term "monovalent configuration" when referring to a heavy chain-only variable region domain means that only one heavy chain-only variable region domain exists, having a single binding site (see Figure 1, Figure A, right arm of the antibody). In contrast, the term "bivalent configuration" when referring to a heavy chain-only variable region domain means that two heavy chain-only variable region domains exist (each having a single binding site) and are linked by a linker sequence (see Figures 1, Figures B and C, right arm of the antibody). Non-limiting examples of linker sequences are further discussed herein and include (but are not limited to) GS linker sequences of different lengths. When the heavy chain-only variable region is in a bivalent configuration, each of the two heavy chain-only variable region domains may have binding affinity for the same antigen or for different antigens (e.g., different epitopes on the same protein; two different proteins, etc.). However, unless otherwise explicitly stated, the heavy chain variable region being described as having a “bivalent configuration” should be understood as containing two identical heavy chain variable region domains linked by a linker sequence, each of which has binding affinity for the same target antigen.

[0109] Aspects of the present invention include antibodies having multispecific configurations, including (but not limited to) bispecific, trispecific, etc. Numerous methods and protein configurations are known and used in bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0110] Various methods for generating multivalent artificial antibodies have been developed by recombinantly fusing variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on the peptide are linked by a peptide linker. A non-limiting example of such a peptide linker is the GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, wherein the sequence is repeated n times, where n is an integer in the range from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. (SEQ ID NO:93)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 may also be used and are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10):1357-69, the entire disclosure of which is incorporated herein by reference.

[0111] The term "triple-chain antibody-like molecule" or "TCA" as used herein refers to an antibody-like molecule comprising, substantially composed of, or composed of three polypeptide subunits, two of which comprise a heavy chain and a light chain of a monoclonal antibody, or a functional antigen-binding fragment of such antibody chain comprising, substantially composed of, or composed of, an antigen-binding region and at least one CH domain. This heavy / light chain is specific for binding to a first antigen. The third polypeptide subunit comprises, substantially composed of, or composed of a heavy-chain-only antibody, which comprises an Fc portion containing, in the absence of a CH1 domain, CH2 and / or CH3 and / or CH4 domains, and one or more antigen-binding domains (e.g., two antigen-binding domains), which bind to epitopes of a second antigen or different epitopes of a first antigen, wherein such binding domains are derived from or sequence-identical to a variable region of the antibody heavy or light chain. A portion of such a variable region may be derived from V H and / or V L Gene segments, D and J H Gene segment or J L Gene segment coding. Variable regions can be rearranged V. H DJ H V L DJ H V H J L or V L J L Gene segment encoding.

[0112] TCA-binding compounds utilize "heavy chain-only antibodies," "heavy chain antibodies," or "heavy chain polypeptides," as used herein, meaning single-chain antibodies containing the heavy chain constant regions CH2 and / or CH3 and / or CH4 but lacking the CH1 domain. In one embodiment, the heavy chain antibody comprises an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In yet another embodiment, the heavy chain antibody comprises an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain antibodies with truncated CH2 and / or CH3 domains are also included herein. In yet another embodiment, the heavy chain comprises an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but lacks a hinge region. Heavy chain-only antibodies may be in dimer form, wherein two heavy chains are covalently or non-covalently linked to each other by disulfide bonds or otherwise, and may optionally include an asymmetric interface between one or more CH domains to facilitate proper pairing between polypeptide chains. Heavy chain antibodies may belong to the IgG subclass, but antibodies belonging to other subclasses (e.g., IgM, IgA, IgD, and IgE subclasses) are also included herein. In certain embodiments, the heavy chain antibody is an IgG1, IgG2, IgG3, or IgG4 subtype, specifically an IgG1 or IgG4 subtype. Non-limiting examples of TCA-binding compounds are described, for example, in WO2017 / 223111 and WO2018 / 052503, the entire disclosure of which is incorporated herein by reference.

[0113] Heavy chain antibodies constitute about a quarter of IgG antibodies produced by camelids (e.g., camels and llamas) (Hamers-Casterman C. et al., Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but contain no light chain. Therefore, the variable antigen-binding moiety is called the VHH domain, and it represents the smallest naturally occurring intact antigen-binding site, only about 120 amino acids in length (Desmyter, A. et al., J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with high specificity and affinity can be generated by immunization against a variety of antigens (van der Linden, RH et al., Biochim. Biophys. Acta. 1431, 37-46 (1999)), and the VHH moiety can be readily cloned and expressed in yeast (Frenken, LGJ et al., J. Biotechnol. 78, 11-21 (2000)). Their expression levels, solubility, and stability are significantly higher than those of the classic F(ab) or Fv fragments (Ghahroudi, MA et al., FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to possess a single VH-like domain in their antibodies, termed 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)).

[0114] The term "CD3" refers to the human CD3 multi-subunit complex. The CD3 multi-subunit complex consists of six distinct polypeptide chains. These chains include the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and a CD3ζ chain homodimer (SwissProt 20963), which associate with the α and β chains of the T cell receptor. Unless otherwise noted, the term "CD3" includes any CD3 variant, isotype, and species homolog that is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides.

[0115] "BCMA×CD3 antibody" is a multispecific heavy chain-only antibody, such as a bispecific heavy chain-only antibody, containing two distinct antigen-binding regions, one of which specifically binds to the antigen BCMA and the other specifically binds to CD3. "PSMA×CD3 antibody" is a multispecific heavy chain-only antibody, such as a bispecific heavy chain-only antibody, containing two distinct antigen-binding regions, one of which specifically binds to the antigen PSMA and the other specifically binds to CD3. "CD19×CD3 antibody" is a multispecific heavy chain-only antibody, such as a bispecific heavy chain-only antibody, containing two distinct antigen-binding regions, one of which specifically binds to the antigen CD19 and the other specifically binds to CD3.

[0116] As used in this article, the term "BCMA" 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 preferentially expressed in differentiated plasma cells. According to UniProt, the extracellular domain of human BCMA consists of amino acids 1-54 (or 5-51).

[0117] The terms “anti-BCMA heavy chain antibody” and “BCMA heavy chain antibody” are used in this article to refer to heavy chain antibodies as defined above that are immune-specifically bound to BCMA.

[0118] As used herein, the term "PSMA" refers to a type II transmembrane protein with N-acetylated α-linked acidic dipeptidase, folic acid hydrolase, and dipeptidyl peptidase activities. The term "PSMA" includes PSMA proteins from any human and non-human animal species, and specifically includes human PSMA as well as PSMA from non-human mammals.

[0119] As used herein, the term "human PSMA" includes any variant, isotype, and species homolog of human PSMA (UniProt Q04609), regardless of its origin or mode of preparation. Therefore, "human PSMA" includes both naturally expressed human PSMA and PSMA expressed on cells transfected with the human PSMA gene.

[0120] 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 interchangeably herein and refer to heavy chain only antibodies as defined above that specifically bind to PSMA (including human PSMA) as defined above. This definition includes (but is not limited to) antibodies produced by transgenic animals (e.g., transgenic rats or mice expressing human immunoglobulins, including those producing human anti-PSMA UniAbs as defined above). TM UniRats of AntibodiesTM Human heavy chain antibodies produced by ).

[0121] As used herein, the terms “CD19” and “differentiation cluster 19” refer to molecules expressed at all stages of B cell development until their eventual differentiation into plasma cells. The term “CD19” includes the CD19 protein of any human and non-human animal species, and specifically includes human CD19 as well as CD19 of non-human mammals.

[0122] As used herein, the term "human CD19" includes any variant, isotype, and species homolog of human CD19 (UniProt P15391), regardless of its origin or mode of preparation. Therefore, "human CD19" includes human CD19 naturally expressed in cells and CD19 expressed in cells transfected with the human CD19 gene.

[0123] 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 heavy chain only antibodies as defined above that specifically bind to CD19 (including human CD19) as defined above. This definition includes (but is not limited to) antibodies produced by transgenic animals (e.g., transgenic rats or mice expressing human immunoglobulins, including UniRats that produce human anti-CD19 UniAb™ antibodies as defined above). TM Human heavy chain antibodies produced by ).

[0124] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after alignment and the introduction of vacancies (if necessary) to achieve the maximum sequence identity percentage, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the amino acid sequence identity percentage, alignment can be performed in various ways well known to those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2.

[0125] "Separated" antibodies are antibodies that have been identified and isolated from and / or recovered from components of their natural environment. Contaminant components of their natural environment are materials that can interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified to (1) greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence as determined by a twist-cup sequencer; or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Separated antibodies include in situ antibodies from recombinant cells, as at least one component of the antibody's natural environment is absent. However, typically, separated antibodies are prepared through at least one purification step.

[0126] The antibodies of this invention include multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher-order independent specific binding affinity (e.g., higher-order multi-epitope specificity), and tetravalent antibodies and antibody fragments. The terms "multispecific antibody," "multispecific heavy-chain-only antibody," "multispecific heavy-chain antibody," and "multispecific UniAb" are also used. TM "In this article, it is used in the broadest sense and covers all antibodies with more than one binding specificity."

[0127] The multispecific antibodies of the present invention specifically include antibodies (i.e., bivalent and bicomplementary) that specifically bind to two or more non-overlapping epitopes on BCMA, PSMA, or CD19 proteins (e.g., human BCMA, human PSMA, or human CD19). The multispecific heavy chain antibodies of the present invention further specifically include antibodies (i.e., bivalent and bicomplementary) that specifically bind to epitopes on BCMA, PSMA, or CD19 proteins (e.g., human BCMA, human PSMA, or human CD19) and epitopes on different proteins (e.g., CD3 proteins, such as human CD3). The multispecific heavy chain antibodies of the present invention further specifically include antibodies (i.e., trivalent and bicomplementary) that specifically bind to two or more non-overlapping or partially overlapping epitopes on BCMA, PSMA, or CD19 proteins (e.g., human BCMA, human PSMA, or human CD19) and epitopes on different proteins (e.g., CD3 proteins, such as human CD3).

[0128] An epitope is a site on the surface of an antigen molecule that a single antibody molecule binds to. Typically, antigens have several or many different epitopes and react with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0129] Epitope mapping is the process of identifying the binding site or epitope of an antibody on its target antigen. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed from a continuous amino acid sequence in a protein. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but aggregate together when the protein folds into its three-dimensional structure.

[0130] "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As noted above, this invention specifically includes heavy chain antibodies with multi-epitope specificity, namely heavy chain antibodies that bind to one or more non-overlapping epitopes on BCMA, PSMA, or CD19 proteins (e.g., human BCMA, human PSMA, or human CD19); and heavy chain antibodies that bind to one or more epitopes on BCMA, PSMA, or CD19 proteins and epitopes on different proteins (e.g., CD3). The terms "non-overlapping epitope" or "non-competitive epitope" of an antigen are defined herein as meaning an epitope that is 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 multi-specific antibodies that target the same antigen do not competitively bind to that antigen and are able to bind to the antigen simultaneously.

[0131] When two antibodies recognize the same or spatially overlapping epitopes, the antibody binds to "substantially the same epitope" as the reference antibody. The most widely used and rapid method for determining whether two epitopes bind to the same or spatially overlapping epitopes is a competitive assay, which can be constructed using labeled antigens or labeled antibodies in all sorts of different formats. 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 radioactive or enzyme labeling.

[0132] As used in this article, the term "valence" refers to the specific number of binding sites in an antibody molecule.

[0133] A monovalent antibody has only one binding site. Therefore, a monovalent antibody is also monospecific.

[0134] "Multivalent" antibodies have two or more binding sites. Therefore, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two, three, and four binding sites, respectively. Thus, the bispecific antibodies of the present invention are at least bivalent, and may be trivalent, tetravalent, or other multivalent. A bivalent antibody according to an embodiment of the present invention may have two binding sites to the same epitope (i.e., bivalent monocomplementary sites) or two different epitopes (i.e., bivalent bicomplementary sites).

[0135] Numerous methods and protein conformations are known and used to prepare bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0136] The term "triple-chain antibody-like molecule" or "TCA" as used herein refers to an antibody-like molecule comprising, substantially composed of, or composed of three polypeptide subunits, two of which comprise a heavy chain and a light chain of a monoclonal antibody, or a functional antigen-binding fragment of such antibody chain comprising an antigen-binding region and at least one CH domain, substantially composed of, or composed of, such heavy chain / light chain. This heavy chain / light chain is specific for binding to a first antigen. The third polypeptide subunit comprises, substantially composed of, or composed of a heavy chain-only antibody, wherein the heavy chain-only antibody comprises an Fc portion containing a CH2 and / or CH3 and / or CH4 domain and an antigen-binding domain in the absence of a CH1 domain, the antigen-binding domain binding to an epitope of a second antigen or a different epitope of a first antigen, wherein such binding domain is derived from or sequence-identical to a variable region of the antibody heavy or light chain. A portion of such a variable region may be derived from V H and / or V L Gene segments, D and J H Gene segment or J L Gene segment coding. Variable regions can be rearranged V. H DJ H V L DJ H V H J L or V L J L Gene segment encoding. TCA protein utilizes heavy chain-only antibodies as defined above.

[0137] The term “chimeric antigen receptor” or “CAR” is used in the broadest sense herein and refers to a modified receptor that has a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) transplanted into a transmembrane domain and an intracellular signaling domain. Typically, a receptor is used to transplant the specificity of a monoclonal antibody onto T cells to generate 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 generate artificial T cell receptors for use in immunotherapy. In one embodiment, “CAR-T cell” means a therapeutic T cell expressing a transgene encoding one or more chimeric antigen receptors, said one or more chimeric antigen receptors comprising at least an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0138] The term "human antibody" is used herein and includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo. The term "human antibody" specifically includes heavy-chain-only antibodies having human heavy-chain variable region sequences produced by transgenic animals (e.g., transgenic rats or mice), specifically those derived from UniRats as defined above. TM The generated UniAbs TM .

[0139] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule containing amino acid sequences from at least two different Ig loci, such as 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 or artificial Fc region and a human individual genotype. Such immunoglobulins can be isolated from the animals of this invention that have been modified to produce such chimeric antibodies.

[0140] As used herein, the term "effective cell" refers to an immune cell that participates in the effector phase of an immune response, which differs from the cognitive and activation phases of the immune response. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells (e.g., natural killer cells) can induce antibody-dependent cytotoxicity (ADCC). For example, monocytes and macrophages expressing Fc receptors participate in the specific killing of target cells and present antigens to other components of the immune system or bind to antigen-presenting cells. In some embodiments, effector cells may phagocytose target antigens or target cells.

[0141] "Human effector cells" are leukocytes that express receptors (e.g., T-cell receptors or FcRs) and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; NK cells are preferred. Effector cells can be isolated from their natural sources, such as from blood or PBMCs as described herein.

[0142] The term “immune cells” is used in the broadest sense herein, including (but not limited to) cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0143] Antibody "effective function" refers to those biological activities attributable to the Fc region of the antibody (either the native Fc region or the Fc region of an amino acid sequence variant). 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).

[0144] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently lyse the target cells. The primary cells that mediate ADCC (i.e., NK cells) 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 evaluate the ADCC activity of the molecule of interest, in vitro ADCC analyses, such as those described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, can be performed. Effector cells that can be used for such analyses include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively or otherwise, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, for example, as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0145] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to dissolve its target in the presence of complement. The complement activation pathway is initiated by binding the first component (C1q) of the complement system to a molecule (e.g., an antibody) that is complexed with a homologous antigen. To evaluate complement activation, a CDC analysis can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0146] "Binding affinity" refers to the strength of the sum 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 indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed by the dissociation constant (Kd). Affinity can be measured using methods known in this technique. Low-affinity antibodies typically bind antigens slowly and tend to dissociate readily, while high-affinity antibodies typically bind antigens rapidly and tend to maintain the binding.

[0147] As used herein, “Kd” or “Kd value” refers to the dissociation constant determined in kinetic mode using a BioLayer Interferometry instrument (Fortebio Inc., Menlo Park, CA) in Octet QK384. For example, a mouse Fc fusion antigen was loaded onto an anti-mouse Fc sensor and then immersed in wells containing antibodies to measure the concentration-dependent association rate (k-association). In the final step, the antibody dissociation rate (k-dissociation) was measured, in which the sensor was immersed in wells containing only buffer. Kd is the ratio of k-dissociation to k-association. (For further details, see Concepcion, J et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0148] The terms “treatment,” “treating,” etc., used herein generally refer to achieving the desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals and includes: (a) preventing the occurrence of a disease in subjects who may be susceptible but have not yet been diagnosed with the disease; (b) suppressing a disease, i.e., halting its development; or (c) alleviating a disease, i.e., causing the disease to subside. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Particular attention is paid to the treatment of ongoing diseases, where the treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Such treatment is intended to be administered before complete loss of function of the invaded tissue. Therapeutic agents may be administered during the symptomatic phase of a disease, and in some cases after the symptomatic phase of a disease.

[0149] "Therapeutic effective amount" is intended to refer to the amount of active agent required to provide a therapeutic benefit to a subject. For example, "therapeutic effective amount" is an amount that induces, improves, or otherwise causes an improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or an improvement in resistance to the condition.

[0150] As used in this article, “prostate cancer” refers to a malignant tumor originating from the glands of the prostate.

[0151] The term "characterized by PSMA expression" broadly refers to any disease or condition in which PSMA expression is associated with or involved in one or more pathological processes characteristic of the disease or condition. Such conditions include (but are not limited to) prostate cancer.

[0152] In the context of this invention, the terms "B-cell sarcoma" or "mature B-cell sarcoma" 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, and Burkitt's lymphoma. This includes follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma (e.g., plasmacytic myeloma), plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodular marginal B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granuloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia, primary effusion lymphoma, and AIDS-related non-Hodgkin's lymphoma.

[0153] The term "characterized by CD19 expression" broadly refers to any disease or condition in which CD19 expression is associated with or involved in one or more pathological processes characteristic of the disease or condition. Such conditions include (but are not limited to) B-cell neoplasms.

[0154] The term "characterized by BCMA expression" broadly refers to any disease or condition in which BCMA expression is associated with or involved in one or more pathological processes characteristic of the disease or condition. Such conditions include (but are not limited to) B-cell sarcomas.

[0155] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to a mammal undergoing treatment evaluation and / or being treated. In the implementation plan, the mammal is a human. The terms “subject,” “individual,” and “patient” cover (but are not limited to) individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects may be humans, but also include other mammals, particularly those that can be used as laboratory models of human diseases (e.g., mice, rats, etc.).

[0156] The term "pharmaceutical formulation" refers to a formulation that is biologically effective for the permitted active ingredient and does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (mediators, additives) are those excipients that are reasonably administered to a subject mammal to provide an effective dose of the active ingredient used.

[0157] "Sterile" preparations are sterile or contain no or substantially no live microorganisms and their spores. "Frozen" preparations are preparations kept at temperatures below 0°C.

[0158] A “stable” formulation is one in which the protein substantially retains its physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical and chemical stability as well as its biological activity during storage. The storage period is typically selected based on the expected shelf life of the formulation. Various analytical techniques for measuring protein stability are available in this field and are reviewed, for example, in *Peptide and Protein Drug Delivery*, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and *Jones. A. Adv. Drug Delivery Rev. 10:29-90* (1993). Stability can be measured by maintaining the protein at a selected temperature for a selected period of time. Stability can be assessed qualitatively and / or quantitatively in a variety of ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); evaluating charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary band electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced antibodies with intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; and assessing antibody bioactivity or antigen-binding function. Instability can involve one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shearing / hydrolysis / fragmentation (e.g., hinge fragmentation), succinimide formation, unpaired cysteine ​​residues, N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0159] II. Detailed description

[0160] Anti-BCMA antibody

[0161] This invention relates to several families of closely related antibodies that bind to human BCMA. The variable regions of these antibody families are described in U.S. Patent Publications Nos. US20190352412, US20200157232, and US20200048348, and PCT Publications Nos. WO2018237037 and WO2019006072, the entire disclosure of which is incorporated herein by reference. Non-limiting selections of representative anti-BCMA heavy chain antibody variable domain sequences are provided in Table 1 below.

[0162] Table 1: Amino acid sequences of variable domains of anti-BCMA heavy chain antibody.

[0163]

[0164]

[0165] The anti-BCMA antibody sequence may be selected from those sequences provided herein for development and therapeutic or other uses (including, but not limited to, use as a multispecific, such as a bispecific antibody). In some embodiments, a bispecific or multispecific antibody is provided, which may have any of the conformations discussed herein, including (but not limited to) TCA. The bispecific antibody contains at least a heavy chain variable region that is specific against proteins other than BCMA.

[0166] When the protein of this invention is a bispecific antibody, one binding portion specifically targets human BCMA, while the other arm can specifically target target cells, tumor-associated antigens, targeting antigens (e.g., integrins), pathogen antigens, checkpoint proteins, etc. Target cells specifically include cancer cells, such as hematologic malignancies, such as B-cell tumors, as discussed below.

[0167] Bispecific antibodies in various formats are within the scope of this invention, including (but not limited to) single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and folds thereof. Specifically, bispecific antibodies described herein include T-cell bispecific antibodies (anti-BCMA × anti-CD3 antibodies) that bind to BCMA (which is selectively expressed on plasma cells (PCs) and multiple myeloma (MM)) and CD3. Such antibodies induce potent T-cell-mediated killing of BCMA-carrying cells and can be used to treat tumors, particularly hematologic malignancies such as B-cell malignancies, as further discussed herein.

[0168] In a preferred embodiment, the bispecific antibody is a TCA comprising: an anti-CD3 VH domain paired with a light chain variable domain (VL), wherein the VH and VL domains together have binding affinity for CD3; a heavy chain variable domain of a heavy chain-only antibody, which has binding affinity for BCMA and is in a monovalent or bivalent configuration; and a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, and T366W mutation (twist), and a second heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (pore). This variant or modified IgG4 Fc domain prevents undesirable Fab exchange, reducing the effector function of the antibody, and also promotes heterodimerization of heavy chain polypeptide subunits to form a bispecific antibody.

[0169] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:58.

[0170] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59.

[0171] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:58, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0172] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0173] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising an anti-CD3 heavy chain containing SEQ ID NO:56, an anti-CD3 light chain containing SEQ ID NO:49, and an anti-BCMA heavy chain containing SEQ ID NO:58.

[0174] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59.

[0175] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:58, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0176] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0177] 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0178] 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0179] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0180] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0181] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:58.

[0182] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59.

[0183] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:58, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0184] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59, wherein the bispecific antibody does not contain the anti-BCMA light chain.

[0185] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising an anti-CD3 heavy chain containing SEQ ID NO:56, an anti-CD3 light chain containing SEQ ID NO:49, and an anti-BCMA heavy chain containing SEQ ID NO:58.

[0186] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59.

[0187] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:58, wherein the bispecific antibody does not contain the anti-BCMA light chain.

[0188] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:56, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:59, wherein the bispecific antibody does not contain the anti-BCMA light chain.

[0189] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0190] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0191] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0192] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0193] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:76.

[0194] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77.

[0195] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:76, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0196] In some embodiments, the present invention comprises a bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0197] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising an anti-CD3 heavy chain containing SEQ ID NO:75, an anti-CD3 light chain containing SEQ ID NO:49, and an anti-BCMA heavy chain containing SEQ ID NO:76.

[0198] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77.

[0199] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:76, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0200] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0201] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific antibody comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0202] 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0203] 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0204] 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0205] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:76.

[0206] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77.

[0207] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:76, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0208] In some embodiments, the present invention comprises a bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77, wherein the bispecific antibody does not contain an anti-BCMA light chain.

[0209] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising an anti-CD3 heavy chain containing SEQ ID NO:75, an anti-CD3 light chain containing SEQ ID NO:49, and an anti-BCMA heavy chain containing SEQ ID NO:76.

[0210] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77.

[0211] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:76, wherein the bispecific antibody does not contain the anti-BCMA light chain.

[0212] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising i) an anti-CD3 heavy chain containing SEQ ID NO:75, ii) an anti-CD3 light chain containing SEQ ID NO:49, and iii) an anti-BCMA heavy chain containing SEQ ID NO:77, wherein the bispecific antibody does not contain the anti-BCMA light chain.

[0213] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0214] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded 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, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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.

[0215] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0216] In some embodiments, the present invention comprises a human monoclonal IgG4 bispecific triple-stranded antibody-like molecule (TCA) comprising (i) a first binding arm to human CD3 and (ii) a second binding arm to human BCMA, the first binding arm comprising a first heavy chain and a light chain, and the second binding arm comprising a bivalent second heavy chain, wherein 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, and wherein the second binding arm does not comprise a light chain.

[0217] Anti-CD19 antibody

[0218] This invention provides a family of closely related antibodies that bind to human CD19. The variable regions of this family of antibodies are described in PCT Publication WO2020018922, the entire disclosure of which is incorporated herein by reference. Anti-CD19 antibody sequences may be selected from those 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 that may have any of the configurations discussed herein, including (but not limited to) TCA. Bispecific antibodies comprise at least a heavy chain variable region that specifically targets proteins other than CD19.

[0219] When the protein of this invention is a bispecific antibody, one binding portion specifically targets human CD19, while the other arm can specifically target target cells, tumor-associated antigens, targeting antigens (e.g., integrins), pathogen antigens, checkpoint proteins, etc. Target cells specifically include cancer cells, such as hematologic malignancies, such as B-cell malignancies, as discussed below.

[0220] Bispecific antibodies in various formats are within the scope of this invention, including (but not limited to) single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, tetra-chain polypeptides, and foldwise polypeptides thereof. Specifically, bispecific antibodies described herein include T-cell bispecific antibodies (anti-CD19 × anti-CD3 antibodies) that bind to both CD19 (which is selectively expressed on mature B cells) and CD3. Such antibodies induce potent T-cell-mediated killing of CD19-expressing cells and can be used to treat tumors, particularly hematologic malignancies such as B-cell tumors, as further discussed herein.

[0221] In a preferred embodiment, the bispecific antibody is a TCA comprising: an anti-CD3 VH domain paired with a light chain variable domain (VL), wherein the VH and VL domains together have binding affinity for CD3; a heavy chain variable domain of a heavy chain-only antibody, which has binding affinity for CD19 and is in a monovalent or bivalent configuration; and a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, and T366W mutation (twist), and a second heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (pore). This variant or modified IgG4 Fc domain prevents undesirable Fab exchange, reducing the effector function of the antibody, and also promotes heterodimerization of heavy chain polypeptide subunits to form a bispecific antibody.

[0222] Anti-PSMA antibody

[0223] This invention provides a family of closely related antibodies that bind to human PSMA. This family of antibodies is exemplified by the provided heavy chain variable region (VH) sequences of SEQ ID NOs: 24 to 54 shown in Table 2. The antibody family provides a variety of benefits that facilitate its use as a clinical therapeutic agent. Antibodies include members with a range of binding affinities, thereby allowing selection of specific sequences with desired binding affinity.

[0224] Table 2. Amino acid sequences of variable domains of anti-PSMA heavy chain antibodies.

[0225]

[0226]

[0227]

[0228]

[0229]

[0230] In a preferred embodiment, the bispecific antibody is a TCA comprising: an anti-CD3 VH domain paired with a light chain variable domain (VL), wherein the VH and VL domains together have binding affinity for CD3; a heavy chain variable domain of a heavy chain-only antibody, which has binding affinity for PSMA and is in a monovalent or bivalent configuration; and a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, and T366W mutation (twist), and a second heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (pore). This variant or modified IgG4 Fc domain prevents undesirable Fab exchange, reducing the effector function of the antibody, and also promotes heterodimerization of heavy chain polypeptide subunits to form a bispecific antibody.

[0231] CD3×target protein triplet antibody-like molecule (TCA)

[0232] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the conformations discussed herein, including (but not limited to) bispecific triple-chain antibody-like molecules. In some embodiments, a multispecific antibody may comprise a heavy chain / light chain pair having binding specificity to a first antigen (e.g., CD3), and a heavy chain-only antibody. In some embodiments, the heavy chain of a heavy chain-only antibody comprises an Fc portion containing CH2 and / or CH3 and / or CH4 domains in the absence of a CH1 domain. In a particular embodiment, a bispecific antibody comprises a heavy chain / light chain pair having binding specificity to an antigen on effector cells (e.g., CD3 protein on T cells), and a heavy chain-only antibody comprising a heavy chain containing an antigen-binding domain having binding specificity to BCMA, PSMA, or CD19.

[0233] In a preferred embodiment, the bispecific antibody is a TCA comprising: an anti-CD3 VH domain paired with a light chain variable domain (VL), wherein the VH and VL domains together have binding affinity for CD3; a heavy chain variable domain of a heavy chain-only antibody, which has binding affinity for BCMA, PSMA, or CD19; and a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, and T366W mutation (twist), and a second heavy chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (pore). This variant or modified IgG4 Fc domain prevents unwanted Fab exchange, reducing the effector function of the antibody, and also promotes heterodimerization of heavy chain polypeptide subunits to form a bispecific antibody.

[0234] In some embodiments, the multispecific antibody comprises a CD3-binding VH domain paired with a light chain variable domain. In some embodiments, the light chain is a fixed light chain. In some embodiments, within the human VH framework, 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 some embodiments, within the human VL framework, 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. The CD3-binding VH domain and the light chain variable domain together 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 with 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 with the heavy chain variable region sequence of SEQ ID NO:43.

[0235] Multispecific antibodies containing the aforementioned CD3-binding VH domain and light chain variable domain possess advantageous properties, for example, as described in PCT Publication No. WO2018 / 052503, the entire contents of which are incorporated herein by reference. Any of the multispecific antibodies and antigen-binding domains described herein that have binding affinity for BCMA, PSMA, or CD19 can be combined with the CD3-binding domain and fixed light chain domain described herein to produce multispecific antibodies that have binding affinity for one or more BCMA epitopes, PSMA epitopes, or CD19 epitopes and CD3.

[0236] Table 3. Amino acid sequences of anti-CD3 heavy and light chains CDR1, CDR2, and CDR3.

[0237]

[0238] Table 4. Amino acid sequences of the variable regions of the anti-CD3 heavy and light chains.

[0239]

[0240] Table 5: Fc region sequences of human IgG1 and IgG4.

[0241]

[0242]

[0243] Table 6: Anti-CD3 antibody sequence.

[0244]

[0245]

[0246]

[0247]

[0248] Table 7: Anti-TAA antibody sequences.

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] In some embodiments, bispecific or multispecific antibodies are provided, which may have any of the conformations discussed herein, including (but not limited to) bispecific triple-chain antibody-like molecules. In some embodiments, the bispecific antibody may comprise at least one heavy chain variable region having binding specificity to BCMA, PSMA, or CD19, and at least one heavy chain variable region having binding specificity to a different protein (e.g., CD3). In some embodiments, the bispecific antibody may comprise a heavy chain / light chain pair having binding specificity to a first antigen and a heavy chain in a monovalent or bivalent conformation of a heavy chain antibody only, said heavy chain comprising an Fc portion containing CH2 and / or CH3 and / or CH4 domains and an antigen-binding domain in the absence of a CH1 domain, said antigen-binding domain binding to an epitope of a second antigen or a different epitope of a first antigen. In one particular embodiment, a bispecific antibody comprises a heavy chain / light chain pair that has binding specificity to an antigen on effector cells (e.g., CD3 protein on T cells), and a heavy chain-only antibody comprises a heavy chain that contains an antigen-binding domain that has binding specificity to BCMA, PSMA, or CD19 and is in a monovalent or bivalent configuration.

[0257] In some embodiments, when the antibody of the present invention is a bispecific antibody, one arm of the antibody (a binding portion or a binding unit) specifically targets human BCMA, human PSMA, or human CD19, while the other arm may specifically target target cells, tumor-associated antigens, targeting antigens (e.g., integrins), pathogen antigens, checkpoint proteins, etc. Target cells specifically include cancer cells, including (but not limited to) cells of solid tumors (e.g., prostate tumors), as discussed below. In some embodiments, one arm of the antibody (a binding portion or a binding unit) specifically targets human BCMA, human PSMA, or human CD19, while the other arm specifically targets CD3.

[0258] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide containing the sequence of SEQ ID NO:43 linked to the sequence of SEQ ID NO:48, an anti-CD3 heavy chain polypeptide containing the sequence of any one of SEQ ID NO:44, 45, 46, 47, 50, 51, 52, 53, 56, or 57, and an anti-BCMA heavy chain polypeptide containing the sequence of any one of SEQ ID NO:58, 59, or 60. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:58, 59, or 60. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:58. In a preferred embodiment, the antibody is a TCA composed of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:58. In one preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:59. In another preferred embodiment, the antibody is a TCA composed of a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:59. In yet another preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:60. In yet another preferred embodiment, the antibody is a TCA composed of a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:60.

[0259] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide containing the sequence of SEQ ID NO:43 linked to the sequence of SEQ ID NO:48, an anti-CD3 heavy chain polypeptide containing the sequence of any one of SEQ ID NO:44, 45, 46, 47, 50, 51, 52, 53, 56, or 57, and an anti-PSMA heavy chain polypeptide containing the sequence of any one of SEQ ID NO:61, 62, 63, 64, 65, or 66. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:61. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:61. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:62. In one preferred embodiment, the antibody is a TCA composed of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:62. In another preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:63. In yet another preferred embodiment, the antibody is a TCA composed of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:63. In yet another preferred embodiment, the antibody is a TCA comprising a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:64. In yet another preferred embodiment, the antibody is a TCA composed of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:64. In yet another preferred embodiment, the antibody is a TCA comprising a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:65. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:65.In a preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:66. In another preferred embodiment, the antibody is a TCA composed of the first polypeptide of SEQ ID NO:49, the second polypeptide of SEQ ID NO:56, and the third polypeptide of SEQ ID NO:66.

[0260] In some embodiments, the antibody comprises an anti-CD3 light chain polypeptide containing the sequence of SEQ ID NO:43 linked to the sequence of SEQ ID NO:48, an anti-CD3 heavy chain polypeptide containing the sequence of any one of SEQ ID NO:44, 45, 46, 47, 50, 51, 52, 53, 56, or 57, and an anti-CD19 heavy chain polypeptide containing the sequence of any one of SEQ ID NO:67, 68, or 69. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:67. In a preferred embodiment, the antibody is a TCA consisting of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:67. In a preferred embodiment, the antibody is a TCA comprising a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:68. In one preferred embodiment, the antibody is a TCA composed of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:68. In another preferred embodiment, the antibody is a TCA comprising a first polypeptide containing SEQ ID NO:49, a second polypeptide containing SEQ ID NO:56, and a third polypeptide containing SEQ ID NO:69. In yet another preferred embodiment, the antibody is a TCA composed of a first polypeptide of SEQ ID NO:49, a second polypeptide of SEQ ID NO:56, and a third polypeptide of SEQ ID NO:69.

[0261] Multispecific antibodies in various formats are within the scope of this invention, including (but not limited to) single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and folds thereof. Specifically, the multispecific antibodies described herein include T-cell multispecific (e.g., bispecific) antibodies that bind to BCMA, PSMA, or CD19 and CD3 (anti-BCMA×anti-CD3 antibody, anti-PSMA×anti-CD3 antibody, anti-CD19×anti-CD3 antibody) and contain a variant human IgG4 Fc domain comprising a first heavy-chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, and T366W mutation (twist), and a second heavy-chain constant region sequence containing S228P mutation, F234A mutation, L235A mutation, T366S mutation, L368A mutation, and Y407V mutation (pore). This variant or modified IgG4 Fc domain prevents unwanted Fab exchange, reduces antibody effector function, and also promotes heterodimerization of heavy chain polypeptide subunits to form bispecific antibodies. These antibodies induce virile T cell-mediated killing of cells expressing BCMA, PSMA, or CD19, respectively.

[0262] Antibody preparation

[0263] The multispecific antibodies of this invention can be prepared by methods known in this art. In a preferred embodiment, the heavy chain antibodies described herein are produced by transgenic animals (including transgenic mice and rats, preferably rats), wherein endogenous immunoglobulin genes are knocked out or deactivated. In a preferred embodiment, the heavy chain antibodies described herein are produced in UniRat... TM Generated in UniRat. TM Different naturally optimized profiles of endogenous immunoglobulin genes were expressed using human immunoglobulin heavy chain translocations to silence endogenous immunoglobulin genes and to express intact human HCAb. Although endogenous immunoglobulin gene loci in rats can be knocked out or silenced using various techniques, in UniRat… TM In this study, zinc finger (endogenous) nuclease (ZNF) technology was used to inactivate the endogenous rat heavy chain J-locus, light chain Cκ locus, and light chain Cλ locus. ZNF constructs used for microinjection into oocytes generated IgH and IgL gene knockout (KO) lines. For details, see, for example, Geurts et al., 2009, Science 325:433. Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941, which reported the characterization of Ig heavy chain knockout rats. The advantage of ZNF technology is that non-homologous end ligation can silence genes or loci by deletions up to several kb and also provides target sites for homologous integration (Cui et al., 2011, Nat Biotechnol 29:64-67). In UniRat... TMHuman heavy chain antibodies produced in this process are called UniAbs. TM They can bind to epitopes that cannot be attacked by conventional antibodies. Their high specificity, affinity, and small size make them ideal for both monospecific and multispecific applications.

[0264] Except UniAbs TM In addition, this article specifically includes heavy-chain-only antibodies lacking the Camelidae VHH framework and mutations, as well as their functional VH regions. Such heavy-chain-only antibodies can be produced, for example, in transgenic rats or mice containing the complete human heavy-chain-only locus as described, for example, in WO2006 / 008548, but other transgenic mammals, such as rabbits, guinea pigs, and rats, are also possible, with rats and mice being preferred. Heavy-chain-only antibodies (including their VHH or VH functional fragments) can also be produced via recombinant DNA technology by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0265] The advantages of combining heavy chain antibody domains with small molecule drugs include: monovalent or multivalent formulations; low toxicity; and cost-effective manufacturing. Due to their small size, these domains are easy to administer, including oral or topical application, and are characterized by high stability, including gastrointestinal stability; and their half-life can be tailored for the desired use or indication. Furthermore, the VH and VHH domains of HCAbs can be manufactured in a cost-effective manner.

[0266] In certain embodiments, the heavy chain antibodies (including UniAbs) of the present invention TM The native amino acid residue at the first position (amino acid position 101 according to the Kabat numbering system) in the FR4 region is substituted with another amino acid residue capable of disrupting the native amino acid residue containing that position or the surface-exposed hydrophobic patch associated with it. Such hydrophobic patches are typically embedded in the interface with the constant region of the antibody light chain, but become surface-exposed in HCAbs, at least in part due to undesirable aggregation and light chain association of the HCAb. The substituted amino acid residue is preferably charged, and 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 antibody derived from a transgenic animal contains a Trp to Arg mutation at position 101. The resulting HCAb preferably exhibits high antigen-binding affinity and solubility under physiological conditions in the absence of aggregation.

[0267] As part of this invention, UniRat proteins that bind to human CD3, BCMA, PSMA, or CD19 are identified in ELISA protein and cell binding assays. TMAnimal-specific human heavy chain antibodies (UniAbs) TM The identified heavy chain variable region (VH) sequences (see, for example, Tables 1 and 2) were positive for protein binding and / or binding to cells expressing target proteins (e.g., CD3, BCMA, PSMA, or CD19), and negative for binding to cells not expressing target proteins.

[0268] Heavy chain antibodies that bind to non-overlapping epitopes on target proteins (e.g., UniAbs) TM This can be identified through competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA) or flow cytometry competitive binding assays. For example, competition between a known antibody binding to a target antigen and the antibody of interest can be utilized. Using this method, a group of antibodies can be separated into antibodies that compete with a reference antibody and those that do not. Non-competitive antibodies are identified as binding to different epitopes that overlap with the epitopes that do not bind to the reference antibody. Typically, one antibody is immobilized, the antigen binds, and the ability of a second labeled (e.g., biotinylated) antibody to bind the captured antigen is tested in an ELISA assay. This can also be performed using surface plasmon resonance (SPR) platforms (including the ProteOn XPR36 (BioRad, Inc.), Biacore 2000, and Biacore T200 (GE Healthcare Life Sciences)) and the MX96 SPR imager (Ibis technologies BV), as well as on biofilm interferometry platforms (e.g., the Octet Red384 and Octet HTX (ForteBio, Pall Inc)). See examples in this document for further details.

[0269] Typically, as determined by standard techniques, such as the competitive binding assay described above, an antibody is considered to "compete" with a reference antibody if it reduces the binding of a reference antibody to the target antigen by approximately 15% to 100%. 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 higher.

[0270] Pharmaceutical compositions, uses and treatment methods

[0271] Another aspect of the invention provides a pharmaceutical composition comprising a mixture of one or more of the multispecific binding compounds of the invention with a suitable pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers as used herein include (but are not limited to) adjuvants, solid carriers, water, buffers, or other carriers used in this art for containing therapeutic components, or combinations thereof.

[0272] 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). TM In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb) having binding specificity to two or more non-overlapping epitopes on a target protein (e.g., CD3, BCMA, PSMA, or CD19). TM In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) heavy chain antibody (e.g., UniAb) that has binding specificity to a target protein (e.g., BCMA, PSMA, or CD19) and binding specificity to a binding target on effector cells (e.g., a binding target on T cells, such as CD3 protein on T cells). TM ).

[0273] The pharmaceutical composition of the antibody used according to the invention is prepared for storage by mixing a protein of desired purity with a pharmaceutically acceptable carrier, excipient, or stabilizer, optionally present (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), for example, in the form of a lyophilized formulation or an aqueous solution. The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration used and includes buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; and preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride). Chloride); phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; 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. TM PLURONICS TM Or polyethylene glycol (PEG).

[0274] Pharmaceutical compositions intended for non-enteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition may be provided in unit dose form (i.e., a dose for a single administration). The formulation depends on the chosen route of administration. The antibodies described herein may be administered intravenously, by infusion, or subcutaneously. For injection administration, the antibodies described herein may be formulated in an aqueous solution, preferably in a physiologically compatible buffer to minimize discomfort at the injection site. The solution may contain carriers, excipients, or stabilizers as discussed above. Alternatively, the antibodies may be in lyophilized form and prepared with a suitable medium (e.g., sterile, pyrogen-free water) prior to use.

[0275] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies of this invention, including UniAbs. TM Subcutaneous antibody preparations are described, for example, in US20160355591 and US20160166689.

[0276] How to use

[0277] The heavy chain antibodies, multispecific antibodies, and pharmaceutical compositions described herein are intended for the treatment of diseases and disorders characterized by the expression of target proteins (e.g., CD3, BCMA, PSMA, or CD19), including (but not limited to) the diseases and disorders further described herein.

[0278] The pharmaceutical compositions containing anti-BCMA antibodies described herein may be used to treat B-cell-related conditions, including B-cell and plasma cell malignancies characterized by BCMA expression or overexpression, as well as autoimmune diseases.

[0279] These B-cell-related diseases include B-cell and plasma cell malignancies as well as autoimmune diseases, including (but not limited to) plasmacytoma, Hodgkin's lymphoma, follicular lymphoma, small non-lytic cell lymphoma, endemic Burkitt's lymphoma, sporadic Burkitt's 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 lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, and pulmonary B-cell vascular lymphoma. Cardiac lymphoma, small lymphocytic lymphoma, B-cell proliferation with uncertain malignant potential, lymphomatoid granuloma, post-transplant lymphoproliferative disorders, immunomodulatory disorders, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis or monoclonal gammaglobulinopathy.

[0280] Plasma cell disorders characterized by BCMA expression include multiple myeloma (MM). MM is a B-cell malignancy characterized by the monoclonal expansion and accumulation of abnormal plasma cells in bone marrow compartments. Current treatments for MM typically induce remission, but almost all patients eventually relapse and die. There is substantial evidence for immune-mediated elimination of myeloma cells in an allogeneic hematopoietic stem cell transplantation setting; however, this approach is highly toxic and rarely results in a cure. Although some monoclonal antibodies have shown promise for treating MM in preclinical studies and early clinical trials, consistent clinical efficacy of any monoclonal antibody therapy for MM has not yet been confirmed. Therefore, there is a great need for new therapies, including immunotherapy for MM (see, for example, Carpenter et al., Clin Cancer Res 2013, 19(8):2048-2060).

[0281] BCMA is known to promote in vivo progression of human multiple myeloma (MM) through overexpression or activation of its proliferative ligand APRIL. BCMA has also been shown to promote in vivo growth of xenografted MM cells with p53 mutations in mice. Since the activity of the APRIL / BCMA pathway plays a crucial role in MM pathogenesis and drug resistance through bidirectional interactions between tumor cells and their supporting bone marrow microenvironment, 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.

[0282] Another B-cell disease involving plasma cells (i.e., those expressing BCMA) is systemic lupus erythematosus (SLE), also known as lupus. SLE is a systemic autoimmune disease that can attack any part of the body and manifests as the immune system attacking the body's own cells and tissues, leading to chronic inflammation and tissue damage. It is a type III hypersensitivity reaction in which antibody-immune complexes precipitate and trigger another immune response (Inaki and Lee, Nat Rev Rheumatol 2010; 6:326-337).

[0283] The anti-BCMA heavy chain antibody (UniAb) of the present invention can be used to develop therapeutic agents for treating multiple myeloma (MM), spontaneous atrophic leukemia (SLE), and other B-cell or plasma cell disorders characterized by BCMA expression (such as those listed above). Specifically, the anti-BCMA heavy chain antibody (UniAb) of the present invention can be used alone or in combination with other MM treatments for the treatment of MM.

[0284] PSMA is a type II transmembrane protein expressed in prostatic epithelial tissue and upregulated in the neovascularization system of prostate cancer and solid tumors. It is also expressed at low levels in healthy tissues (e.g., brain, kidneys, and salivary glands), but its overexpression in malignant prostate tissue makes it an attractive target for therapeutic treatment of prostate cancer. Given its high expression in the malignant neovascularization system, it may also be associated with therapy or imaging 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). Additionally, radionuclide conjugates specifically targeting PSMA are being investigated for imaging and treatment of prostate cancer (e.g., Hofman et al., 2018, PMID:29752180).

[0285] In one respect, the PSMA heavy chain antibodies in this paper (e.g., UniAbs) TM The drug composition can be used to treat conditions characterized by PSMA expression, including (but not limited to) prostate cancer and solid tumors.

[0286] CD19 is a cell surface receptor expressed on all human B cells but not found on plasma cells. It has a relatively large 240-amino acid cytoplasmic tail. The extracellular Ig-like domain is separated by a potentially disulfide-linked 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. Like CD20 and CD22, the restricted expression of CD19 in the B cell lineage makes it an attractive target for therapeutic treatment of B-cell malignancies. Given its observed expression in a variety of hematologic malignancies, CD19 is a promising target for antibody-based therapeutics.

[0287] In one respect, the CD19 heavy chain antibody (e.g., UniAbs) in this article TM The drug and pharmaceutical composition can be used to treat hematologic malignancies characterized by CD19 expression, including (but not limited to) diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia (CLL), and B-cell acute lymphoblastic leukemia (ALL).

[0288] Diffuse large B-cell lymphoma (DLBCL or DLBL) is the most common form of non-Hodgkin's lymphoma in adults (Blood 1997 89(11):3909-18), with an estimated annual incidence of 7 to 8 cases per 100,000 people in the US and UK. It is characterized by an aggressive cancer that can occur in almost any part of the body. The etiology of DLBCL is unknown, and it can originate from normal B cells as well as from the malignant transformation of other types of lymphoma or leukemia cells. Treatment typically involves chemotherapy and radiation, and has resulted in an average five-year overall survival of approximately 58% in adults. Although some monoclonal antibodies have shown promise in treating DLBCL, consistent clinical efficacy has not yet been proven. Therefore, there is a great need for new DLBCL therapies, including immunotherapy.

[0289] On the other hand, the CD19 heavy chain antibodies in this article (e.g., UniAbs) TM The drug and pharmaceutical composition can be used to treat autoimmune diseases characterized by pathogenic B cells expressing CD19, including (but not limited to) systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS).

[0290] The effective dosage of the compositions of the present invention for treating diseases varies depending on many different factors, including the method of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. Typically, the patient is human, but non-human mammals such as companion animals (e.g., dogs, cats, horses, etc.) and laboratory mammals (e.g., rabbits, mice, rats, etc.) can also be treated. The therapeutic dosage can be titrated to optimize safety and efficacy.

[0291] Dosage levels can be easily determined by a competent clinician and can be modified as needed, such as according to the patient's response to the therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dose varies depending on the host being treated and the specific administration method. Dosage units typically contain between about 1 mg and 500 mg of active ingredient.

[0292] In some embodiments, the therapeutic dose of the agent may range from about 0.0001 to 100 mg / kg of host body weight, more typically from 0.01 to 5 mg / kg of host body weight. For example, the dose may be 1 mg / kg of body weight or 10 mg / kg of body weight or in the range of 1-10 mg / kg. Exemplary treatment regimens require administration once every two weeks, once a month, or once every 3 to 6 months. The therapeutic entity of the invention is typically administered in multiple doses. The interval between individual doses may be weekly, monthly, or annually. The interval may also be irregular, as indicated by measuring the blood level of the therapeutic entity in the patient. Alternatively, the therapeutic entity of the invention may be administered with a continuous release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the peptide in the patient.

[0293] Typically, compositions are prepared as injectable formulations, in the form of liquid solutions or suspensions; they can also be prepared in solid forms suitable for solutions or suspensions in liquid media prior to injection. The pharmaceutical compositions described herein are suitable for intravenous or subcutaneous administration following direct or reconstituted solid (e.g., lyophilized) formulations. Formulations may also be emulsified or encapsulated in liposomes or microparticles (e.g., polylactic acid, polyglycolic acid, or copolymers) to enhance adjuvant effects, as discussed above. (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 accumulated injectable or implantable formulations that can be formulated to allow for sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practice (GMP) requirements of the U.S. Food and Drug Administration.

[0294] The toxicity of the antibodies and antibody structures described herein can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the LD50 (the dose that is lethal to 50% of the population) or LD100 (the dose that is lethal to 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. Data obtained from these cell culture analyses and animal studies can be used to formulate a dose range that is non-toxic to humans. The dosage of the antibodies described herein is preferably within a range of cyclic concentrations that include effective doses with minimal or no toxicity. The dosage may vary within this range depending on the dosage form and route of administration used. Individual physicians may select the precise formulation, route of administration, and dosage based on the patient's condition.

[0295] Compositions intended for administration typically contain antibodies or other agents (e.g., another ablative agent) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as buffered saline solutions. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized using well-known, conventional sterilization techniques. Compositions may contain pharmaceutically acceptable adjuvants, such as pH adjusters and buffers, toxicity modifiers, etc., to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the specific administration modality chosen and the patient's needs (e.g., Remington's Pharmaceutical Science (15th edition, 1980) and Goodman and Gillman, The Pharmacological Basis of Therapeutics (edited by Hardman et al., 1996)).

[0296] Pillboxes containing the active agents and formulations thereof, as well as instructions for use, are also within the scope of this invention. Pillboxes may further contain at least one other agent, such as a chemotherapeutic agent. Pillboxes typically include markings indicating the intended use of the contents. As used herein, the term "markings" includes any written or recorded material on, provided with, or otherwise accompanying the pillbox.

[0297] The present invention will now be fully described, and those skilled in the art will understand that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0298] Example

[0299] Example 1: Heterodimer formation

[0300] Heterodimer formation was analyzed using both non-reducing and reducing SDS-PAGE analyses to determine whether antibodies according to embodiments of the invention, containing multiple mutations in their hinge and Fc regions as well as knob-in-pore mutations, could be successfully expressed and assembled into the desired heterodimer combination. For this purpose, the antibody construct was expressed in recombinant CHO cell cultures. The harvested cell culture was then purified by protein A affinity chromatography to analyze the different antibody fragments produced. The protein A elution pools were then analyzed on both reducing and non-reducing gels to visualize the different types.

[0301] The results of these analyses show that Figure 2 Figures A and B Figure 3 Figures A and B and Figure 24Furthermore, it demonstrates that the percentage of heterodimer formation for antibody species including those with knob-in-hole mutations is excellent, even when effector silencing mutations (F234A, L235A) and mutations preventing Fab arm exchange (S228P) are also present in the heavy chain sequence. Figure 25 The provided data evaluates the purified CD19 construct to analyze the percentages of high molecular weight (HMW) and low molecular weight (LMW) species, as well as the percentage of monomers.

[0302] Example 2: Fcγ receptor binding via biomembrane layer interference (BLI) technology

[0303] The Fcγ receptor-IgG interaction was analyzed using a Ni-NTA biosensor (ForteBio) on the Octet platform. The Ni-NTA biosensor features a nickel (Ni2+)-filled QIAGEN Tris-NTA pre-immobilized to its tip. The Ni-NTA binds to an HIS tag linked to the recombinant protein. In this format, the Fcγ receptor protein is loaded onto the biosensor as a ligand and then associates with IgG. Antibodies according to embodiments of the invention were studied to analyze the degree of interaction between their Fc regions and the Fcγ receptor protein immobilized on the biosensor.

[0304] Here, the Fcγ receptor is the human Fcγ receptor I / CD64 (Acro Biosystems). The antibody concentrations tested included 2× serial dilutions ranging from 100 nM to 1.6 nM. The results of these studies are shown in... Figure 4 Figures A-D Figure 5 Figures A-E Figure 6 Figures A-D Figure 7 Figures A-E and Figure 26 Figures A-D show that the binding of the silenced Fc receptor antibody to human FcγR1 is significantly inhibited, even when knob-in-hole mutations and Fab arm exchange mutations are also present in the Fc region.

[0305] Specifically, Figure 4 Figure A shows the results of a bispecific CD3×BCMA (monovalent) IgG1 antibody without KiH mutations or silencing mutations. The data confirm that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Figure 4 Figure B shows the results of a bispecific antibody used in Figure A, but now including a silencing mutation in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced due to the presence of the silencing mutation. Figure 4Figure C shows the results for a bispecific CD3×BCMA (monovalent) IgG1 antibody that includes the KiH mutation but excludes the silent mutation. These data demonstrate that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a manner very similar to that observed using the antibody in Figure A. Figure 4 Figure D shows the results of a bispecific antibody used in Figure C, but now including a silencing mutation in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced with the presence of the silencing mutation, even when the KiH mutation is included.

[0306] Figure 5 Figure A shows the results of a bispecific CD3×BCMA (monovalent) IgG4 antibody that does not contain the KiH mutation or the silent mutation, but includes the S228P mutation that prevents Fab arm exchange. The data confirm that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Figure 5 Figure B shows the results of the same bispecific antibody used in Figure A, but now including silent mutations (F234A, L235A) in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced due to the presence of silent mutations, even in the presence of the S228P mutation. Figure 5 Figure C shows the results for bispecific CD3×BCMA (monovalent) IgG4 antibodies, including KiH and S228P mutations but excluding silencing mutations. These data demonstrate that the antibodies interact with the Fcγ receptor immobilized on the biosensor in a manner very similar to that observed using the antibodies in Figure A. Figure 5 Figure D shows the results of bispecific antibodies used in Figure C, but now including silent mutations (F234A, L235A) in the CH2 domain, as well as S228P and KiH mutations. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced with the presence of silent mutations, even when S228P and KiH mutations are included. Figure E shows the results of bispecific CD3×BCMA (bivalent) IgG4 antibodies including S228P mutations, silent mutations F234A and L235A, and a KiH mutation in the CH3 domain. These data demonstrate that the interaction between this antibody and the Fcγ receptor immobilized on the biosensor is significantly reduced, even in the presence of S228P and KiH mutations.

[0307] Figure 6 Figure A shows the results of a bispecific CD3×PSMA (monovalent) IgG1 antibody without KiH mutations or silencing mutations. The data confirm that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Figure 6Figure B shows the results of a bispecific antibody used in Figure A, but now including a silencing mutation in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced due to the presence of the silencing mutation. Figure 6 Figure C shows the results for a bispecific CD3×PSMA (monovalent) IgG1 antibody that includes the KiH mutation but excludes the silent mutation. These data demonstrate that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a manner very similar to that observed with the antibody in Figure A. Figure 6 Figure D shows the results of a bispecific antibody used in Figure C, but now including a silencing mutation in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced with the presence of the silencing mutation, even when the KiH mutation is included.

[0308] Figure 7 Figure A shows the results of a bispecific CD3×PSMA (monovalent) IgG4 antibody that does not contain the KiH mutation or the silent mutation, but includes the S228P mutation that prevents Fab arm exchange. The data confirm that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Figure 7 Figure B shows the results of the same bispecific antibody used in Figure A, but now including silent mutations (F234A, L235A) in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced due to the presence of silent mutations, even in the presence of the S228P mutation. Figure 7 Figure C shows the results for bispecific CD3×PSMA (monovalent) IgG4 antibodies that include KiH and S228P mutations, but exclude silencing mutations. These data demonstrate that the antibodies interact with the Fcγ receptor immobilized on the biosensor in a manner very similar to that observed using the antibodies in Figure A. Figure 7 Figure D shows the results of bispecific antibodies used in Figure C, but now including silent mutations (F234A, L235A) in the CH2 domain, as well as S228P and KiH mutations. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced with the presence of silent mutations, even when S228P and KiH mutations are included. Figure E shows the results of bispecific CD3×PSMA (bivalent) IgG4 antibodies including S228P mutations, silent mutations F234A and L235A, and a KiH mutation in the CH3 domain. These data demonstrate that the interaction between this antibody and the Fcγ receptor immobilized on the biosensor is significantly reduced, even in the presence of S228P and KiH mutations.

[0309] Figure 26Figure A shows the results of a bispecific CD3×CD19 (monovalent) IgG4 antibody that does not contain the KiH mutation or the silent mutation, but includes the S228P mutation that prevents Fab arm exchange. The data confirm that the antibody interacts with the Fcγ receptor immobilized on the biosensor. Figure 26 Figure B shows the results of the same bispecific antibody used in Figure A, but now including silent mutations (F234A, L235A) in the CH2 domain. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced due to the presence of silent mutations, even in the presence of the S228P mutation. Figure 26 Figure C shows the results for a bispecific CD3×CD19 (monovalent) IgG4 antibody that includes KiH and S228P mutations but excludes silencing mutations. These data demonstrate that the antibody interacts with the Fcγ receptor immobilized on the biosensor in a manner very similar to that observed with the antibody in Figure A. Figure 26 Figure D shows the results of bispecific antibodies used in Figure C, but now including silent mutations (F234A, L235A) in the CH2 domain, as well as S228P and KiH mutations. These results demonstrate that the interaction between the antibody and the Fcγ receptor on the biosensor is significantly reduced with the presence of silent mutations, even when S228P and KiH mutations are included.

[0310] In short, Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 26 The data provided confirm that the VH region sequence of the bispecific antibody has no effect on the functional properties of the IgG4 Fc mutation described herein. Therefore, the IgG4 Fc modifications (S228P; F234A, L235A; T366W, T366S, L368A, and Y407V) described herein can be implemented in antibodies with different VH sequences (i.e., different binding targets) to achieve reduced Fab arm exchange (S228P), reduced effector activity (F234A, L235A), and correct heterodimerization (T366W; T366S, L368A, and Y407V).

[0311] Example 3: Anti-PSMA UniAbs TM Flow cytometry analysis of binding with PSMA-positive and negative cells

[0312] Binding to PSMA-positive cells was evaluated using flow cytometry (Guava easyCyte 8HT, EMD Millipore) with LNCaP cell lines (ATCC: CRL-1740), 22Rv1 cell lines (ATCC CRL-2505), PC3 cell lines (ATCC CRL-1435) stably transfected to express human PSMA, or the DU-145 cell line (ATCC HTB-81). In short, a series of purified UniAbs were used. TM The dilution was used to stain 50,000 target cells at 4°C for 30 minutes. After incubation, the cells were washed twice with flow cytometry buffer (1×PBS, 1% BSA, 0.1% NaN3) and stained with goat F(ab')2 anti-human IgG (Southern Biotech, catalog number 2042-09) bound to R-phycoerythrin (PE) to detect antibody binding. After incubation at 4°C for 20 minutes, the cells were washed twice with flow cytometry buffer and mean fluorescence intensity (MFI) was measured by flow cytometry. Background signal was determined using the MFI of cells stained with secondary antibodies only, and the binding of each antibody was converted to background by fold. Binding to cynomolgus monkey PSMA-positive cells was determined using the same protocol with the following modifications: target cells were Freestyle 293-F cells (ThermoFisher R79007) transiently transfected to express the extracellular domain of cynomolgus monkey PSMA. In some experiments, GraphPad Prism 7 was used to calculate the EC50 value.

[0313] Table 8 summarizes the target-binding activities of the anti-PSMA heavy chain antibodies (HCAbs) described in this paper. Column 1 indicates the clone ID of the HCAb. Column 2 indicates the binding to LNCaP cells measured as a fold increase over the background MFI signal.

[0314] Table 8: Binding with PSMA-expressing cell lines

[0315]

[0316]

[0317] like Figure 8 As shown in Figures A and B, the differences in binding with cynomolgus monkey PSMA support the differences in human PSMA epitopes identified by HCAb 346181 and HCAb 345497.

[0318] Example 4: Composition of dual complementary sites and bivalent anti-PSMA antibody

[0319] As shown in Table 9, anti-PSMA clone ID 350123 is composed of clone ID 346181, which is linked to clone ID 345497 with the bridging sequence GGGGSGGGGS (SEQ ID NO: 71). Clone ID 350122 is composed of two repeats of clone ID 346181 linked by the same linker sequence. Clone ID 350123 is doubly complementary because it consists of two anti-PSMA domains that recognize different epitopes on PSMA. Clone ID 350122 is bivalent but not doubly complementary because it consists of the same anti-PSMA domains tandemly. Schematic illustrations of the various triple-stranded antibody-like molecules (TCAs) are shown in Figures A-C of FIG1.

[0320] Table 9: Description of the amino acid sequences of bivalent anti-PSMA antibodies with double complementary sites.

[0321]

[0322] Example 5: Killing of PSMA-positive prostate tumor cells mediated by multispecific antibodies via T cell redirection

[0323] Analysis using resting T cells

[0324] Target cells were seeded at 15,000 cells / well in 96-well plates and incubated overnight at 37°C. After incubation, incremental amounts of multispecific antibody were added to resting human T cells at a 10:1 effector-to-target cell ratio, and the cells were incubated at 37°C for 48 or 72 hours (48 hours for analyses using LNCaP, MDA-PCa-2b, and PC3-PSMA cells, and 72 hours for analyses using 22Rv1 cells). Cell death was measured using the WST-1 cell proliferation reagent (Sigma catalog number: 11644807001) or flow cytometry. In some experiments, small samples of each supernatant were collected after incubation but before analyzing target cell viability and reserved for cytokine production analysis. When analyzing cell viability using the WST-1 reagent, the stock solution was diluted 1:10 and added to each well, and incubated at 37°C for 90 minutes. The absorbance was then measured at 450 nm (reference 690 nm), and the specific solubility percentage was calculated.

[0325] If target cell viability is analyzed by flow cytometry, target cells are labeled with the membrane dye DiR (ThermoFisher D12731) before starting the analysis. After incubation with T cells and antibodies, the supernatant is retained for cytokine analysis or discarded. The wells are then washed once to collect dead tumor cells and T cells, which are transferred to a flow cytometry plate. The remaining attached tumor cells are trypsinized and then added to the corresponding wells in the flow cytometry plate. Dead cells are stained with connexin-V reagent and flow cytometry (BD FACSCelesta) is performed to quantify the percentage of dead tumor cells in each sample, gated by DiR staining. Spontaneous cell death is normalized using wells containing untreated target cells. In some experiments, a negative control antibody containing the same CD3 targeting arm as in the PSMA×CD3 multispecific molecule is used, but the tumor targeting arm is replaced with VH, which specifically targets the HIV protein gp120.

[0326] Figure 9 Demonstrates T cell-mediated lysis of PSMA-positive cells using unstimulated T cells. Unstimulated human T cells were incubated with PSMA-expressing cells (LNCaP) and different concentrations of multispecific antibodies. The double-complementary anti-PSMA×CD3 antibody (350 × 123 × CD3) was superior to the single-complementary PSMA×CD3 antibody (346 × 181 × CD3).

[0327] Analysis using pre-activated T cells

[0328] Human pan-T cells were preactivated for three days using plate-bound OKT3 and IL-2, followed by incubation for another day in fresh IL-2. Target cells were trypsinized, loaded with calcein-AM (ThermoFisher C3100MP), mixed with activated T cells to a 20:1 E:T ratio, and added to the wells of a 96-well plate. A series of different multispecific antibody dilutions were added, followed by incubation at 37°C for 4 hours. The supernatant was then transferred to a black 96-well plate, and absorbance was measured at 480 nm / 520 nm ex / em to quantify calcein release. Target cells not incubated with T cells were used to normalize spontaneous calcein release from intact tumor cells. Adding 2% Triton-X to control wells containing target cells allowed for the calculation of the calcein signal corresponding to maximum cell lysis. Using this value, each experimental well was reported as a % relative to maximum cell lysis. Data analysis was performed using GraphPad Prism 7.

[0329] Figure 10Demonstrates T-cell-mediated PSMA-positive cell lysis using pre-activated T cells. Pre-activated human T cells were incubated with human PSMA-expressing cells (LNCaP) and varying concentrations of multispecific antibodies. Tumor cell death was measured by calcein release and normalized to spontaneous tumor cell release in the absence of T cells. The bicomponent anti-PSMA×CD3 antibody (350123×CD3) was superior to two monocomponent PSMA×CD3 antibodies.

[0330] Figure 11 The results showed that multispecific antibodies did not lyse PSMA-negative cells. Preactivated human T cells were incubated with PSMA-negative prostate cancer cells (DU145) and different concentrations of multispecific antibodies. None of the antibodies tested lysed these cells.

[0331] Figure 12 The PSMA×CD3 multispecific antibody was shown to bind to both PSMA-positive and PSMA-negative cells. The multispecific anti-PSMA×anti-CD3 antibody showed binding to PSMA-positive prostate tumor cells (22Rv1) but not to PSMA-negative prostate tumor cells (DU145). The double complementary site molecule (350123) showed the strongest target cell binding.

[0332] Figure 13 Illustration of T cell-mediated PSMA-positive cell lysis. Figure 13 The data shows that binding to PSMA via two different epitopes can increase cell killing compared to the bivalent but monospecific form of the antibody.

[0333] Example 6: A single complementary PSMA×CD3 bispecific antibody induces the production of fewer cytokines than a single complementary PSMA×CD3 multispecific antibody.

[0334] Resting T cells were used to analyze cytokine production in tumor cytotoxicity assays. The design of these assays is detailed elsewhere. After the assay was completed (72 hours of incubation for assays using 22Rv1 cells, and 48 hours of incubation for assays using all other cell lines), the supernatant was collected. IL-2 (Biolegend 431804) and IFNγ (Biolegend 430104) were detected using ELISA kits according to the manufacturer’s protocol. The experimental supernatant was diluted before analysis in the ELISA kits so that the cytokine levels would fall within the linear portion of the standard curve provided with each kit. In some cases, cytokines may not be detected in the wells, and the values ​​will be reported as less than or equal to the lower limit of quantitation.

[0335] Figure 14(Figures A, B, and C) show a comparison of T cell-mediated PSMA-positive cell lysis and cytokine production. Multispecific PSMA×CD3 antibody induced T cell-mediated lysis of the PSMA-positive prostate cancer cell line LNCaP. Compared to a single complementary molecule (346181), a double complementary molecule (350123) stimulated more potent tumor cell killing and induced higher levels of cytokines (i.e., interferon-γ (IFNγ) and interleukin-2 (IL-2)), such as through… Figure 14 As illustrated in Figures B and C.

[0336] Table 10 shows T cell-mediated lysis and cytokine production against four PSMA-positive prostate tumor cell lines. In the in vitro tumor cell cytotoxicity assay, unstimulated T cells and a series of doses of the antibody were used to test a PSMA×CD3 multispecific antibody against a group of four PSMA-positive tumor cell lines. Tumor cell death % and the highest kill % achieved were calculated and reported by EC50 after 72 hours (22Rv1) or 48 hours (MDA-PCa-2b, LNCAP, PC3-PSMA). The supernatants from these wells were collected and cytokines, namely interferon-γ (IFNγ) or interleukin-2 (IL-2), were analyzed by ELISA. Compared to the double-complementary molecule, the single-complementary molecule (3461881) induced approximately equivalent levels of tumor cytotoxicity against all four cell lines tested, but had a higher cytokine production EC50 and, in most cases, stimulated lower levels of maximum cytokine production.

[0337] Table 10: T cell-mediated lysis and cytokine production in four PSMA-positive prostate tumor cell lines.

[0338]

[0339] Example 7: PSMA×CD3 multispecific antibody induces T cell proliferation

[0340] PSMA-positive tumor cells were seeded at 25,000 cells / well in 96-well plates and grown overnight at 37°C. Human pan-T cells isolated from resting PBMCs (Miltenyi130-096-535) were labeled with the lineage tracer dye CFSE (ThermoFisher C34554) according to the manufacturer's instructions. 100,000 labeled pan-T cells were then added to wells containing tumor cells, followed by a series of antibody dilutions, and incubated at 37°C and 8% CO2. After 5 days of incubation, the cells were gently mixed and transferred to flow cytometry plates. Cell pelleting was performed, and the supernatant was removed. The cells were then stained on ice for 20 minutes with anti-CD8 bound to APC (Biolegend 301049) and anti-CD4 bound to PE (Biolegend 317410). The cells were then washed and resuspended in flow cytometry buffer for analysis (BD FACSCelesta). Cells were gated based on anterior and lateral dispersion and CD4 or CD8 expression. The percentage of proliferating T cells in the entire T cell population and CD4 and CD8 subgroups was calculated, as indicated by CD4 or CD8 positive staining and low or negative CFSE signaling. Flow cytometry data were analyzed using FlowJo and plotted in GraphPad Prism 7.

[0341] Figure 15 (Figures A, B, C, and D) show that PSMA×CD3 multispecific antibodies stimulate T cell proliferation in the presence of PSMA-positive tumor cells, and that single complementary PSMA bispecific antibodies preferentially activate CD3 T cells. Multispecific antibodies were incubated with PSMA-expressing tumor cells and T cells labeled with the lineage-tracing dye CFSE. After 5 days of incubation, T cell proliferation and the composition of proliferating T cells (CD8+ to CD4+) were analyzed by flow cytometry. Figures A and B show total T cell proliferation, while Figures C and D indicate the ratio of CD8+ to CD4+ T cells in the proliferation wells. The horizontal dashed line indicates the CD8:CD4 ratio of unstimulated T cells, which is approximately 1:2 (actual value = 0.64). The single complementary site PSMA×CD3 bispecific antibody (346181) preferentially activates CD8 T cells (the CD8:CD4 ratio after expansion is about 2:1), while the double complementary site PSMA×CD3 multispecific antibody (350123) does not preferentially activate CD8+ T cells (the CD8:CD4 ratio is about 1:1).

[0342] Example 8: Inhibition of prostate tumor growth in a xenograft model induced by multispecific antibodies

[0343] Ten million 22Rv1 cells were subcutaneously implanted into the right lower abdomen of 5-6 week old male immunodeficient CIEA-NOG mice (Taconic), followed by a tail vein injection of 10 million human PBMCs one day after tumor implantation. Starting one day after tumor implantation, animals received 100 μg of a multispecific antibody or drug via tail vein injection on days 1, 5, 9, and 13. Tumor volume was quantified using calipers and recorded for up to 25 days.

[0344] Figure 16 Results are shown in the 22Rv1 tumor xenograft model. The double complementary site PSMA×CD3 molecule (350123) showed inhibition of 22Rv1 tumor growth in the tumor xenograft model. Three mice were tested in each treatment group, and tumor volume changes in each animal are plotted in cubic millimeters. Animals received PBMCs on day 1 post-tumor implantation and were treated with antibodies on days 1, 5, 9, and 13. Two of the three animals treated with the multispecific antibody showed a delay in tumor progression.

[0345] Example 9: Analysis of T cell activation

[0346] CD69 is a cell surface marker on T cells that is upregulated upon stimulation, thus serving as an indicator of T cell activation. In this experiment, CD69 activation was assessed under three different conditions: 1) total peripheral blood mononuclear cells (PBMCs) without BCMA coating; 2) pan-T cells with BCMA coating; and 3) pan-T cells without BCMA coating. PBMCs were isolated from the erythrocyte sedimentation rate (ESR) amber layer using Ficoll (1.077 g / ml density), and refrigerated PBMCs were thawed and analyzed at 2 × 10⁻⁶. 6 Cells / mL were incubated at 37°C for 24 hours in RPMI 1640 supplemented with 10% FBS. On day 2, pan-T cells were isolated from the incubated PBMCs using the Miltenyi negative selection kit, and the isolated cells were used in the second and third analytical conditions. For the first analytical condition, PBMCs were counted and plated in the analytical plate.

[0347] For cells evaluated under antigen-coated conditions, 96-well plates were coated with recombinant BCMA protein (human BCMA protein, Fc tag, Acro Biosystems, catalog BC7-H5254) at a concentration of 1 μg / mL, recombinant PSMA protein (recombinant human PSMA / FOLH1 protein, from RND systems, catalog 4234-ZN-0101) at a concentration of 10 μg / mL, or recombinant CD19 protein (human CD19 protein, His tag, Acro Biosystems, catalog CD9-H52H2) at a concentration of 10 μg / mL. Bispecific antibodies were analyzed using a 12-point dose profile and 3-fold dilutions, with a maximum dose of 300 nM. The bispecific antibodies and T cells were resuspended in RPMI 1640 supplemented with 10% FBS and incubated for 18 hours. Pan-T cells were effector cells and seeded at 100K cells / well.

[0348] For cells not evaluated under antigen-coated conditions, bispecific antibodies were incubated with cells using a 12-point dose curve and a 3-fold dilution. 300 nM of bispecific antibody was the highest concentration tested in this analysis. Samples were incubated at 37°C for 18 hours in RPMI 1640 supplemented with 10% FBS. Pan-T cells isolated from PBMCs were effector cells, seeded at 100 K cells / well.

[0349] For all experimental conditions, cells were washed and labeled with cell surface T cell antibodies. The following antibodies were used to label (1) CD4-positive T cells (FITC anti-human CD4 antibody), (2) CD8-positive T cells (PE anti-human CD8a antibody), and (3) CD69 activation (Alexa Fluor647 anti-human CD69 antibody) (Biolegend). Cells were then analyzed using an appropriate template on a BD Celesta to measure CD69 activation.

[0350] The results of the T cell activation study are shown in the figure below: Figures 17-18 , Figure 27 Figures A-B (without BCMA antigen coating, using PBMC); Figure 30 Figures A and B (without PSMA wrapping); and Figure 33 Figures A-B (without CD19 coating).

[0351] Figure 17This graph shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. Generally, bispecific antibodies containing the IgG1 Fc sequence exhibit CD4+ T cell activation at lower concentrations. Bispecific antibodies containing the IgG4 Fc sequence exhibit CD4+ T cell activation at higher concentrations. It should be noted that CD4+ T cell activation achieved with IgG4 Fc bispecific antibodies containing PAA and KiH mutations is lower, confirming that the introduction of PAA and KiH mutations reduces the BCMA-independent activation of T cells by these bispecific antibodies.

[0352] Figure 18 This graph shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. Similar to CD4+ T cells, bispecific antibodies containing the IgG1 Fc sequence exhibit CD8+ T cell activation at lower concentrations. Bispecific antibodies containing the IgG4 Fc sequence exhibit CD8+ T cell activation at higher concentrations. It should be noted that CD8+ T cell activation achieved with IgG4 Fc bispecific antibodies containing PAA and KiH mutations is lower, confirming that the introduction of PAA and KiH mutations reduces the BCMA-independent activation of T cells by these bispecific antibodies.

[0353] Figure 27 Figure A shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. CD4+ T cell activation achieved with the IgG4 Fc bispecific antibody containing PAA and KiH mutations was similar to that of the negative control (gp120, CD3(F2B)), confirming that the introduction of PAA and KiH mutations reduces the BCMA-independent activation of T cells by these bispecific antibodies. Figure 27 Figure B shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. Similar to CD4+ T cells, CD8+ T cell activation induced by the IgG4 Fc bispecific antibody containing PAA and KiH mutations was similar to the negative control, confirming that the introduction of PAA and KiH mutations reduces the BCMA-independent activation of T cells by these bispecific antibodies.

[0354] Figure 30Figure A shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. CD4+ T cell activation achieved with the IgG4 Fc bispecific antibody containing PAA and KiH mutations was similar to that of the negative control (gp120, CD3(F2B)), confirming that the introduction of PAA and KiH mutations reduces the PSMA-independent activation of T cells by these bispecific antibodies. Figure 30 Figure B shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. Similar to CD4+ T cells, CD8+ T cell activation induced by the IgG4 Fc bispecific antibody containing PAA and KiH mutations was similar to the negative control, confirming that the introduction of PAA and KiH mutations reduces the PSMA-independent activation of T cells by these bispecific antibodies.

[0355] Figure 33 Figure A shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. CD4+ T cell activation achieved with the IgG4 Fc bispecific antibody containing PAA and KiH mutations was significantly lower than that observed with other antibody constructs lacking PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations reduces the CD19-dependent activation of T cells by these bispecific antibodies. Figure 33 Figure B shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. Similar to CD4+ T cells, CD8+ T cell activation induced by the IgG4 Fc bispecific antibody containing PAA and KiH mutations was significantly lower than that observed from other antibody constructs without PAA and KiH mutations. This confirms that introducing PAA and KiH mutations reduces the CD19-independent activation of T cells by these bispecific antibodies.

[0356] Results of CD8+ T cell activation using pan-T cells isolated from resting PBMCs under antigen coating are provided. Figure 19 , Figure 28 Figure B Figure 31 Figure B and Figure 34 Figure B shows the results of CD4+ T cell activation using pan-T cells isolated from resting PBMCs under antigen coating. Figure 28 Figure A Figure 31 Figure A and Figure 34 In Figure A, the antigen coating concentrations for BCMA and PSMA were 1 μg / mL, while the antigen coating concentration for CD19 was 10 μg / mL.

[0357] Figure 19 This graph shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs shown in the legend as a function of bispecific antibody concentration. Unlike the experiment without BCMA coating, all bispecific antibodies exhibited CD8+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD8+ T cell activation was achieved using IgG4Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD8+ T cell activation activity of these molecules.

[0358] Figure 28 Figure B shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. Unlike the experiment without BCMA coating, all bispecific antibodies exhibited CD8+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD8+ T cell activation was achieved using IgG4 Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD8+ T cell activation activity of these molecules.

[0359] Figure 31 Figure B shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. Unlike the experiment without PSMA coating, all bispecific antibodies exhibited CD8+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD8+ T cell activation was achieved using IgG4 Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD8+ T cell activation activity of these molecules.

[0360] Figure 34 Figure B shows the percentage of CD8+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. Unlike the experiment without CD19 coating, all bispecific antibodies exhibited CD8+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD8+ T cell activation was achieved using IgG4 Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD8+ T cell activation activity of these molecules.

[0361] Figure 28Figure A shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. Unlike the experiment without BCMA coating, all bispecific antibodies exhibited CD4+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD4+ T cell activation was achieved using IgG4 Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD4+ T cell activation activity of these molecules.

[0362] Figure 31 Figure A shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. Unlike the experiment without PSMA coating, all bispecific antibodies exhibited CD4+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD4+ T cell activation was achieved using IgG4 Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD4+ T cell activation activity of these molecules.

[0363] Figure 34 Figure A shows the percentage of CD4+CD69+ T cells in the bispecific antibody constructs illustrated in the legend as a function of bispecific antibody concentration. Unlike the experiment without CD19 coating, all bispecific antibodies exhibited CD4+ T cell activation at similar concentrations for antigen-coated cells. It should be noted that CD4+ T cell activation was achieved using IgG4 Fc bispecific antibodies containing PAA and KiH mutations, confirming that the introduction of PAA and KiH mutations does not eliminate the CD4+ T cell activation activity of these molecules.

[0364] Results of CD8+ T cell activation using pan-T cells isolated from resting PBMCs without antigen coating are provided. Figure 20 , Figure 29 Figure B Figure 32 Figure B and Figure 35 Figure B shows the results of CD4+ T cell activation using pan-T cells isolated from resting PBMCs without antigen coating. Figure 29 Figure A Figure 32 Figure A and Figure 35 In Figure A.

[0365] Figure 20 This is a graph showing the percentage of CD8+CD69+ T cells in the bispecific antibody constructs as a function of bispecific antibody concentration, as illustrated in the legend. These results confirm that, for all bispecific antibody molecules tested, CD69 activation in CD8+ T cells is BCMA-dependent.

[0366] Figure 29 Figures A and B show the percentages of CD4+CD69+ T cells and CD8+CD69+ T cells in the bispecific antibody constructs illustrated in the legend, respectively, as a function of bispecific antibody concentration. These results confirm that, for all bispecific antibody molecules tested, CD69 activation in both CD4+ T cells and CD8+ T cells is BCMA-dependent. At higher concentrations of the bispecific antibody construct excluding silencing mutations, CD69 activation in both CD4+ T cells and CD8+ T cells was slightly increased.

[0367] Figure 32 Figures A and B show the percentage of CD4+CD69+ T cells and CD8+CD69+ T cells in the bispecific antibody constructs shown in the legend, respectively, as a function of bispecific antibody concentration. These results confirm that, for all bispecific antibody molecules tested, CD69 activation in CD4+ T cells and CD8+ T cells is PSMA-dependent.

[0368] Figure 35 Figures A and B show the percentages of CD4+CD69+ T cells and CD8+CD69+ T cells in the bispecific antibody constructs illustrated in the legend, respectively, as a function of bispecific antibody concentration. These results confirm that for all bispecific antibody molecules tested, CD69 activation in both CD4+ T cells and CD8+ T cells is CD19-dependent. At higher concentrations of the bispecific antibody construct excluding silencing mutations, CD69 activation in both CD4+ T cells and CD8+ T cells was slightly increased.

[0369] Example 10: Lysis of tumor cells

[0370] This study analyzed the ability of anti-CD3× anti-BCMA bispecific antibodies to retarget and kill three different BCMA+ tumor cell lines and one BCMA-negative cell line via activated primary T cells. In this experiment, tumor cells were mixed with activated pan-T cells at a 10:1 E:T ratio, and the bispecific antibody was added simultaneously. The results were shown in... Figure 21 Figures A-D are shown. Figure A shows the killing effect on RPMI-8226 cells, Figure B shows the killing effect on NCI-H929 cells, Figure C shows the killing effect on U-266 cells, and Figure D shows the killing effect on K562 cells (i.e., the negative control). The x-axis shows the concentration of the antibody used, and the y-axis shows the percentage of tumor cell lysis 6 hours after antibody addition.

[0371] The release level of IL-2 cytokine was measured after resting human T cells were cultured with various tumor cell lines and escalating doses of anti-CD3×anti-BCMA bispecific antibody. Figure 22Figure A shows the release of IL-2 after stimulation by RPMI-8226 cells. Figure 22 Figure B shows the release of IL-2 after stimulation by NCI-H929 cells. Figure 22 Figure C shows IL-2 release stimulated by U-266 cells, and Figure 22 Figure D shows the release of IL-2 after stimulation with K562 cells (i.e., negative control).

[0372] The release levels of IFN-γ cytokines were measured after resting human T cells were cultured with various tumor cell lines and escalating doses of anti-CD3×anti-BCMA bispecific antibodies. Figure 23 Figure A shows the release of IFN-γ after stimulation by RPMI-8226 cells. Figure 23 Figure B shows the release of IFN-γ after stimulation by NCI-H929 cells. Figure 23 Figure C shows the release of IFN-γ stimulated by U-266 cells, and Figure 23 Figure D shows the release of IFN-γ after stimulation by K562 cells (i.e., negative control).

[0373] This application includes the following implementation scheme:

[0374] 1. An isolated multispecific antibody comprising:

[0375] The first heavy chain polypeptide subunit contains the human IgG4 constant region containing mutations of S228P, F234A, L235A, and T366W; and

[0376] The second heavy chain polypeptide subunit contains the human IgG4 constant region containing mutations of S228P, F234A, L235A, T366S, L368A, and Y407V.

[0377] 2. The isolated multispecific antibody as described in embodiment 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 lacks the CH1 domain.

[0378] 3. The isolated multispecific antibody as described in embodiment 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.

[0379] 4. The isolated multispecific antibody as described in any one of embodiments 1 to 3, further comprising a first binding moiety having binding specificity to CD3, the first binding moiety comprising:

[0380] The heavy chain variable structural domain comprises a CDR1 sequence containing the sequence of SEQ ID NO:36, a CDR2 sequence containing the sequence of SEQ ID NO:37, and a CDR3 sequence containing the sequence of SEQ ID NO:38; and

[0381] The light chain variable structural domain comprises a CDR1 sequence containing the sequence of SEQ ID NO:39, a CDR2 sequence containing the sequence of SEQ ID NO:40, and a CDR3 sequence containing the sequence of SEQ ID NO:41.

[0382] 5. The isolated multispecific antibody as described in Scheme 4, wherein:

[0383] The CDR1, CDR2, and CDR3 sequences in the heavy chain variable structural domain of the first binding portion exist in the human VH framework; and

[0384] The CDR1, CDR2, and CDR3 sequences in the light chain variable structural domain of the first binding portion are present in the human Vκ framework.

[0385] 6. The isolated multispecific antibody as described in Scheme 5, wherein:

[0386] The heavy chain variable domain of the first binding portion contains a sequence having at least 95% identity with SEQ ID NO:42; and

[0387] The light chain variable structural domain of the first binding portion contains a sequence that has at least 95% identity with SEQ ID NO:43.

[0388] 7. The isolated multispecific antibody as described in Scheme 6, wherein:

[0389] The heavy chain variable structural domain of the first binding portion includes the sequence of SEQ ID NO:42; and

[0390] The light chain variable structural domain of the first binding portion contains the sequence of SEQ ID NO:43.

[0391] 8. The isolated multispecific antibody as described in any one of embodiments 1 to 7 further comprises a second binding moiety having binding specificity to proteins other than CD3.

[0392] 9. The isolated multispecific antibody as described in embodiment 8, wherein the second binding portion comprises a single heavy chain variable region in a monovalent or bivalent configuration.

[0393] 10. The isolated multispecific antibody as described in embodiment 9, wherein the first binding portion comprises a light chain polypeptide subunit and a heavy chain polypeptide subunit, and wherein the second binding portion comprises a heavy chain polypeptide subunit.

[0394] 11. The isolated multispecific antibody as described in embodiment 10, wherein the light chain polypeptide subunit of the first binding portion comprises a light chain constant domain.

[0395] 12. The isolated multispecific antibody as described in any one of embodiments 8 to 11, wherein the protein other than CD3 is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0396] 13. The isolated multispecific antibody as described in embodiment 12, wherein the TAA is B cell maturation antigen (BCMA).

[0397] 14. The isolated multispecific antibody as described in embodiment 12, wherein the TAA is CD19.

[0398] 15. The isolated multispecific antibody as described in embodiment 12, wherein the TAA is prostate-specific membrane antigen (PSMA).

[0399] 16. A pharmaceutical composition comprising a multispecific antibody as described in any one of embodiments 1 to 15.

[0400] 17. A polynucleotide encoding a multispecific antibody as described in any one of embodiments 1 to 15.

[0401] 18. A vector comprising the polynucleotide as described in embodiment 17.

[0402] 19. A cell comprising a carrier as described in embodiment 18.

[0403] 20. A method for generating a multispecific antibody as described in any one of embodiments 1 to 15, comprising growing cells as described in embodiment 19 under conditions that allow expression of the multispecific antibody and isolating the multispecific antibody from the cells.

[0404] 21. A treatment method comprising administering to an individual in need an effective dose of a multispecific antibody as described in any one of embodiments 1 to 15 or a pharmaceutical composition as described in embodiment 16.

[0405] 22. The use of the multispecific antibody as described in any one of embodiments 1 to 15 for the preparation of a medicament for treating a disease or condition in an individual in need.

[0406] 23. The multispecific antibody as described in any one of embodiments 1 to 15 or the pharmaceutical composition as described in embodiment 16, used in the therapy of an individual in need.

[0407] 24. A method of treating a disease or ailment characterized by BCMA expression, comprising administering to an individual in need an effective dose of a multispecific antibody as described in embodiment 13 or a pharmaceutical composition comprising a multispecific antibody as described in embodiment 13.

[0408] 25. The method as described in embodiment 24, wherein the disease is an autoimmune disease.

[0409] 26. The method as described in embodiment 24, wherein the disease is cancer.

[0410] 27. The method as described in embodiment 26, wherein the cancer is myeloma.

[0411] 28. The method of embodiment 27, wherein the myeloma is multiple myeloma.

[0412] 29. A method of treating a disease or ailment characterized by PSMA expression, comprising administering to an individual in need an effective dose of a multispecific antibody as described in embodiment 15 or a pharmaceutical composition comprising a multispecific antibody as described in embodiment 15.

[0413] 30. The method as described in embodiment 29, wherein the disease is cancer.

[0414] 31. The method as described in embodiment 29, wherein the cancer is prostate cancer.

[0415] 32. A method of treating a disease or ailment characterized by CD19 expression, comprising administering to an individual in need an effective dose of a multispecific antibody as described in embodiment 14 or a pharmaceutical composition comprising a multispecific antibody as described in embodiment 14.

[0416] 33. The method as described in embodiment 32, wherein the disease is diffuse large B-cell lymphoma (DLBCL).

[0417] 34. The method as described in embodiment 32, wherein the disease is acute lymphoblastic leukemia (ALL).

[0418] 35. The method of embodiment 32, wherein the condition is non-Hodgkin's lymphoma (NHL).

[0419] 36. The method of embodiment 32, wherein the condition is systemic lupus erythematosus (SLE).

[0420] 37. The method of embodiment 32, wherein the condition is rheumatoid arthritis (RA).

[0421] 38. The method as described in embodiment 32, wherein the condition is multiple sclerosis (MS).

[0422] 39. A kit for treating a disease or condition in an individual in need, comprising a multispecific antibody as described in any one of embodiments 1 to 15 or a pharmaceutical composition as described in embodiment 16 and instructions for use.

[0423] 40. The medicine box as described in embodiment 39 further comprises at least one other reagent.

[0424] 41. The kit as described in embodiment 40, wherein the at least one other reagent comprises a chemotherapeutic agent.

[0425] 42. A bispecific triple-stranded antibody-like molecule, comprising:

[0426] The first polypeptide subunit comprises:

[0427] The light chain variable domain (VL) containing the sequence of SEQ ID NO:43; and

[0428] Light chain constant structural domain (CL);

[0429] The second polypeptide subunit comprises:

[0430] The heavy chain variable domain (VH) of the sequence containing SEQ ID NO:42; and

[0431] Heavy chain constant domain (CH) containing the sequence of SEQ ID NO:72 or 73;

[0432] The light chain variable structural domain and the heavy chain variable structural domain together form a first bonding portion bonded to CD3; and

[0433] The third polypeptide subunit comprises:

[0434] Binding to heavy chain-only variable regions of proteins other than CD3 in monovalent or divalent configurations; and

[0435] Heavy chain constant domain (CH) containing the sequence of SEQ ID NO:54 or 55.

[0436] 43. The bispecific triplet antibody-like molecule as described in embodiment 42, wherein the third polypeptide subunit comprises a heavy-chain-only variable region in a divalent configuration that binds to BCMA.

[0437] 44. The bispecific triple-stranded antibody-like molecule as described in embodiment 43, comprising:

[0438] The first polypeptide subunit contains the sequence of SEQ ID NO:49;

[0439] The second polypeptide subunit contains the sequence of SEQ ID NO:56; and

[0440] The third polypeptide subunit contains the sequence of SEQ ID NO:58.

[0441] 45. A pharmaceutical composition comprising a bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44.

[0442] 46. ​​A polynucleotide encoding a bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44.

[0443] 47. A carrier comprising the polynucleotide as described in embodiment 46.

[0444] 48. A cell comprising a carrier as described in embodiment 47.

[0445] 49. A method for generating a bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44, comprising growing cells as described in embodiment 48 under conditions that allow expression of the bispecific triple-stranded antibody-like molecule and isolating the bispecific triple-stranded antibody-like molecule from the cells.

[0446] 50. A treatment method comprising administering to an individual in need an effective dose of a bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44 or a pharmaceutical composition as described in embodiment 45.

[0447] 51. The use of the bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44 for the preparation of a medicament for treating a disease or condition in an individual in need.

[0448] 52. A bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44 or a pharmaceutical composition as described in embodiment 45, for use in the therapy of an individual in need.

[0449] 53. A method of treating a disease or ailment characterized by BCMA expression, comprising administering to an individual in need an effective dose of a bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44 or a pharmaceutical composition as described in embodiment 45.

[0450] 54. The method as described in embodiment 53, wherein the disease is an autoimmune disease.

[0451] 55. The method as described in embodiment 53, wherein the disease is cancer.

[0452] 56. The method as described in embodiment 55, wherein the cancer is myeloma.

[0453] 57. The method of embodiment 56, wherein the myeloma is multiple myeloma.

[0454] 58. A kit for treating a disease or condition in an individual in need, comprising a bispecific triple-stranded antibody-like molecule as described in any one of embodiments 42 to 44 or a pharmaceutical composition as described in embodiment 45 and instructions for use.

[0455] 59. The medicine box as described in embodiment 58 further comprises at least one other reagent.

[0456] 60. The kit as described in embodiment 59, wherein the at least one other reagent comprises a chemotherapeutic agent.

[0457] This application also includes the following implementation schemes:

[0458] 1. A bispecific monoclonal antibody that binds to human CD3δε and human BCMA, comprising:

[0459] (i) A first polypeptide subunit comprising the amino acid sequence of SEQ ID NO:49;

[0460] (ii) A second polypeptide subunit comprising the amino acid sequence of SEQ ID NO:56.

[0461] The first and second polypeptide subunits together form the first binding moiety to human CD3δε; and

[0462] (iii) The third polypeptide subunit that binds to human BCMA, which contains the amino acid sequence of SEQ ID NO:58.

[0463] 2. The bispecific monoclonal antibody as described in embodiment 1, wherein the first polypeptide subunit comprises a light chain.

[0464] 3. The bispecific monoclonal antibody as described in embodiment 1, wherein the second polypeptide subunit comprises a heavy chain.

[0465] 4. The bispecific monoclonal antibody as described in embodiment 1, wherein the third polypeptide subunit comprises a divalent heavy chain.

[0466] 5. The bispecific monoclonal antibody as described in embodiment 4, wherein the bivalent heavy chain comprises a heavy chain-only variable region in a bivalent configuration.

[0467] 6. The bispecific monoclonal antibody as described in embodiment 4, wherein the divalent heavy chain lacks the CH1 domain.

[0468] 7. A bispecific monoclonal antibody binding to human CD3δε and human BCMA, comprising:

[0469] (i) A first polypeptide subunit comprising the amino acid sequence of SEQ ID NO:49;

[0470] (ii) A second polypeptide subunit comprising the amino acid sequence of SEQ ID NO:75.

[0471] The first and second polypeptide subunits together form the first binding moiety to human CD3δε; and

[0472] (iii) The third polypeptide subunit that binds to human BCMA, which contains the amino acid sequence of SEQ ID NO:76.

[0473] 8. The bispecific monoclonal antibody as described in embodiment 7, wherein the first polypeptide subunit comprises a light chain.

[0474] 9. The bispecific monoclonal antibody as described in embodiment 7, wherein the second polypeptide subunit comprises a heavy chain.

[0475] 10. The bispecific monoclonal antibody as described in embodiment 7, wherein the third polypeptide subunit comprises a divalent heavy chain.

[0476] 11. The bispecific monoclonal antibody as described in embodiment 10, wherein the bivalent heavy chain comprises a heavy chain-only variable region in a bivalent configuration.

[0477] 12. The bispecific monoclonal antibody as described in embodiment 10, wherein the bivalent heavy chain lacks the CH1 domain.

[0478] 13. A human monoclonal IgG4 bispecific antibody, comprising:

[0479] (i) binding to the first binding arm of human CD3δε; and

[0480] (ii) Binding to the second binding arm of the human BCMA.

[0481] The first binding arm comprises a first heavy chain and a light chain, the first heavy chain comprising the amino acid sequence of SEQ ID NO:56, and the light chain comprising the amino acid sequence of SEQ ID NO:49; and

[0482] The second binding arm comprises a divalent second heavy chain, which contains the amino acid sequence of SEQ ID NO:58.

[0483] 14. The human monoclonal IgG4 bispecific antibody as described in embodiment 13, wherein the second binding arm does not contain a light chain.

[0484] 15. The human monoclonal IgG4 bispecific antibody as described in embodiment 13, wherein the bivalent second heavy chain comprises a heavy chain-only variable region in a bivalent configuration.

[0485] 16. The human monoclonal IgG4 bispecific antibody as described in embodiment 13, wherein the bivalent second heavy chain lacks the CH1 domain.

[0486] 17. A human monoclonal IgG4 bispecific antibody, comprising:

[0487] (i) binding to the first binding arm of human CD3δε; and

[0488] (ii) Binding to the second binding arm of the human BCMA.

[0489] The first binding arm comprises a first heavy chain and a light chain, the first heavy chain comprising the amino acid sequence of SEQ ID NO:75, and the light chain comprising the amino acid sequence of SEQ ID NO:49; and

[0490] The second binding arm comprises a divalent second heavy chain, which contains the amino acid sequence of SEQ ID NO:76.

[0491] 18. The human monoclonal IgG4 bispecific antibody as described in embodiment 17, wherein the second binding arm does not contain a light chain.

[0492] 19. The human monoclonal IgG4 bispecific antibody as described in embodiment 17, wherein the bivalent second heavy chain comprises a heavy chain-only variable region in a bivalent configuration.

[0493] 20. The human monoclonal IgG4 bispecific antibody as described in embodiment 17, wherein the bivalent second heavy chain lacks the CH1 domain.

[0494] 21. A pharmaceutical composition comprising a bispecific antibody and a pharmaceutically acceptable carrier as described in any one of embodiments 1 to 20.

[0495] 22. A polynucleotide encoding a bispecific antibody according to any one of embodiments 1 to 20.

[0496] 23. A carrier comprising the polynucleotide described in embodiment 22.

[0497] 24. A cell comprising the carrier described in embodiment 23.

[0498] 25. A method for generating a bispecific antibody, comprising growing the cells of embodiment 24 under conditions that allow the expression of the bispecific antibody and isolating the bispecific antibody from the cells.

[0499] 26. A kit comprising a bispecific antibody according to any one of embodiments 1 to 20 and instructions for use.

[0500] 27. A method of treating a subject with BCMA-expressing cancer, comprising administering to the subject a therapeutically effective amount of the bispecific antibody according to any one of embodiments 1 to 20.

[0501] 28. The use of the bispecific antibody as described in any one of embodiments 1 to 20 for the treatment of a subject with BCMA-expressing cancer.

[0502] 29. Use of the bispecific antibody as described in any one of embodiments 1 to 20 in the preparation of a medicament for treating a subject in need of a cancer expressing BCMA.

[0503] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the appended claims define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A bispecific monoclonal antibody that binds to human CD3 and human BCMA, comprising: (i) A first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 49; (ii) A second polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 56, The first and second polypeptide subunits together form the first binding portion that binds to human CD3; and (iii) A third polypeptide subunit that binds to human BCMA, comprising the amino acid sequence of SEQ ID NO: 58; The bispecific monoclonal antibody does not include the light chain that binds to human BCMA.

2. A bispecific monoclonal antibody that binds to human CD3 and human BCMA, comprising: (i) A first polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 49; (ii) A second polypeptide subunit comprising the amino acid sequence of SEQ ID NO: 75, The first and second polypeptide subunits together form the first binding portion that binds to human CD3; and (iii) A third polypeptide subunit that binds to human BCMA, comprising the amino acid sequence of SEQ ID NO:76; The bispecific monoclonal antibody does not include the light chain that binds to human BCMA.

3. A human monoclonal IgG4 bispecific antibody, comprising: (i) binding to the first binding arm of human CD3; and (ii) Binding to the second binding arm of the human BCMA, The first binding arm comprises a first heavy chain and a light chain, the first heavy chain comprising the amino acid sequence of SEQ ID NO: 56, and the light chain comprising the amino acid sequence of SEQ ID NO: 49; and The second binding arm comprises a divalent second heavy chain, which contains the amino acid sequence of SEQ ID NO: 58; the second binding arm does not contain a light chain.

4. A human monoclonal IgG4 bispecific antibody, comprising: (i) binding to the first binding arm of human CD3; and (ii) Binding to the second binding arm of the human BCMA, The first binding arm comprises a first heavy chain and a light chain, the first heavy chain comprising the amino acid sequence of SEQ ID NO: 75, and the light chain comprising the amino acid sequence of SEQ ID NO: 49; and The second binding arm comprises a divalent second heavy chain, which contains the amino acid sequence of SEQ ID NO: 76; the second binding arm does not contain a light chain.

5. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. A polynucleotide encoding the bispecific antibody according to any one of claims 1 to 4.

7. A vector comprising the polynucleotide of claim 6.

8. A cell comprising the carrier of claim 7.

9. A method for generating a bispecific antibody, comprising growing the cells of claim 8 under conditions that allow expression of the bispecific antibody and isolating the bispecific antibody from the cells.

10. A medicine box comprising the bispecific antibody according to any one of claims 1 to 4 and instructions for use.

11. The bispecific antibody of any one of claims 1 to 4, for the treatment of a subject in need of BCMA-expressing myeloma.

12. The bispecific antibody of any one of claims 1 to 4, for the treatment of a subject with BCMA-expressing multiple myeloma.

13. Use of the bispecific antibody as described in any one of claims 1 to 4 in the preparation of a medicament for treating BCMA-expressing myeloma in subjects of need.

14. The use according to claim 13, wherein the myeloma is multiple myeloma.

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